Thioredoxins and gene regulation
in the Drosophila germline
av
Malin J. Svensson
Akademisk avhandling
Som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet för
avläggande av filosofie doktorsexamen i molekylär biologi framlägges till
offentlig granskning i sal B, byggnad 1D, 9 tr, fredagen den 9:e mars, kl. 10.00.
Avhandlingen kommer att försvaras på engelska.
Examinator:
Prof. Elisabeth Sauer-Eriksson
Fakultetsopponent:
Prof. emeritus David Roberts, Genetics Unit, Biochemistry
Department, Magdalen College, Oxford, Storbritannien
Umeå Centre for Molecular Pathogenesis
Umeå University
Umeå 2007
Organisation
UMEÅ UNIVERSITY
Umeå Centre for Molecular Pathogenesis
SE-90187 Umeå, Sweden
Document type
DOCTORAL DISSERTATION
Date of publication
March 2007
Author
Malin J. Svensson
Title
Thioredoxins and gene regulation in the Drosophila germline
Abstract
The process of spermatogenesis is in many organisms one of the most dramatic cellular
transformations - a normal round diploid cell is ultimately transformed into a needle
shaped haploid cell with tightly packaged cell machinery. In Drosophila melanogaster this
process involves several characteristic stages, one of these being the primary spermatocyte stage, which is the stage prior to meiosis. This stage is characterized by a loose
chromatin structure in the nucleus and exceptionally high rates of transcription and
translation to produce essentially all the mRNAs and proteins that are needed later during
spermatid formation. Two proteins that are expressed in high levels in primary
spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF).
Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a
mechanism that is utilized in many different contexts. In this thesis I show that the TrxT
gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome
loops in primary spermatocytes. TrxT is located right next to deadhead (dhd), a gene that
encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A
third thioredoxin in Drosophila is the ubiquitously expressed Thioredoxin-2 (Trx-2). I
have found that flies lacking Trx-2 are viable but have shorter life spans than wild type
flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress.
Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what
could be expected. All three thioredoxins are conserved among Drosophilids and the
striking genomic organization of TrxT and dhd is generally conserved.
The gene name Painting of fourth originates from the finding that POF stains the
4th chromosome of Drosophila in a banded pattern on salivary gland chromosomes. I show
in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in
genus Drosophila and that POF co-localizes with both a protein and a histone modification
associated with dosage compensation in species where POF also binds the male X. POF is
expressed ubiquitously in both males and females, but at very high levels in male testes. I
show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing
spermatids and that a lack of POF in the male germline causes a global down-regulation of
chromosome 4 genes. These results combined suggest a function of POF in the first known
case of chromosome-wide gene regulation of an autosome.
Key words: Drosophila, thioredoxin, sex-specific, Pof, chromosome 4, gene regulation
Language: English
ISBN: 978-91-7264-255-3
Number of pages: 166
Thioredoxins and gene regulation
in the Drosophila germline
Malin J. Svensson
Umeå Centre for Molecular Pathogenesis
Umeå University
Umeå 2007
Umeå Centre for Molecular Pathogenesis
Umeå University
SE-90187 Umeå, Sweden
Copyright © 2007 by Malin J. Svensson
ISBN 978-91-7264-255-3
Printed by Solfjädern Offset AB
To Björn and to my family
“The most exciting phrase to hear in science,
the one that heralds new discoveries, is not
’Eureka!’ (I found it!) but ’That’s funny ...’ “
Isaac Asimov
CONTENTS
CONTENTS
LIST OF PAPERS ................................................................................................ 6
ABBREVIATIONS ............................................................................................... 7
PROLOGUE.......................................................................................................... 8
INTRODUCTION................................................................................................. 9
THIOREDOXINS........................................................................................... 9
Biochemistry ....................................................................................... 10
Physiological roles .............................................................................. 11
Thioredoxins in different cellular compartments ......................... 11
Thioredoxins and disease ............................................................. 12
Thioredoxins in the mammalian germline.................................... 13
Thioredoxins in Drosophila................................................................ 14
deadhead ...................................................................................... 14
Thioredoxin-2 ............................................................................... 15
ThioredoxinT ................................................................................ 16
GENE REGULATION ................................................................................. 17
Cellular memory ................................................................................. 17
The PcG and trxG complexes....................................................... 18
Chromosome-wide gene regulation................................................... 18
Dosage compensation in mammals .............................................. 20
Dosage compensation in C. elegans............................................. 20
Dosage compensation in Drosophila ........................................... 21
Painting of fourth in Drosophila .................................................. 23
Gene regulation in the male germline of Drosophila ....................... 24
Spermatogenesis........................................................................... 24
The Y chromosome ....................................................................... 25
Testis specific gene regulation ..................................................... 26
4
CONTENTS
AIMS .................................................................................................................... 28
MATERIALS AND METHODS ....................................................................... 29
METHODS USED IN THIS THESIS.......................................................... 29
In vitro generation of zeste mutants........................................................... 29
Protein aggregation experiments............................................................... 29
RESULTS AND DISCUSSION ......................................................................... 30
PAPERS I-V ................................................................................................. 30
TrxT encodes a male-specific thioredoxin ........................................ 30
TrxT locates to Y-chromosome loops in primary
spermatocytes............................................................................... 30
Trx-2 mutants have shorter life spans .............................................. 31
Thioredoxin redundancy or specialized functions?...................... 32
TrxT, dhd and Trx-2 are conserved in Drosophilids ........................ 33
TrxT has an extremely variable C-terminal domain .................... 33
Regulatory motifs in the region between TrxT and dhd ............... 34
Pof in genus Drosophila suggests autosome-specific gene
regulation ............................................................................................ 35
Deletion mutants in the Pof promoter region are lethal .............. 36
Pof in spermatogenesis ....................................................................... 37
Pof upregulates chromosome 4 genes in the male testes ............. 37
NEW RESULTS........................................................................................... 39
Thioredoxins can break Zeste aggregates ........................................ 39
CONCLUSIONS ................................................................................................. 42
SUMMARY IN SWEDISH ................................................................................ 43
ACKNOWLEDGEMENTS ............................................................................... 44
REFERENCES.................................................................................................... 46
PAPER I-V
5
LIST OF PAPERS
LIST OF PAPERS
This thesis is based on the following papers, which in the text will be referred to
by their Roman numerals (I-V):
I
Svensson, M.J., Chen, J.D., Pirrotta, V. and Larsson, J. (2003) The
ThioredoxinT and deadhead gene pair encode testis- and ovary-specific
thioredoxins in Drosophila melanogaster. Chromosoma 112:133-143.
II
Svensson, M.J. and Larsson, J. (2007) Thioredoxin-2 affects lifespan and
oxidative stress in Drosophila. Hereditas In press
III
Svensson, M.J., Stenberg, P. and Larsson, J. (2007) Organization and
regulation of sex-specific thioredoxin encoding genes in genus Drosophila.
Manuscript
IV
Larsson, J., Svensson, M.J., Stenberg, P. and Mäkitalo, M. (2004) Painting
of fourth in genus Drosophila suggests autosome-specific gene regulation.
Proc. Natl. Acad. Sci. USA 101:9728-9733.
V
Svensson, M.J., Stenberg, P., Johansson, A-M. and Larsson, J. (2007)
Painting of fourth in Drosophila spermatogenesis. Manuscript
Papers I, II and IV are reproduced with permission from the publishers.
6
ABBREVIATIONS
ABBREVIATIONS
achi/vis
aly
AP-1
can
comr
DCC
dpy
dhd
ECFP
EYFP
GR
H4K16
HP1
mia
mle
mof
MSL
NF-κB
nht
PcG
Pof
Ref-1
ROS
roX
rye
sa
sdc
Sptrx
TAF
TBP
TGR
topi
TPx
Trx-2
TrxG
TrxR
TrxT
Txl
Xa/Xi
Xic
Xist
z
ZIP
achintya/vismay
always early
Activator protein 1
cannonball
cookie monster
Dosage compensation complex
dumpy
deadhead
Enhanced Cyan Fluorescent Protein
Enhanced Yellow Fluorescent Protein
Glutathione reductase
Histone 4 at Lysine 16
Heterochromatin protein 1
meiosis I arrest
maleless
males absent on first
Male specific lethal
Nuclear factor-κB
no hitter
Polycomb Group
Painting of fourth
redox factor 1
reactive oxygen species
RNA on the X chromosome
ryan express
spermatocyte arrest
sex determination and dosage compensation
sperm thioredoxin (mammalian)
TBP-associated factor
TATA-binding protein
thioredoxin/glutathione reductase
matotopetli
Thioredoxin peroxidase
Thioredoxin-2
Trithorax Group
Thioredoxin reductase
ThioredoxinT
Thioredoxin-like
Activated/inactivated X chromosome
X inactivation center
Xi-specific transcript
zeste
Zeste Interacting Protein
7
PROLOGUE
PROLOGUE
It’s hard to tell how it all really started, but if Thomas Hunt Morgan hadn’t found
that white eyed fly one day in 1910, this particular thesis would probably never
have been written. But he did, and in only a few years the fledgling Drosophila
research field took a large step forward. But why did these tiny fruit flies become
so popular for doing research on? Well, for one, they are very convenient to
cultivate. You basically only need some flies, some vials and some rotting fruit
and you’re good to go. Now, rotting fruit is perhaps not the nicest thing to be
around, so nowadays we use a mix of potato-mash, yeast and agar, but the
principle of it is the same. Two other reasons why Drosophila became so popular
have to do with Morgan and his fellow researchers. First, they happily shared all
their fly strains with anyone that wanted them, thereby setting the standard for an
entire research community. Second, a large amount of the early publications on
Drosophila were findings and methods that set the foundation for a large spectrum
of genetic tools, that even to this day are being used. Although the first
Drosophilists were mostly interested in genetics and heredity, the scientific area of
Drosophila development soon drew the interest of an increasing amount of
scholars. These two fields combined eventually resulted in the discovery of a type
of genes that are essential for laying down the body plan in the fly embryo. These
genes were called Hox-genes and were later also discovered in mammals and other
animals. Mutations in many of the Hox-genes produced peculiar phenotypes, like
legs instead of antennae and four wings instead of two. To make sure that the Hoxgenes are not on or off at the wrong time two other groups of genes, the Polycomb
Group and the Trithorax Group, are there to maintain the active or silenced state.
These in turn need regulators and interaction partners of their own, one of them
being the gene zeste. Now zeste happens to be, in its own way, related to the very
beginning of this story, since certain mutations in it does strange things to the
same gene that caused the white phenotype found by Morgan almost one hundred
years ago. But what has all of this got to do with how this thesis came to be?
Well… In the late 90’s a talented young scientist had just earned his PhD and was
in need of a new and interesting project to focus his research on. As it happened
the Professor that had been the opponent at his dissertation was interested in a
collaboration, and so our young scientist went for two month-long visits to the
Professor’s lab. When he came back he brought with him a handful of newly
identified Drosophila genes from a yeast two-hybrid screen for proteins that
interacted with Zeste. The genes were referred to as ZIPs (Zeste Interacting
Proteins) followed by a number, since hardly anything was known about them yet.
The young scientist conducted experiments on several of the genes, but soon two
of them, ZIP16 and ZIP42, stood out as particularly promising. One day a student
looking for an exam-project turned up... and the rest, as they say, is history.
8
INTRODUCTION
INTRODUCTION
Since the work this thesis is based on was divided into two separate parts, this
introduction will also be divided into two parts. But as you will see in later
sections of this thesis, the two parts are perhaps not as separate as they may seem
at first glance.
THIOREDOXINS
The first thioredoxin (Trx) was discovered in 1964, in the bacteria Escherichia
coli, and along with it thioredoxin reductase (TrxR), a flavoenzyme that maintains
thioredoxin in its active, i.e. reduced, state (Laurent et al. 1964; Moore et al.
1964). Only a few years after, thioredoxin was discovered in other bacteria, yeast
and rat (Orr & Vitols 1966; Moore 1967; Gonzalez Porqué et al. 1970). Since then
thioredoxins have been reported in practically every organism studied, including
plants, and in the last ten years the field has essentially exploded with publications
of thioredoxins in new organisms and new biological contexts. But to understand
what thioredoxins are and what they do, one first needs to know a little bit about
the basic biochemistry of thioredoxins.
Figure 1. Schematic view of the thioredoxin protein structure. Alpha helices are
represented by blocks and beta sheets are represented by arrows. N indicates the Nterminal and C indicates the C-terminal. The two cysteines of the active site are shown as
large dots.
9
INTRODUCTION
Biochemistry
Thioredoxins are a family of small thiol proteins characterized by a similar
structure (referred to as the thioredoxin fold) and a conserved active site, trp-cysgly-pro-cys (WCGPC) (Holmgren 1989). In some cases, one or two of the noncysteine amino acids are exchanged for some other amino acid, but WCGPC is the
“classical” and most common active site motif. The typical thioredoxin is a protein
with a size of approximately 12 kDa (Gromer et al. 2004). The basic thioredoxin
fold, a consensus structure model shared with other similar proteins, like for
instance glutaredoxin and DsbA, consists of three alpha helixes and four beta
sheets. Thioredoxins have an additional alpha helix, beta sheet and large connecting loop in the N-terminal part of the protein, with the active site located in the Nterminal end of the second alpha helix, close to the second beta sheet (Figure 1)
(Martin 1995). The basic biochemical function of thioredoxins is to reduce or
oxidize disulfides in other proteins using the two cysteines of its conserved active
site. This essentially means that it creates or breaks inter- or intra-molecular
disulfide bonds. The most common mode of action for thioredoxins is reduction
and in this process the protein itself becomes oxidized and thereby becomes
inactivated. To restore thioredoxin to its active state, Thioredoxin reductase
(TrxR) reduces the thioredoxin disulfide through a reaction that converts NADPH
to NADP+ (Figure 2) (Holmgren 1989).
Figure 2. The thioredoxin system. Thioredoxin (Trx) in its active form (Trx-(SH)2)
reduces disulfides in its target proteins, and thereby becomes oxidized (Trx-S2) and hence
inactivated. To reactivate the oxidized thioredoxin, Thioredoxin reductase (TrxR) reduces
the thioredoxin disulfide in a reaction that turns NADPH into NADP+.
Thioredoxin reductases can be roughly divided into two classes: those containing
selenocysteine, like mammalian TrxRs, but also TrxR-1 from C. elegans and
TrxR-2 from Chlamydomonas, and those containing ordinary cysteine, for
example TrxR from Drosophila, Anopheles, C. elegans (TrxR-2) and Plasmodium.
TrxRs come in two sizes - large (~55 kDa) and small (~35 kDa). A typical
example of a small thioredoxin reductase is TrxR from E. coli, while for example
10
INTRODUCTION
mammals, Drosophila and Plasmodium all have large TrxRs (Gromer et al. 2004).
The conservation among TrxRs is quite high. Drosophila TrxR-1, for example,
shares 56 % sequence identity with the human TrxR-1 and 49 % identity with C.
elegans TrxR-1 on amino acid level (Kanzok et al. 2001).
Thioredoxins have been shown to be involved in many different types of reactions.
For instance, thioredoxin was first reported as an electron donor for ribonucleotide
reductase, a protein that is necessary for the production of deoxyribonucleotides
used in the synthesis of DNA (Orr & Vitols 1966). Later on thioredoxin was
shown to function as a structural component of DNA polymerase in the T7
bacteriophage (Huber et al. 1987). It has also been shown to be important for
maintaining intracellular proteins in a reduced state (Fernando et al. 1992; Stewart
et al. 1998) and to function as a hydrogen donor for 3’-phosphoadenyl-sulphate
(PAPS) reductase in E. coli (Lillig et al. 1999). In the literature in recent years,
thioredoxins have also been increasingly implicated in the cellular system protecting against reactive oxygene species (ROS) and oxidative stress (Nonogaki et al.
1991; Ritz et al. 2000; Jee et al. 2005). A common target for thioredoxins is the
peroxiredoxins, proteins with the major function of reducing hydrogen peroxide
(H2O2) (Hofmann et al. 2002). Peroxiredoxins rely on various thiol proteins and
thiol substances for their reducing functions. Peroxiredoxins that rely exclusively
on thioredoxins are called thioredoxin peroxidases (TPx:s). These are however
only a few of the functions that have been described for thioredoxins, countless
more thioredoxins and biological roles have been reported.
Physiological roles
Thioredoxins in different cellular compartments
Thioredoxins are often described as essentially cytoplasmic proteins that are
responsible for the maintenance of the redox-status of other proteins, but already
in 1989 thioredoxins specifically located in mitochondria of both animals and
plants were reported (Bodenstein-Lang et al. 1989) and ten years later a mitochondrial thioredoxin system in yeast was also published (Pedrajas et al. 1999). In
plants there are more than 20 different thioredoxins (Gelhaye et al. 2005). Among
these are thioredoxins specific for both the mitochondria and the chloroplasts. The
mitochondrial thioredoxin system in Arabidopsis, consists of AtTRX-o1, a novel
thioredoxin with regards to sequence and intron structure, and a thioredoxin
reductase, AtNTRB, with high similarity to the cytosolic thioredoxin reductase
AtNTRA (Laloi et al. 2001; Reichheld et al. 2005). The chloroplast-specific
thioredoxin system is made up of thioredoxins, ferredoxin/thioredoxin reductase
11
INTRODUCTION
and the iron-sulphur protein ferredoxin. This system is very important for the
light-regulation of carbon metabolism (Schürmann 2003).
In mammals, the mitochondrial thioredoxin system is comprised by one
thioredoxin (Trx-2) and one thioredoxin reductase (TrxR2). The mitochondrial
thioredoxin has an important function as hydrogen donor for peroxiredoxin
(Miranda-Vizuete et al. 2000). Mitochondrial peroxiredoxin is a thioredoxindependant protein involved in regulation of cell proliferation and in protection
against the oxidative stress caused by the hydrogen peroxide that is produced by
the mitochondrial metabolism (Immenschuh & Baumgart-Vogt 2005). But the
mitochondrion is not the only cellular compartment in mammals, besides the
cytoplasm, where thioredoxins can be found. Mammalian Trx-1 translocates to the
nucleus when cells are exposed to oxidizing radiation in the form of UV-radiation
but also when exposed to nitric oxide (Hirota et al. 1999; Arai et al. 2006). In the
nucleus thioredoxin enhances the DNA binding capacity of the transcription factor
NF-κB (Hirota et al. 1999) and activates the AP-1 transcription factor trough
interactions with redox factor-1 (Ref-1) (Hirota et al. 1997; Wei et al. 2000). NFκB is a well known regulator of the immune response, but is also involved in
regulating the response to different forms of stress, like ROS and UV-radiation
(Kratsovnik et al. 2005). Thioredoxin has also been found to have functions in the
extracellular compartment. A truncated form of human Trx-1 (in those days
referred to as eosinophil cytotoxicity-enhancing factor, but later referred to as
Trx80) functions as a cytokine, regulating the proinflammatory functions of
human eosinophils (Silberstein et al. 1993). It also works as a chemokine for
monocytes, T lymphocytes and polymorphonuclear leukocytes (Bertini et al.
1999).
Thioredoxins and disease
The thioredoxin that undeniably has been most investigated with regards to human
diseases is human Trx-1. It is a mainly cytosolic protein that under certain
conditions can be translocated into the nucleus or be secreted for extracellular
functions. Changes in Trx-1-levels have been associated with a number of
different diseases. In HIV infected individuals thioredoxin levels are elevated in
blood serum (Nakamura et al. 1996, 2001). This may be due to the increase in
oxidative stress that has repeatedly been reported for HIV-patients (Pace & Leaf
1995). Although full length Trx-1 suppresses production of the virus, Trx80, the
truncated form of Trx-1, counteracts this effect and enhances HIV-production
(Newman et al. 1994). Increased thioredoxin levels have also been found in
rheumatoid arthritis and related diseases (Maurice et al. 1999). Thioredoxin seems
to be a double edged sword in many studied human diseases, not only in HIV, as
mentioned above, but also in cancer and other diseases. In early stages it can
12
INTRODUCTION
counteract the disease through protection against induced oxidative stress, while in
later stages it promotes the disease by increasing cell proliferation (Iwata et al.
1997). Many carcinogens exert their deleterious mechanisms through free radicals
and ROS, a step where the antioxidative properties of thioredoxin and the other
components of the thioredoxin system has the potential to protect cells through
elimination of harmful radicals (Gromer et al. 2004). Thioredoxin thereby plays a
preventive role in the mechanisms that can lead to cancer, but when the growth of
a tumor once has started the positive effects of Trx-1 on cell proliferation and its
protective effects against the immune system promotes further tumor growth and
thereby worsens the condition (Andersson et al. 1996; Grogan et al. 2000).
Interest in thioredoxins and the thioredoxin system as targets for a range of
therapeutic measures in a variety of diseases has increased dramatically in recent
years. Although very promising due to the extensive number of conditions where
the thioredoxin system has been implicated, the diverse and sometimes ambiguous
functions of thioredoxins in vivo complicates matters and demands further
research and extended knowledge before thioredoxins can serve as large scale
therapeutic targets.
Thioredoxins in the mammalian germline
The mammalian testis-specific thioredoxin system is composed of a thioredoxin
reductase (TGR) and four thioredoxins or thioredoxin-like proteins, Sptrx-1,
Sptrx-2, Sptrx-3 and Txl-2. The first three are testis/spermatid specific while the
last, Txl-2 is associated with microtubules in flagella and cilia (Miranda-Vizuete et
al. 2004). TGR differs from most other thioredoxin reductases in that it is not
exclusively reducing disulfides in thioredoxins, but also reduces glutathione (Sun
et al. 2001). This is accomplished by two separate domains in the protein, a small
N-terminal glutaredoxin-like domain and a C-terminal thioredoxin reductase
domain. TGR is highly expressed in the testis, especially at the site of mitochondrial sheath formation in early spermatids, but it is also expressed elsewhere, for
example in the heart, the lung and the liver (Su et al. 2005). Sptrx-1 was the first
spermatid specific mammalian thioredoxin to be identified (Miranda-Vizuete et al.
2001). It is a 486 amino acid protein, with two distinct domains. The N-terminal
domain is constituted of a 15-residue motif repeated 23 times. This is a novel
domain predicted to have a coiled-coil structure, while the second domain can be
described as a typical thioredoxin (Jimenez et al. 2002). Sptrx-1 protein is
expressed in elongating spermatids, specifically located to longitudinal columns of
the fibrous sheath of the sperm tail (Yu et al. 2002). The second sperm specific
thioredoxin to be discovered, Sptrx-2, is a 588 amino acid protein (Sadek et al.
2001). It consists of two parts, just like Sptrx-1, but in this case, the thioredoxin is
located in the N-terminal part of the protein and then followed by a repeat of three
NDP kinase domains. Sptrx-2 has been suggested to be a structural part of the
13
INTRODUCTION
fibrous sheath, since enzymatic activity of neither the thioredoxin nor NDP kinase
domains could be found in vitro and the protein locates to the principal piece of
the flagellum (Miranda-Vizuete et al. 2003). Txl-2, the third thioredoxin with a
strong localization to testis and spermatids, is very similar to Sptrx-2 but differs in
two regards. Like Sptrx-2 it has an N-terminal thioredoxin, however, the thioredoxin domain is only followed by one NDP kinase domain (Sadek et al. 2003).
Another thing that differs from the other spermatid specific thioredoxins is that,
although Txl-2 is expressed at high levels in spermatids and located to the microtubule-based spermatid manchette and axoneme, it is also expressed in cilia of the
lung and at low levels in various other tissues (Sadek et al. 2003). The fourth, and
as of yet, last mammalian spermatid specific thioredoxin, Sprtx-3, stands out from
the others in that it lacks any extra domains besides the thioredoxin domain
(Miranda-Vizuete et al. 2004). The Sptrx-3 gene is located only 50 kB downstream of Trx-1 in the human genome and shares large sequence similarity with
Trx-1, from which it is believed to have originated through a duplication event
(Jiménez et al. 2004). Sptrx-3 is located in the Golgi apparatus in elongating
spermatids and pachytene spermatocytes, but also briefly located in the developing
achrosome. It is important to keep in mind that 3 of the four mammalian testis
specific thioredoxins would probably not be classified as thioredoxins due to their
large extra domains, but rather thioredoxin-like proteins, if they were to be
conventionally named.
Thioredoxins in Drosophila
In most organisms there are two separate systems for thioredoxins and glutaredoxins and they function side by side, performing similar tasks with the same
basic function of donating electrons. However, in Drosophila melanogaster, as
well as in Anopheles gambiae, there is no gene coding for glutathione reductase
(GR) (Kanzok et al. 2001; Bauer et al. 2003a). It is therefore quite likely that the
thioredoxin system (and thioredoxin reductase especially) substitutes for GR in
reducing the oxidized form of glutathione in both fruit flies and mosquitoes.
Drosophila thioredoxin reductase (TrxR) has been extensively characterized in
vitro (Bauer et al. 2002, 2003b), however very few studies have attempted any
studies of DmTrxR in vivo (Orr et al. 2003).
deadhead
The first thioredoxin gene to be discovered in Drosophila melanogaster was the
female specific thioredoxin deadhead (dhd), also referred to as DmTrx-1. dhd was
found in a screen for recessive female-sterile mutations in genes that mapped
within a certain region (4E1-2 - 4F11-12) on the X chromosome (Salz et al. 1994).
14
INTRODUCTION
Female homozygous dhd mutants were shown to lay eggs that seemed morphologically normal, but that in a majority of cases failed to initiate development. In
10 % of the cases, where embryos of later stages could be found, the embryos had
asynchronous nuclear divisions. At developmental stage ten these embryos had
areas of the cortex that lacked nuclei and some embryos also had nuclei that varied
in size, suggesting polyploidy. These phenotypes together suggested an arrest in
meiosis, but it was later shown by another research group to be an arrest
immediately after meiosis, but before the S-M cycles (Page & Orr-Weaver 1996;
Elfring et al. 1997). In 5 % of the cases the embryos developed far enough to
secrete cuticle, but had severe head defects. Male fertility was not affected in these
mutants. Using Northern blots and in situ hybridizations Salz and colleagues show
that dhd RNA is expressed mainly in the female ovaries after oogenesis stage nine,
specifically expressed in the nurse cells, but not in the somatic follicle cells (Salz
et al. 1994). They also show that dhd RNA is maternally contributed to the
embryo, where it is evenly distributed and prevails until the four hour stage.
However, another research group have later shown that maternally contributed dhd
RNA is enriched in the pole cells in stage five embryos, and that the embryo itself
expresses dhd RNA in the pole cells and the embryonic gonad at stage ten and
sixteen (Mukai et al. 2006).
The DHD protein consists of 107 amino acids and is 32 % and 36 % identical to
yeast and human thioredoxins, respectively. The two cysteines of its conserved
active site are necessary for the function of the protein in vivo, which suggests that
the protein has a redox-function in the fly body. However, DHD does not play a
role in DNA synthesis (Pellicena-Palle et al. 1997). DHD has been biochemically
characterized in vitro and was found to be a 12.4 kD protein with a classical
thioredoxin fold (Kanzok et al. 2001). Kanzok and colleagues have also shown
that DHD is able to efficiently reduce glutathione disulfide and that it is
effectively reduced by Thioredoxin reductase (TrxR), but that it does not function
as a substrate for Thioredoxin peroxidase (TPx-1) (Kanzok et al. 2000, 2001;
Bauer et al. 2002). The details of DHD’s physiological functions in vivo are still
largely unknown.
Thioredoxin-2
The next Drosophila thioredoxin gene to be cloned and published was
Thioredoxin-2 (Trx-2 or DmTrx-2) (Bauer et al. 2002). The Trx-2 protein has been
biochemically characterized; it was found to be able to reduce glutathione as
efficiently as DHD. It was also found to be oxidized by TrxR and to be able to
function as a substrate for TPx-1 (Bauer et al. 2002). Bauer and colleagues also
showed that Trx-2 is expressed in larvae, adults of both sexes and the embryonically derived Schneider cell line. The difference in potential as substrate for TPx-1
15
INTRODUCTION
between Trx-2 and DHD, coupled with a fairly low amino acid sequence identity
(only 38 %) between the two proteins and the difference in expression pattern
suggests a difference in functionality (Bauer et al. 2002). Trx-2 has also been
crystallized in both reduced an oxidized form (Figure 3) (Wahl et al. 2005). Trx-2
was shown to be structurally very similar to thioredoxins from many other
organisms (considering the large evolutionary time span since the divergence of
ancestral species), including E. coli and humans, which is consistent with the
similarities in amino acid sequence (34 % and 47 % identity, respectively).
Despite the large similarities with human thioredoxin, oxidized Trx-2 did not form
dimers under experimental conditions, and was thereby deemed not likely to form
dimers in vivo, in contrast to the human thioredoxin (Wahl et al. 2005).
Figure 3. Ribbon plot of oxidized Drosophila melanogaster Trx-2. α1-α4 indicates the
four alpha-helices and β1- β5 indicates the five beta-sheets. Alpa-helices and beta-sheets
are numbered from the N to the C terminus. C32 and C35 indicate the redox-active
disulfide. Adapted from Wahl et al. (2005).
ThioredoxinT
The third thioredoxin gene that was discovered in Drosophila is ThioredoxinT
(TrxT). It was found with a yeast two-hybrid screen for proteins that interact with
the PcG-group protein Zeste (Chen 1992), and was in the initial stages of this
work referred to as ZIP42 (short for Zeste-interacting protein 42).
16
INTRODUCTION
GENE REGULATION
Gene regulation can be thought of as the multitude of different ways in which the
genes of an organism can be controlled. There are many different systems and
mechanisms controlling the ‘wheres’ and ‘whens’ of the expression of an
organism’s genes. The most important basal mechanism that genes in general
encounter is transcription, where the genetic information is copied into RNA, the
medium that is utilized as a template in the mechanism that converts gene
information into a functional protein. Many of the mechanisms that regulate genes
are in some way related to transcription, either by directly affecting the
transcription machinery by helping it or interfering with it, or more indirectly
affecting it by attracting it or repelling it. A good example of a category of
proteins that directly affect transcription is the transcription factors. These are
proteins that regulate the initiation of transcription and the binding of RNA
polymerase (the major component in the transcription machinery) to DNA (Turner
2001). Transcription factors can either be enhancers or repressors, that is, they
either assist or block RNA polymerase binding. Many transcription factors bind
both DNA (often in the promoter or enhancer region of a gene) and other
transcription factors. Transcription and transcription factors are regulated both by
the chromatin structure of the gene region that they bind, by epigenetic markers
present on the DNA and by the presence of other DNA binding proteins. Two of
the important indirect mechanisms that contribute to gene regulation are the
cellular memory that maintains correct expression of the genes governing
development and the chromosome-wide gene regulation often associated with
dosage compensation.
Cellular memory
In basically all higher organisms (from yeast to plants and animals) there is a
category of genes called the homeobox genes (Levine et al. 1984; Kaufmann
1993; Williams 1998). These genes all have a conserved domain, the homeobox,
which in its protein form can bind to DNA. The Homeobox genes are generally
transcription factors. In nematodes and higher animals, a certain category of the
homeobox genes are called homeotic genes or Hox genes (after the Hox gene
cluster in which they are situated) (Garber et al. 1983; Scott et al. 1983; Way &
Chalfie 1988). The homeotic genes are very important to any organism that has
them since they determine the layout of the organism’s body plan and they have
been described as the master regulators of development (Turner 2001). Hox genes
are highly conserved through evolution and are generally organized in conserved
clusters in the genome. The first homeotic genes, before they were even called
Hox genes, were found in Drosophila (Lewis 1978). There are two clusters of
17
INTRODUCTION
homeotic genes in Drosophila: the Antennapedia complex (ANT-C) and the
Bithorax complex (BX-C) (Lewis 1978; Lewis et al. 1980a-b). The Antennapedia
complex is responsible for the segment identity of the head and the thorax and
consists of labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced
(Scr) and Antennapedia (Antp). The Bithorax complex is responsible for the
segments in the abdomen and consists of Ultrabithorax (Ubx), abdominal-A (abdA) and Abdominal-B (Abd-B). If all homeotic genes would be active all the time,
every bodysegment in an organism would look exactly the same. To make sure
that only the right homeotic genes are on at the right time there is a transient
regulation performed by gap genes and segmentation genes, that activates or
silences the homeotic genes. To maintain the activated or silenced states of the
homeotic genes there is a controlling system commonly referred to as the cellular
memory. The cellular memory generally consists of two systems, one that
mediates maintenance of the active state and another that mediates maintenance of
the repressed state.
The PcG and trxG complexes
The cellular memory system in Drosophila that maintains the correct expression
of the homeotic genes is made up of two basic units; the Polycomb Group (PcG)
complex, which is responsible for maintaining the inactive state of genes that have
initially been turned off, and the trithorax Group (trxG) complex, which is
responsible for maintaining the active state of genes that have initially been turned
on. The complexes bind specific regulatory elements in the vicinity of the
homeotic genes, where they exert their activating or silencing functions. These
regulatory elements are commonly called PcG or TrxG response elements
(PREs/TREs) (Simon et al. 1993). Both of these protein complexes contain a
number of different proteins. Some examples of genes coding for PcG components
are Polycomb (Pc), Posterior sex combs (Psc) , polyhomeotic (ph), Enhancer of
zeste (E(z)), extra sexcombs (esc), and Suppressor of zeste 12 (Su(z)12), and
examples of trxG component genes are trithorax (trx), brahma (brm), absent,
small or homeotic discs 1 and 2 (ash-1, ash-2) and zeste (z), and there are many
more (reviewed by Ringrose & Paro 2004, and Schwarts & Pirrotta 2007). It is not
known exactly how the PcG and trxG complexes maintain the silenced or
activated states of the homeotic genes, despite decades of research, but histone
methylation has been repeatedly shown to be involved in both cases. Both mono,
di and tri-methylation of different lysines of the histone tails have been shown for
the PcG complexes (Schwarts & Pirrotta 2007). Histone de-acetylation has also
been correlated with PcG silencing., while acetylation has been connected to trxG.
In recent years, more and more reports suggest that silencing is the default state
and at least two of the TrxG proteins have been proposed to be repressors of
silencing rather than true activators (Poux et al. 2002; Klymenko & Müller 2004).
18
INTRODUCTION
Chromosome wide gene regulation
In most organisms the loss of one of the two copies of any chromosome leads to
lethality (with some exceptions). This is because the total transcript dose of all
expressed genes on that chromosome goes down to half that of the normal level
and the combined effect of all these drastic alterations in transcript level is too
much for the cell to handle. In many organisms however, males and females have
different numbers of sex-determining chromosomes, i.e. one sex is homogametic
(e.g. XX for mammalian females and ZZ for male birds) while the other is heterogametic (e.g. XY for mammalian males and ZW for female birds). In most cases
the Y or W chromosome has lost the majority of genes it perhaps once had in
common with its partner chromosome and mainly contains sex-determining genes
and/or genes that are exclusively involved in fertility. The heterogametic sex is
thereby effectively haploid for all the genes on the X or Z chromosome. This
would present a problem for the cell if it were not for the phenomenon of dosage
compensation.
Dosage compensation is the way in which an organism compensates for the
inequality in gene dosage on the sex-chromosome common to both sexes. This has
been solved in many different ways through evolution, the best known being those
of mammals, nematodes and fruit flies. The modes of dosage compensation in
these three organisms differ quite a lot. However, recent research in the field
suggests that they are all based on an initial general up-regulation of the X
chromosomes, followed by specific mechanisms to equalize the dose between the
sexes (Gupta et al. 2006). In mammals, one X chromosome becomes inactivated
(X-inactivation), while in C. elegans transcription on both hermaphrodite X
chromosomes is downregulated to half (hypo-transcription), in both cases compensating for a general up-regulation of the X chromosomes compared to the autosomes (Figure 4). Drosophila differs from the other two in that the dosage
compensation occurs in males, where transcription of the male X is upregulated
two-fold (hyper-transcription) (Figure 4).
Figure 4. Three variants of dosage compensation. A) X-inactivation in mammals. B)
Hypotranscription in the nematode Caenorhabditis elegans. C) Hypertranscription in the
fruit fly Drosophila melanogaster.
19
INTRODUCTION
Dosage compensation in mammals
In mammalian dosage compensation there are basically three important steps: i.
up-regulation of the X chromosome in males and females, ii. inactivation of one of
the female X chromosomes and iii. maintenance of silencing of the inactivated X
(Xi) (Heard & Disteche 2006). It has been known for a long time that one of the
female X chromosomes becomes inactivated, forming the Barr body (Lyon 1961).
However, it was not until resent years that it became clear that there is also upregulation of the X chromosomes in relation to the autosomes, i.e. step one listed
above (Adler et al. 1997; Gupta et al. 2006; Nguyen & Disteche 2006). This upregulation is particularly strong in the brain and absent in haploid germ-cells. It is,
as of yet, not known what is controlling this up-regulation, whether it is caused by
a protein complex or by evolutionary changes in the regulatory regions of X genes
as an indirect response to loss of the corresponding genes on the Y chromosome
(Nguyen & Disteche 2006).
Much more is known about what factors are controlling the inactivation and
maintenance of silencing of one of the female’s X chromosomes. For X
inactivation to occur at all, a cell must have (at least) two copies of a complex X
chromosome region called the X inactivation centre (Xic) (Rastan & Robertson
1985). The Xic, which is also involved in the “chromosome counting” that
determines whether X inactivation should occur at all (Alexander & Panning
2005), contains the Xist gene, which codes for a non-coding RNA (Brown et al.
1991). The Xist RNA is expressed exclusively from the inactivated chromosome
and coats its entire length (Brown et al. 1992; Clemson et al. 1996). Partially
overlapping the Xist gene lies Tsix, which codes for an Xist antisense RNA that is
involved in early regulation of Xist (Lee et al. 1999). The mechanism for this is
not fully known, but transient colocalization of the two Xics, suggesting crosstalk, has recently been shown (Bacher et al. 2006). Other elements located in the
Xic, e.g. Xite, have also been connected with Xist regulation (Ogawa & Lee 2003).
The major function of Xist RNA is to draw silencing complexes to the inactivated
X, for example PRC1 and PRC2 (Plath et al. 2003). These are involved in histone
modifications of various kinds, but other modifications, for instance DNA
methylation, are also typical for the Xi. Some examples of histone modifications
are the hypoacetylation of histone H4 (Keohane et al. 1996), the enrichment of H3
Lys-27 trimethylation (Plath et al. 2003; Silva et al. 2003) and H2A K119
monoubiquitylation (de Napoles et al., 2004; Fang et al. 2004). Many of these
modifications have been, or are suggested to be, connected with irreversible
silencing of the Xi (Kohlmaier et al. 2004).
20
INTRODUCTION
Dosage compensation in C. elegans
In the nematode C. elegans both the sex of an individual and the dosage compensation is determined by the ratio of X chromosomes to autosomes. All ratios below
2/3 result in male development, while ratios of 3/4 and above result in hermaphrodite development and dosage compensation in the form of down-regulation of
both X chromosomes (Cline & Meyer 1996). In C. elegans a dosage compensation
complex (DCC) has been identified, a complex that in turn consists of two subcomplexes with several members, some of which are also involved in sex determination. One subcomplex, the DPY-complex (named after genes with the dumpy
phenotype) consists of DPY-26, DPY-27, DPY-28 and MIX-1, while the other
subcomplex, SDC (named after a group of genes found in a screen for sex determination and dosage compensation mutants) is composed of SDC-1, SDC-2, SDC3, DPY-21 and DPY-30 (Lucchesi et al. 2005). The DPY-complex can be
described as an adapted form of the condensin complex (the MIX-1 protein is for
example a member of both complexes). The condensin complex is a chromosome
associated complex necessary for sister chromatid resolution and DNA compaction, both during mitosis and meiosis (Hagstrom & Meyer 2003). Recruitment
of the DPY-complex to X chromosomes requires at least three of the components
of the SCD-complex: SDC-2, SDC-3 and DPY-30.
The C. elegans Dosage Compensation Complex is initially recruited to the X
chromosome at a number of recruitment sites and later spread in cis along the
chromosome (Csankovszki et al. 2004). The DCC not only binds to the X chromosome, it also locates to the her-1 gene, where it acts as a switch for hermaphrodite
development by a 20-fold repression of her-1 (Davis & Meyer 1997). The master
switch for both dosage compensation and sex determination is the gene xol-1. The
functional role of xol-1 is to repress sdc-2, thereby also blocking her-1 repression,
resulting in simultaneous repression of dosage compensation and activation of
male differentiation (Miller et al. 1988). It was not known until very recently that
also the nematode X chromosomes are generally upregulated compared to the
autosomes, in both males and females (Gupta et al. 2006). Very little has so far
been reported in the subject, but the suggested up-regulation would certainly
provide an important and logical piece in the puzzle, since it is otherwise hard to
explain how an organism can survive with a general X chromosomal transcription
level corresponding to that of a single autosome, i.e. leading to haploinsufficiency.
With a general up-regulation of the male and female X chromosomes, a dosage
compensation system that halves the transcription level of the female X:s would
lead to an equalized transcription level of all chromosomes, in both sexes.
21
INTRODUCTION
Dosage compensation in Drosophila
Contrary to mammals and nematodes, the fruit fly has a form of dosage compensation that affects males only. In Drosophila the single male X is upregulated twofold, equalizing the expression from the male X to that of the two X chromosomes
in females (Larsson & Meller 2006). The core dosage compensation complex
(DCC) in Drosophila consists of the five proteins MSL-1, MSL-2, MSL-3, MLE
and MOF and either one of two non-coding RNAs. The genes encoding the five
proteins are named after their characteristic male-lethal phenotypes when mutated:
male specific lethal -1, -2 and -3, maleless and males absent on first. mle was first
discovered by Fukunaga and coworkers (1975) but was not really connected with
dosage compensation of the single male X chromosome until 1980 (Belote &
Lucchesi 1980b). The same year, Belote and Lucchesi had published the finding of
two additional sex specific lethals: msl-1 and msl-2 (Belote & Lucchesi 1980a),
but it took many years before the two remaining components were found;
characterization of msl-3 was first published by Gorman and coworkers (1995)
and mof was found by Hilfiker and coworkwers (1997). The two non-coding
RNAs, roX1 and roX2, were published in the same issue of Cell (Meller et al.
1997; Amrein & Axel 1997). They were later shown to be essential components of
the dosage compensation machinery (Franke & Baker 1999).
All MSL proteins, except MSL-2, are expressed also in females. This means that
MSL-2 acts as a master switch for the entire complex. In the presence of the sexdetermining protein SXL (which is the component that acts first in the cascade
leading to female-specific development) MSL-2 is repressed, but in its absence
MSL-2 is expressed, thereby enabling complex formation (Bashaw & Baker 1995,
1997). If MSL-2 is ectopically expressed in females, the dosage compensation
system is switched on, resulting in up-regulation of the female X chromosomes,
virtually rendering them transcriptionally tetraploid, which leads to lethality
(Kelley et al. 1995). The first step in the formation of the DCC is an interaction
between MSL-1 and MSL-2, via MSL-1s coiled-coil domain and a RING finger in
MSL-2 (Lucchesi et al. 2005), enabling both binding of the other components and
binding of the complex to DNA (Lyman et al. 1997; Copps et al. 1998). Next, the
histone acetyltransferase MOF binds to MSL-2 and MSL-3 associates with MSL-1
after acetylation by MOF (Gu et al. 1998; Scott et al. 2000; Buscaino et al. 2003;
Morales et al. 2004). The fifth protein subunit, MLE, is an ATP dependant
DNA/RNA helicase that only seems to associate with the other proteins through
one of the two roX RNAs (Richter et al. 1996; Lee et al. 1997). All five DCC
proteins are essential for male survival, while either one of roX1 or roX2, but not
both, can be removed without effect (Meller & Rattner 2002; Deng et al. 2005).
22
INTRODUCTION
The dosage compensation complex is targeted to the male X via 25-35 entry sites,
the roX genes being two of the first sites where the complex binds (Kelley et al.
1999; Gu et al. 2000; Demakova et al. 2003). From these sites, binding of the
complex spreads in cis to sites with lower affinity. Recent publications suggest
that the complex binds to actively transcribed genes (Alekseyenko et al. 2006).
MSL localization to the X can be seen as a banded pattern along the whole
chromosome when visualized with antibody staining against any of the complexcomponents. Intriguingly, the MSL complex does not locate to the X chromosome
in the male germline, even though it has been shown to be dosage compensated
(Rastelli & Kuroda 1998; Gupta et al. 2006). Essentially nothing is so far known
about either the mechanism or the components that might be involved in the upregulation of the X chromosome in the male germline.
Painting of fourth in Drosophila
The tiny, largely heterochromatic, 4th chromosome in Drosophila is the only
autosome that can be present in only one copy without causing lethality (Hochman
1976). This has often been explained by the fact that the 4th chromosome is so
small; it only contains about one hundred annotated genes, compared to several
thousand each for the other chromosomes. It was also the most plausible explanation until the gene Painting of fourth (Pof) was discovered by Larson and coworkers (2001). Pof is situated in region 60E on chromosome 2R and encodes a
novel protein with an RNA-binding domain (RRM1). Pof RNA levels are the
highest in males, with lower levels in pupae and larvae. Pof RNA is also present in
females but at much lower levels and there is hardly any maternal contribution of
Pof RNA. Antibody staining of POF protein on salivary gland polytene chromosomes reveals a specific localization of POF to the 4th chromosome, hence the
name Painting of fourth (Larsson et al. 2001). The staining is equally strong in
males and females, despite the difference in Pof RNA levels in male and female
flies. Larsson and coworkers could, through a number of translocations between
the 4th and some of the other chromosomes, show that binding of POF to chromosome 4 was dependent on a proximal region of the chromosome. The binding then
spreads in cis over the remainder of the chromosome. The spreading is also
dependent on sequences or chromatin structures specific to chromosome 4, since it
will not spread into autosomal pieces translocated to the 4th, but can spread to
translocated chromosome 4 regions in trans.
Different numbering systems of the chromosomes have been used in different
Drosophila species. Therefore it is better to use Muller’s classification of
Drosophilid chromosomes, where D. melanogaster’s 4th chromosome corresponds
to the F-element (Muller 1940). In the related species Drosophila busckii the Felement is part of the X-chromosome. In males, but not females, of this species
23
INTRODUCTION
POF staining is seen along the entire X-chromosome, suggesting an involvement
of POF in the dosage compensation system of D. busckii (Larsson et al. 2001).
Since spreading of POF in D. melanogaster is dependent on chromosome 4
specific sequences or structures, Larsson and colleagues suggested that the DINE1 element, which is a short repetitive sequence highly enriched in chromosome
four but also in other heterochromatic chromosome regions (Locke et al. 1999), is
a possible candidate as a target for POF. A DINE-probe did however not specifically hybridize to the X chromosome in D. busckii, either suggesting that the DINE
element is not involved in POF targeting or that the element has diverged to a
point where a D. melanogaster DINE-probe will no longer cross-hybridize with a
D. busckii DINE fragment. A few years later it was shown that a group of short
sequence motifs, which highly contributed to separating the 4th chromosome from
the other chromosomes in a multivariate sequence signature analysis, resided
within DINE-1 elements (Stenberg et al. 2005). Regions of chromosome 4 with
low numbers of these motifs correlate well with regions where POF does not bind
on polytene chromosomes. This suggests that DINE-1 elements, or sequence
motifs colocalized with them, are directly or indirectly involved in POF’s binding
to chromosome four (Stenberg et al. 2005). All of these findings together suggest
that POF is part of a regulatory complex for the 4th chromosome, which is quite
extraordinary since it is the first autosome specific regulatory complex.
Gene regulation in the male germline of Drosophila
Spermatogenesis
The Drosophila male reproductive tract consists of several different organs; the
testes, the testicular ducts, the seminal vesicles, the accessory glands, the anterior
ejaculatory duct, the ejaculatory bulb and the posterior ejaculatory duct, with the
major part of spermatogenesis carried out in the testis (Fuller 1993). Located in the
tip of the testis is a cluster of cells, referred to as the germinal proliferation center.
This cluster consists of three different cell types; the apical cells, the germline
stem cells and the cyst progenitor cells. The first step in spermatogenesis is when
one of the stem cells goes through a radially oriented mitotic division, resulting in
two daughter cells, one stem cell and one primary spermatogonial cell (Figure 5).
The spermatogonial cell goes through another mitotic division followed by further
mitosis in its daughter cells (four rounds of mitosis in total), ultimately resulting in
a cyst of 16 primary spermatocytes (Fuller 1993). The primary spermatocytes
grow and mature. During this phase many genes that were previously silenced are
activated and expressed at high levels and essentially all the RNA and proteins
that are needed later during spermatogenesis are produced, since transcription is in
essence shut down at the onset of meiosis. In these cells the Y chromosome,
24
INTRODUCTION
forming large loop-like structures, is an especially prominent feature when viewed
with light microscopy. The growth phase is followed by meiosis, producing a cyst
of 64 haploid early spermatids. The early spermatids go through extensive differentiation, radically transforming them from round cells with ‘normal’ internal
architecture to highly elongated cells with tightly packaged cell machinery. A
morphologically very characteristic sub-stage in spermatid differentiation is the
onion stage, where the individual mitochondria have fused and become packaged
into a dense sphere, giving the cell the look of a bowl with two marbles in it, one
black (the mitochondrial sphere) and one white (the nucleus). In the later part of
spermatid differentiation, elongation of the spermatid takes place. During this
phase the flagellar axoneme is assembled, eventually forming the main part of the
sperm tail. The very last stage of spermatogenesis includes the individualization
and coiling of the mature sperm (Fuller 1993).
Figure 5. Schematic view of Drosophila spermatogenesis. Adapted from Fuller (1993).
The Y chromosome
The Drosophila Y chromosome is metacentric, with a long and a short arm.
Sequence homology between the Y and the X, both in genes and repetitive
sequences, is extremely low, so low that it has been proposed that the Y chromosome does not evolutionarily stem from the X, but from a B-chromosome. This is
supported by the fact that the majority of the few known genes on the Y chromosome have ancestral genes on the autosomes and not on the X (Carvalho 2002). A
very high number of them are also genes involved in male fertility. Recent data
regarding the Y chromosome in D. pseudoobscura suggests that the D. melano-
25
INTRODUCTION
gaster Y and the D. pseudoobscura Y are not homologous. Out of 15 genes and
pseudogenes on the D. pseudoobscura Y none had homologs on the D. melanogaster Y, while the six known D. melanogaster Y genes all had orthologs on
D. pseudoobscura autosomes, suggesting that the D. pseudoobscura Y has arisen
de novo from autosomes (Carvalho & Clark 2005).
In most Drosophila cells the Y chromosome is highly heterochromatic, but in the
primary spermatocytes of the male testis the Y chromosome takes on a huge
floated form. These peculiar structures are called the Y chromosome loops and
have been identified as the three fertility factors ks-1, kl-3 and kl-5 (Bonaccorsi et
al. 1988). The fertility factors (kl-5, kl-3, kl-2 and kl-1 on the long arm of the Y
chromosome and ks-1 and ks-2 on the short arm), are gigantic genes with
numerous and large introns containing repetitive sequences, but also a few smaller
genes. Only a handful of genes have been identified on the D. melanogaster Y
chromosome; e.g. kl-2 and kl-3 encode dynein heavy chain polypeptides and ks-1
and ks-2 have been suggested to correspond to an occludin-related gene and a
coiled-coils gene respectively (Carvalho et al. 2000, 2001). The Y chromosome
loops have been suggested to function as storage compartments for proteins that
are expressed in large quantities in the primary spermatocytes but not needed until
later in spermatogenesis (Hennig et al. 1989), but some of the proteins reported to
bind to the Y have also been suggested to be involved in regulation of some of the
Y genes (Marhold et al. 2002).
Testis specific gene regulation
There are two separate systems in Drosophila ensuring correct expression and
translational timing of testis specific genes (White-Cooper et al. 1998). The first
system, which is responsible for transcriptional activation of a large number of
testis specific genes, is comprised of the meiotic arrest genes always early (aly),
achintya/vismay (achi/vis), cookie monster (comr) and matotopetli (topi). The first
aly allele was found in a screen for mutations induced by transposable elements
(Cooley et al. 1988). aly was later shown to be a homolog of a C. elegans gene,
lin-9 (White-Cooper et al. 2000), a gene involved in regulating vulval cell fate
specification (Ferguson & Horvitz 1989). The two tandem-repeat genes achi and
vis encode TALE-class homeodomain transcription factors and are homologs of
human TGIF (Ayyar et al. 2003). comr was found in an EMS-screen for male
sterile mutations and encodes a novel acidic protein (Jiang & White-Cooper 2003).
comr and aly are mutually dependent. topi was found in the same screen as comr
and encodes a testis-specific Zn-finger protein (Perezgasga et al. 2004). The
protein products of the four genes are believed to be part of a protein complex that
regulates the transcription of approximately one thousand genes specifically
expressed in the testes.
26
INTRODUCTION
The second system, which is responsible for another set of testis specific genes
(among them genes required for spermatid differentiation), is comprised of five
TAF-homologs: cannonball (can), no hitter (nht), spermatocyte arrest (sa),
meiosis I arrest (mia) and ryan express (rye). The can gene was discovered in a
screen for male-sterile mutations and encodes a homolog of dTAF5 (Lin et al.
1996; Hiller et al. 2004). nht, mia, sa and rye were all found in a search for second
homologs of known TAF-genes (Hiller et al. 2004). TAFs (TBP-associated factor)
are proteins that together with TBP (TATA-binding protein) form the general
transcription factor TFIID (Albright & Tjian 2000). nht encodes a dTAF4 homolog, sa encodes a homolog of dTAF8, mia a homolog of dTAF6 and rye a homolog of dTAF12 (Hiller et al. 2004). These are believed to form a complex that is
necessary for the regulation of genes involved in terminal spermatid differentiation. One example is the twine/cdc25 phosphatase, which is necessary for proper
chromosomal segregation during the transit from G2 to M in meiosis (WhiteCooper et al. 1993).
27
AIMS
AIMS
The aims of the work presented in this thesis have been:
ƒ
To clone the TrxT gene and investigate the expression patterns of the two sexspecific Drosophila melanogaster thioredoxins.
ƒ
To create mutants in the Drosophila melanogaster Trx-2 gene and investigate
the mutant phenotypes and potential redundancy.
ƒ
To investigate the conservation of TrxT, DHD and Trx-2 and the TrxT – dhd
gene region in other insect species.
ƒ
To create mutants in the Pof gene and investigate whether POF’s localization
to chromosome 4 is conserved in evolution.
ƒ
To investigate POF’s expression and function in the male germline.
ƒ
To investigate the connection between Zeste and thioredoxins.
28
MATERIAL AND METHODS
MATERIALS AND METHODS
The methods used in the papers are described in the material and methods section
of each paper.
METHODS USED IN THIS THESIS
In vitro generation of zeste mutants
Mutated versions of zeste, in which the two cysteine-encoding codons were
replaced with serine-encoding codons, were constructed using a Quick-change
Site-directed Mutagenesis kit, according to the instructions of the manufacturer
(Stratagene), and the primers 5´-GAACAACTGCGGAAGTCCTCCACTTCA
TGAGG-3´, 5´-CCTCATGAAGTGGAGGACTTCCGCAGTTGTTC-3´, 5´-GC
TGAACTGCGCTCCAAGGAGCAGCAG-3´ and 5´-CTGCTGCTCCTTGGAG
CGCAGTTCAGC-3´.
Protein aggregation experiments
Proteins were translated in vitro using a TNT® Quick Coupled Transcription
/Translation System (Promega) according to the manufacturer’s protocol. 35Slabeled methionine was used to label and detect the proteins. Two types of
experiments were performed. In the first, 6 µl of Zeste was mixed with 4 µl of
either mock (i.e. a sample with no added DNA), DHD, TrxT or Trx-2 and 5 µl
buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.8). In the second, 8 µl of Zeste,
Zc520s, Zc571s or Zc520sc571s was mixed with 4 µl samples of either mock or
DHD and 3 µl buffer, and 4 µl Zc520s was mixed with 4 µl Zc571s, 4 µl mock or
DHD and 3 µl buffer. All samples were incubated for 90 min and then centrifuged
for 10 min at 16000g at 4° C. The resulting pellets and supernatants were
separated into different tubes. Each pellet was washed with 30 µl buffer and
centrifuged as previously. The buffer was removed and the pellet was dissolved in
30 µl 1 × loading buffer (4 M Urea, 100 mM Tris-HCl pH 7.6, 2 % SDS, 5 %
BME, 5 % Ficoll). The supernatant was mixed 1:1 with 15 µl 2 × loading buffer.
All samples were denatured at 100° C for 5 min and then separated on SDS-PAGE
gels. The gels were fixed in fixation buffer (1:2:7 acetic acid: methanol: dH2O) for
5 min, dried in a GelDryer at 80° C for 60 min, then exposed and the labeling in
Zeste bands was quantified using a PhosphorImager.
29
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
PAPERS I-V
TrxT encodes a male-specific thioredoxin (Paper I)
The female-specific thioredoxin gene dhd was for many years the only known sexspecific thioredoxin in Drosophila (Salz et al. 1994), until we published the
cloning and characterization of TrxT, a male-specific thioredoxin gene (Paper I).
We found that the TrxT gene is located right next to the dhd gene, with only 471
bp separating them. Salz and colleagues had previously shown that dhd encodes a
thioredoxin specifically expressed in the female ovaries and that flies null mutant
for dhd are female sterile. When we tested flies that were null mutants for TrxT we
found that they were both viable and fertile and we have not been able to find any
other obvious phenotypes in these flies. With a number of different Northern blots
we could show that TrxT is expressed exclusively in males, starting in larvae and
culminating in the adult, and that expression of both TrxT and dhd is not
dependent on correct X/Y chromosome dosage, but is dependent on proper
development of the gonads (since neither TrxT nor dhd is correctly expressed in
intersex animals or pseudomales).
TrxT locates to Y-chromosome loops in primary spermatocytes
To examine TrxT and DHD expression in vivo we constructed fly strains that
carried TrxT-EYFP and DHD-ECFP expressing constructs and examined live
preparations of their testes and ovaries. We found that both TrxT and DHD are
predominantly nuclear. DHD specifically locates to nurse cell nuclei and the
oocyte karyosome. TrxT can first be seen in nuclei of primary spermatocytes (the
growth stage between mitosis and meiosis) and persists in the nucleus until
spermatid individualization. In primary spermatocytes TrxT locates specifically to
the two Y-chromosome loops corresponding to the ks-1 and kl-5 fertility factors.
Y-loops have among other things been suggested to function as storage compartments for proteins needed in later stages of spermatogenesis (Hennig et al. 1989).
This could mean that the TrxT binding to ks-1 and kl-5 is a storage phase and that
TrxT is needed at a later stage, for instance during spermiogenesis (the transformation of post-meiosis cells to finished sperms). A possible role for TrxT could
be to ensure correct folding of certain proteins or to assist in dissolving protein
complexes that are stabilized by disulfide bonds. Another possibility is that TrxT
is involved in regulating the Y chromosome fertility factors. dMBD2/3, a protein
believed to be involved in epigenetic silencing, has for instance also been shown
to bind to at least one of the Y-loops (Marhold et al. 2002). Yet another possible
role for TrxT in the testis could be to protect against reactive oxygen species
30
RESULTS AND DISCUSSION
(ROS) in the gonads. In humans, oxidative stress has been suggested as one of the
major reasons for male infertility and loss of sperm motility (Aitken & Baker
2006). This function would however most likely have to be carried out prior to or
at the latest during sperm individualization, since TrxT does not seem to be
present in or on mature sperm, but can be found in excessive amounts in the waste
bag, where all material not needed in the mature sperm ends up (results not
shown).
Trx-2 mutants have shorter life spans (Paper II)
Since TrxT null mutants have shown no obvious phenotype, we suspected that
there might be redundancy between TrxT and the third known Drosophila
thioredoxin, the ubiquitously expressed Trx-2, which is also expressed in nuclei of
primary spermatocytes, as we have previously shown (Paper I). Trx-2 has
previously been biochemically characterized and crystallized (Bauer et al. 2002;
Wahl et al. 2005), but hardly anything was previously known about Trx-2’s
function in vivo. To investigate the possible redundancy and to learn more about
what Trx-2 is doing in the fly body we constructed deletion mutations in the Trx-2
gene. We recovered fourteen deletion mutants and one revertant. The mutant
alleles produce transcripts, but no proteins that can be recognized by our Trx-2
antibody. All of the mutants are probably null mutants for Trx-2, but one of them
is a guaranteed null mutant since it lacks the region that codes for the thioredoxin
active site. When examining the mutant phenotypes we found that Trx-2 mutant
flies are viable but have shorter life spans than control flies. The mutants are
equally sensitive to oxidative stress as control flies, but over-expression of Trx-2
mediates an increased resistance to oxidative stress. When we examined double
mutant flies (TrxT; Trx-2 and dhd; Trx-2) we found that the double mutants had
life spans similar in length to control flies, but they were slightly more sensitive to
oxidative stress.
It is very striking that one can remove three major thioredoxins in Drosophila
without causing lethality or other similarly strong phenotypes, especially since
Drosophila lacks glutathione reductase and consequently should be completely
reliant on the thioredoxin system for reduction of glutathione and for other
common glutaredoxin-system functions (Kanzok et al. 2001). The strong
dependence on the thioredoxin system is made apparent by the lethality seen in
Drosophila thioredoxin reductase (TrxR-1) mutants (Missirlis et al. 2002). The
entire thioredoxin system does however not seem to be as essential as TrxR, since
both Trx-2, TrxT and DHD can be removed without any ensuing lethality. Neither
are Trx-2 mutants any more sensitive to oxidative stress than controls, but the fact
31
RESULTS AND DISCUSSION
that the double mutant is slightly more sensitive to oxidative stress could perhaps
suggest that either TrxT or DHD, or both, are in some way connected to protection
against oxidative stress in the gonads. Considering this, it might be surprising that
a knock-down of the cytosolic Drosophila thioredoxin peroxidase TPx-1 (using
RNAi) causes increased sensitivity to oxidative stress (Radyuk et al. 2003), while
over-expression of TPx mediates resistance to oxidative stress (Missirlis et al.
2003). The over-expression results for Drosophila TPx are however in line with
our over-expression results for Trx-2. Over-expression of thioredoxins has also in
many other cases been shown to increase resistance to oxidative stress: overexpression of DHD has recently been shown to increase resistance to paraquat, a
substance that is commonly used to induce oxidative stress (Chen et al. 2004).
Similar results have been shown in both mammalian cell-lines and mice (Mitsui et
al. 2002; Byun et al. 2005).
Thioredoxin redundancy or specialized functions?
The negative effect of Trx-2 deletion on life span but not on oxidative stress could
suggest that there is extensive redundancy between different thioredoxins or
thioredoxin-like proteins in Drosophila, but it could also mean Trx-2 has
specialized functions with a stronger connection to systems affecting life span than
to systems affecting oxidative stress tolerance, i.e. Trx-2 is perhaps not at all
responsible for reducing TPx. This would however mean that another thioredoxin
is responsible for reducing the cytosolic TPx. Neither TrxT nor DHD could be
candidates for this since both are exclusively expressed in the gonads. This leaves
us with the other thioredoxin-like proteins identified in our database search. The
two major candidates, based on active site amino acid sequence, would be Txl and
the protein encoded by CG13473. Both are possible, but Txl seems less likely
since it has a large extra domain that equals the thioredoxin domain in size. The
Txl ortholog in humans has recently been suggested to have a function in sugar
starvation stress response, suggesting a quite different role for Txl in the cell
machinery (Jiménez et al. 2006). Essentially nothing is known about CG13473, as
can be guessed by the anonymous designation. It would be very interesting to
create and analyze mutations in CG13473 and to investigate the expression profile
of the CG13473 encoded protein. It could also be very informative to make double
mutants of CG13473 and Trx-2 and analyze their response to oxidative stress. It
should also be mentioned that two of the identified thioredoxin-like proteins are
suggested glutaredoxins/-related proteins: clot is a glutaredoxin-related protein
that is necessary for reduction of glutathione in the production of drosopterins, the
red pigments of the Drosophila eye (Giordano et al. 2003), and Grx-1 has been
classified as a glutaredoxin on Flybase (http://flybase.org), by sequence identity
alone. Grx-1 could very well act as reducer for a number of the other
32
RESULTS AND DISCUSSION
peroxiredoxins that have been found in Drosophila (Rodriguez et al. 2000;
Radyuk et al. 2001).
TrxT, dhd and Trx-2 are conserved in Drosophilids (Paper III)
In our first thioredoxin-study we found that the TrxT, DHD and Trx-2 proteins in
D. melanogaster have a fairly high amino acid sequence identity to each other and
we have also found that TrxT and dhd, the two sex-specific genes are located right
next to each other, transcribed in opposite directions (Paper I). We were intrigued,
both by the likeness of the three proteins and the unusual gene organization of
TrxT and dhd and therefore decided to look for the three thioredoxin genes in other
insect species. We found that all three thioredoxins had conserved putative orthologs in all of the twelve Drosophilid species in the genome database (Paper III).
Additionally, putative orthologs for Trx-2 were found in the more distantly related
species Anopheles gambiae (mosquito) and Tribolium castaneum (red flour
beetle). We also found that the organization of TrxT and dhd is conserved in the
Drosophilids, with two interesting exceptions. In one species, D. ananassae, the
dhd gene seems to have been subjected to retro-transposition at some point in
time, since dhd is located more than 1 Mbp away from TrxT and the neighboring
genes snf and CG4198 in this species. In the second species, D. willistoni, dhd also
seems to have been subjected to retro-transposition since it is located immediately
downstream of TrxT in this particular species (inserted between snf and TrxT), as
opposed to upstream in all the other cases. Additionally, two extra copies of the
dhd gene are present in the D. willistoni genome, suggesting more retro-transposition events. These results, combined with data from phylogenetic comparisons
of the three thioredoxins, showing that TrxT and DHD cluster together despite the
fact that TrxT shares the highest sequence identity with Trx-2, clearly suggest that
Trx-2 is the ancestral gene, which at some point gave rise to TrxT, which in turn
gave rise to dhd, most likely through retro-transposition.
TrxT has an extremely variable C-terminal domain
When comparing the twelve Drosophila TrxT orthologs we realized that the Cterminal domain (an additional domain after the common thioredoxin domain) of
TrxT is hyper-variable, i.e. has very low amino acid sequence identity between
different Drosophila species. A thioredoxin-like protein in Drosophila that also
has an additional domain is Txl, with an extra domain of roughly 190 amino acids.
This domain is, contrary to TrxT’s extra domain, as conserved as Txl’s thioredoxin domain. In fact, the entire Txl protein is conserved, from C. elegans, to
Drosophila, to humans (Miranda-Vizuete & Spyrou 2000). The extra domains of
Txl orthologs have no homologies to other known protein domains, but human Txl
33
RESULTS AND DISCUSSION
has been implicated in cell response to glucose starvation in a human cell line
(Jiménez et al. 2006). Considering the large difference in conservation level of the
extra domains of TrxT and Txl, and considering how different their extra domains
are in amino acid sequence, it seems unlikely that these two domains should be
related in function. There are, however, also other known thioredoxins with extra
domains, especially in humans. Many of these are exclusively expressed in the
male testis and bind specific compartments of the mature sperm tail (MirandaVizuete et al. 2004). Many of the extra domains of the mammalian sperm specific
thioredoxins have however been determined to be nucleosidediphosphate kinase
(NDPk) domains.
The very high variability of the extra domain of TrxT would suggest that the part
of TrxT that encodes the extra domain is evolving at a very high rate, while the
rest of the gene evolves at neutral rate. Since the extra domain of TrxT is so
extremely variable, it can not be accurately aligned and the evolutionary rate can
therefore not even be determined. A certain type of genes that are often highly
variable, even between closely related species, are genes involved in sexual
conflict and male-female co-evolution (Swanson & Vacquier 2002). A good
example of this type of quickly evolving genes, is the seminal protein Ovulin (also
referred to as Acp26Aa) (Tsaur & Wu 1997; Wong et al. 2006). Ovulin is
expressed in the male accessory gland, secreted into the sperm soup and
transferred to the female together with the sperm during copulation (Herndon &
Wolfner 1995). Inside the female oviduct Ovulin is processed by components
supplied by both male and female (Park & Wolfner 1995). The processed protein
then stimulates egg-laying (Heifetz et al. 2000). The hyper-variability of TrxT’s
C-terminal domain suggests that TrxT might have a connection to sexual conflict,
possibly having a role in regulating seminal fluid proteins or protecting them from
premature cysteine oxidation. A possible role for TrxT in regulation or protection
of seminal fluid proteins would however require TrxT either to be incorporated
into the sperm tail or to be transferred to the sperm soup in some other way. We
have so far no indications that that is the case, but it would certainly be an
interesting question to pursue.
Regulatory motifs in the region between TrxT and dhd
With our TrxT-EYPF and DHD-ECFP constructs we have previously shown that
the short region between TrxT and dhd is enough to ensure correct expression of
both genes (Paper I). This means that the regulatory sequences of TrxT and dhd
essentially have to be situated somewhere within this region. We decided to
investigate this by searching for conserved motifs in the 150 first bases upstream
of TrxT and dhd in different Drosophila species. We found two conserved
candidate motifs that were specific for TrxT and three that were specific for dhd.
34
RESULTS AND DISCUSSION
When comparing the motifs with upstream regions of other testis and ovary
specific genes we could essentially not find the motifs in any of the examined
genes. This could suggest that the TrxT-dhd gene pair is unique when it comes to
their regulatory motifs and that their regulatory motifs might be specific for these
particular genes. It would however be quite surprising since Perezgasga and
coworkers (2004) have shown that transcription of TrxT is regulated by the
meiotic arrest genes topi and comr. Both of these genes are believed to be part of a
complex with proteins encoded by the meiotic arrest genes aly and achi/vis
(White-Cooper et al. 2000; Ayyar et al. 2003). This complex regulates the
transcription of approximately one thousand genes in the testes (Perezgasga et al.
2004). Nothing is known, so far, about what genes might be regulating dhd. It
should be stressed that it is possible that the true regulatory motifs of TrxT and dhd
are not at all identical to the conserved motifs that we have found. The true motifs
might for example be too degenerated between the species for us to find them with
this method. It is also possible that transcription factors regulating these genes are
varying between different Drosophilids, even though we see the same expression
pattern of TrxT and dhd in all of the species we have examined.
Pof in genus Drosophila suggests autosome-specific gene regulation (Paper
IV)
For many years it has been widely known that the male X chromosome in
Drosophila is “painted” by the components of the dosage compensation complex.
This phenomenon of “chromosome painting”, where a protein or proteins bind a
chromosome along its entire length, was for a long time believed to be exclusive
for the X chromosome, until it was shown that the 4th chromosome in D. melanogaster is painted by the protein encoded by the Painting of fourth gene (Larsson et
al. 2001). In Drosophila busckii, a related species where the F-element (the
general designation for 4th chromosome corresponding element in other fruit flies)
is part of the X, POF was shown to bind the entire X chromosome in males,
suggesting a connection of Pof to the dosage compensation complex in D. busckii.
To investigate this connection further we stained male and female polytene
chromosomes from Scaptodrosophila lebanonensis and from several species in
genus Drosophila with antibodies against POF, MSL3 and acetylated H4K16
(Paper IV). We find H4K16 acetylation (a histone modification specific to the
DCC-component MOF) on the male X in all of the examined species and MSL3
staining in all species except S. lebanonensis and D. busckii. The fact that H4K16
acetylation is found in all of the examined species suggests that a similar dosage
compensation system is used, even though MLS3 can not be recognized by our
antibody in two of the species. In the distantly related S. lebanonensis, where the
35
RESULTS AND DISCUSSION
F-element is part of the X just as in D. busckii, we find no POF staining. Neither
can we find any POF staining in D. willistoni, where the F-element is part of one
of the autosomes. Staining of D. busckii polytene chromosomes reveals that POF
colocalizes with H4K16 acetylation, strengthening the hypothesis that POF is
connected to dosage compensation in certain species. In all examined species
where the F-element exists as a unique dot chromosome (as in D. melanogaster)
POF binds to the F-element in both sexes, but in two cases (D. ananassae and D.
malerkotliana) POF also binds to the male X. Double staining with antibodies
against POF and MSL3 in D. ananassae reveals that the two proteins colocalize on
the male X, while POF is the only one that stains the F-element. To determine
whether the POF’s chromosome-specificity depends upon the protein sequence of
POF in that particular species, the Pof-gene from D. ananassae was chosen and a
DaPof-EYFP construct was made and injected into D. melanogaster embryos.
DaPOF-EYFP stains the 4th chromosome, but it does not locate to the male X in D.
melanogaster. This ultimately suggests that POF has had a connection to, and has
possibly even been part of, the dosage compensation complex during Drosophila
evolution and that POF targeting to a specific chromosome is determined by an
external source, for instance another protein or a non-coding RNA, and not by the
POF protein sequence per se.
Deletion mutants in the Pof promoter region are lethal
Staining of POF in various different cell types revealed that POF is located to the
4th chromosome in generally all cells (Paper IV). To investigate whether POF has
an essential function we created deletion mutations in the Pof gene region using Pelement excision. All deletions in the Pof promoter region resulted in lethality and
female sterility (as seen in germline clones), which led us to draw the conclusion
that Pof is essential for fly viability. This is however not the case. At the time
when the mutants reported in Paper IV were created, it was not known that a
second gene is located in the Pof promoter region. Later releases of the D. melanogaster genome annotation indicated that a gene designated CG33228 was located
in this region. The CG33228 gene organization and mRNA sequence was characterized and confirmed by Johansson and colleagues (2007). They constructed
new deletion mutants in the coding region of Pof and could conclude that
CG33228 deletion mutants are lethal and female sterile, while Pof deletion
mutants are viable. This may at first have seemed as a setback, but when they
crossed haplo-4th flies with Pof-mutant flies they found that a complete lack of
POF combined with only one copy of chromosome 4 leads to lethality. This
phenotype could be rescued by a transgenic Pof-construct (Johansson et al. 2007).
These findings are very interesting since the haplosufficieny of Drosophila’s 4th
chromosome has previously been explained by its small size and low number of
genes. The results clearly suggest that Pof has an important function in regulating
36
RESULTS AND DISCUSSION
the 4th chromosome in Drosophila. It also adds credibility to the hypothesis that
POF has an evolutionary connection to the MSL complex.
Pof in spermatogenesis (Paper V)
Previous publications on Painting of fourth have indicated that expression of Pof
RNA and POF protein is high in the male gonads (Larsson et al. 2001). We were
intrigued by this since the X chromosomal MSL-complex is not present in the
male germline, even though it has been shown that the male X is dosage compensated also in the germline (Rastelli & Kuroda 1998; Gupta et al. 2006). To look
for POF expression in the male germline we stained male testes with an antibody
against POF and also looked at testes from males carrying a POF-EYFP expressing construct. We found that POF expression is concentrated to a few small foci in
early primary spermatocyte nuclei and that the number of foci increase the older
the primary spermatocytes get and POF staining eventually becomes evenly
distributed within the nucleus (Paper V). The POF localization is lost during
meiosis, but returns during the spermatid maturation stages, culminating with a
strong staining in the highly condensed elongated spermatid nuclei, just prior to
individualization. No POF staining is seen in mature sperms. It is still unknown
what structures POF might be binding to in the primary spermatocytes and in the
elongated spermatid nuclei. The 4th chromosome is generally believed to localize
together with the X chromosome within the spermatocyte nuclei, but it is also
possible that the 4th chromosome is de-heterochromatinized, just as the normally
heterochromatic Y chromosome that forms large decondensed structures (the Yloops) in spermatocyte nuclei.
Pof upregulates chromosome 4 genes in the male testes
It is tempting to speculate on what function Pof might be serving in the male
germline. A function for Pof in regulating genes on the 4th chromosome in the
male germline is obviously the first thing that comes to mind. This idea might not
seem very controversial at first glance, considering previous results on Pof, but
when considering that the MSL-complex is not expressed in the testes, it becomes
increasingly so. It is also possible that POF is regulating genes on the X chromosome, the Y chromosome or particular groups of genes that are specifically regulated in a certain manner in the testes etc. To test this idea we performed a microarray analysis on testis tissue from wildtype and Pof mutant males. The genes
examined were categorized into twelve groups: six chromosomes (X, 2L, 2R, 3L,
3R and 4), heterochromatic chromosome regions (Xh, 2h, 3h and Yh), genes with
unknown location and a subset of testis specific genes. We found that a lack of Pof
in the male germline causes a global down-regulation of genes on the 4th
37
RESULTS AND DISCUSSION
chromosome when compared to levels in wildtype testes. We could find no
significant up- or down-regulation of any of the other examined groups. These
results are completely in line with the microarray findings of Johansson and coworkers (2007), where they show that loss of Pof causes a global down-regulation
of the 4th chromosome genes in first instar larvae. Intriguingly, they also show that
a loss of heterochromatin protein-1 (HP1) causes a global up-regulation of genes
on chromosome 4 and that binding of POF and HP1 to chromosome 4 is interdependent (Johansson et al. 2007). This suggests a balancing mechanism to ensure
correct expression levels of chromosome 4 genes, involving POF and HP1 as
partners with opposite functions: up-regulation performed by POF and downregulation performed by HP1. The proposed balancing mechanism is reminiscent
of the proposed dosage compensation mechanisms in mammals and C. elegans,
where initial global up-regulation of the X chromosomes is balanced by inactiveation of one X in females and down-regulation of both X:s in hermaphrodites
respectively. This similarity gives more credibility to the idea that the twofold upregulation of the X chromosome in Drosophila males performed by the MSLcomplex is being balanced by a, so far unknown, protein system or complex.
An interesting phenomenon specific to the X chromosome in the male germline is
the condensation and/or inactivation of the X chromosomes earlier than the
autosomes prior to meiosis. This phenomenon is called precocious X inactivation
and has been observed in many organisms, including C. elegans, Drosophila and
mammals (Lifschytz & Lindsley 1972; Wu & Xu 2003). Precocious X inactivation
seems to be limited to organisms where males are the heterogametic sex, since it
has not been observed in either birds or butterflies, where it is the females that are
heterogametic (Wu & Xu 2003). Interestingly, in Drosophila, translocations
between the X and the autosomes result in dominant male sterility in more than 75
% of the cases, but no male sterility is seen in translocations between the 4th and X
(Lindsley 1965; Lindsley & Tokuyasu 1980). It has been proposed that X:A
specific sterility is caused by a disturbance in the precocious X inactivation by the
translocated chromosome. The disturbance could either be that the translocated
autosome becomes inactivated too soon or that the precocious inactivation of the
translocated X is delayed or stopped. The reason why X:4 translocations do not
cause male sterility has been believed to be either that the 4th is too small to
influence the precocious inactivation or that the 4th has different regulatory properties than the other autosomes (Lindsley & Tokuyasu 1980). The 4th chromosome is
decidedly more heterochromatic than the other autosomes, and so the heterochromatic state of the 4th might be too similar to the inactivated state during
meiosis to be able to excert any activating influence over the translocated X. But
what of the translocated 4th? Since POF is up-regulating chromosome 4 genes also
in the male germline, it is definitely possible that POF protects against the
38
RESULTS AND DISCUSSION
inactivating effect of the translocated X. This hypothesis would be very interesting
to test by introducing X:4 translocations in a Pof mutant background. If the X:4
males are sterile in Pof mutant background it would suggest that POF protects the
4th chromosome from inproper inactivation. This would add even more support for
the notion of POF as an important player in chromosome-wide gene regulation.
NEW RESULTS
Thioredoxins can break Zeste aggregates
All the three Drosophila thioredoxins have previously been shown to be present in
nuclei of the germline (Paper I). We were interested in the possibility that thioredoxins may play a role in cellular memory or gene regulation in the Drosophila
germline, and since TrxT was first identified in a yeast-two-hybrid screen for
Zeste-interacting proteins (Chen 1992), we decided to investigate the possible
interactions between Zeste and thioredoxins. Pirrotta and colleagues have shown
that Zeste forms large aggregates both in vitro (Bickel & Pirrotta 1990) and in vivo
(Chen & Pirrotta 1993a), and its aggregation is believed to be an important
element of the mechanism underlying many of the zeste mutant phenotypes seen in
vivo. In our initial assays of the interactions between thioredoxins and Zeste, TrxT
was shown to dramatically increase the mobility of a Zeste/DNA complex when
separated by gel electrophoresis (results not shown). This led us to speculate that
thioredoxins might be able to break Zeste aggregates. Our approach to test this
hypothesis was to translate zeste and the three Drosophila thioredoxins in vitro,
mix Zeste with TrxT, DHD, Trx-2 or a mock sample separately, and to test the
aggregation capacity of Zeste under these four different conditions in a pelletation
assay previously developed to monitor Zeste aggregation (Bickel & Pirrotta 1990;
Rosen et al. 1998). In this assay, aggregated Zeste protein will form a pellet, while
non-aggregated Zeste remains in the supernatant. We found that DHD could
clearly break Zeste aggregates in vitro, as seen from the low amount of Zeste in
the pellet, while both TrxT and Trx-2 had much weaker effects in this assay
(Figure 7A).
The C-terminal hydrophobic region of Zeste has been shown to be necessary for
aggregation of the protein (Chen et al. 1992; Chen & Pirrotta 1993a-b). This
hydrophobic region is predicted to have an alpha-helical structure and three
leucine zippers, two of them aligned in a row on one side and one on the opposite
side. Aggregation of Zeste has been proposed to involve an initial dimerization
followed by multimerization of the dimers. There are only two cysteines in this
hydrophobic region, one situated two amino acids from the C-terminus, the other
39
RESULTS AND DISCUSSION
situated approximately in the middle of the hydrophobic region, at the end of one
of the leucine zippers (Figure 6). We hypothesized that the two cysteines located
in this hydrophobic region are involved in the aggregation process by forming
disulfide bonds, thereby stabilizing the aggregation, and that the thioredoxins can
break Zeste aggregates by reducing these disulfide bonds.
Figure 6. Hypothetical aggregation model of Zeste’s hydrophobic region. Two Zeste
molecules dimerize at the double leucine zipper. The C-terminal cysteine could act as a
stabilizer. Zeste dimers form aggregates through multimerization at the other leucine
zipper, where the second cysteine could act as another stabilizer.
To test this hypothesis we created zeste clones that were mutated at one or both of
the positions encoding these cysteines. We then performed the same pelletation
assay as above, but this time with the four different versions of Zeste (wildtype Z,
Zc520s, Zc571s and Zc520sc571s) and with only a mock sample or DHD added.
Both mutant Zeste forms aggregated much more weakly than the wild type protein
when mock sample was added, and their low degrees of aggregation could be
completely inhibited by adding DHD. When both single mutants were mixed in a
sample, aggregation was also weaker than wild type, but stronger than with either
of the single mutants alone. However, the strongest inhibition was observed in
assays with the double mutant, in which virtually no aggregation was detected
(Figure 7B). We conclude that thioredoxins can affect Zeste aggregation and that
the two cysteines c520 and c571 are important for the Zeste aggregation process,
which provides a possible mechanistic link to the thioredoxins. Further support
and proof for the hypothesized aggregation model remains to be shown.
40
RESULTS AND DISCUSSION
Figure 7. Zeste – thioredoxin interactions. A. Thioredoxins can break Zeste aggregates in
vitro. Proteins were translated in vitro and used in a pelletation assay separating the
aggregated form of Zeste in the pellet (P) from the non aggregated form in the supernatant
(S). The amount of Zeste in the pellet is indicated as percent of total. The panels are
PhosphorImager captures of SDS-PAGE gels. Added thioredoxin or mock protein is
indicated. B. Zeste proteins with cysteine substitutions aggregate less than wild type Zeste
in vitro. The same method was used as in A.
41
CONCLUSIONS
CONCLUSIONS
ƒ
TrxT encodes a testis-specific thioredoxin that specifically locates to the Ychromosome loops ks-1 and kl-5 in primary spermatocytes.
ƒ
dhd encodes an ovary-specific thioredoxin that specifically locates to nurse
cell nuclei and the oocyte karyosome.
ƒ
Trx-2 mutants are viable but have shorter life spans than control flies.
ƒ
Trx-2 mutants are as sensitive to oxidative stress as control flies, but overexpression of Trx-2 mediates an increased resistance to oxidative stress.
ƒ
All three thioredoxin genes can be removed without causing lethality or any
other strong phenotypes.
ƒ
The genomic organization of TrxT and dhd is conserved in ten out of twelve
Drosophilid species.
ƒ
The C-terminal domain of TrxT is hyper variable.
ƒ
POF binding to the F-element (chromosome 4 in D. melanogaster) is
conserved in genus Drosophila.
ƒ
POF colocalizes with MSL-3 and H4K16ac in species where POF binds the
male X.
ƒ
POF is present in the nuclei of primary spermatocytes, but also in nuclei of
maturing spermatids.
ƒ
Lack of POF in the male germline causes a global down-regulation of
chromosome 4 genes.
ƒ
Thioredoxins can break Zeste aggregates in vitro and Zeste proteins that lack
the two cysteines in the hydrophobic domain have a very low aggregation
capacity.
42
SUMMARY IN SWEDISH
SUMMARY IN SWEDISH
Spermatogenesen är i många organismer en av de mest dramatiska förvandlingar
som en cell kan genomgå – en vanlig, rund, diploid cell omvandlas till en
nålformad, haploid cell med ett tätt packat cellmaskineri. I bananfluga, Drosophila
melanogaster, innebär denna process flera karaktäristiska stadier. Ett av dessa är
det primära spermatocyt-stadiet, som infaller innan cellen påbörjar meios-delning.
Stadiet karaktäriseras av en uppluckrad kromatinstruktur i cellens kärna och
ovanligt höga transkriptions- och translationshastigheter, för att producera allt det
mRNA och de proteiner som behövs senare under spermatidomvandlingen. Två av
de proteiner som uttrycks i höga nivåer i primära spermatocyter är ThioredoxinT
(TrxT) och Painting of fourth (POF).
Thioredoxiner är små proteiner som har som funktion att reducera
disulfidbryggor i andra proteiner, en mekanism som används i många olika
fysiologiska sammanhang. I denna avhandling visar jag att TrxT-genen kodar för
ett testikelspecifikt thioredoxin som binder specifikt till Y-kromosom-loopar i
primära spermatocyter. TrxT-genen ligger precis bredvid deadhead (dhd), en gen
som kodar för ett hon-specifikt thioredoxin som är lokaliserat till cellkärnorna i
flugans äggstockar. Ett tredje thioredoxin i Drosophila är det allmänt uttryckta
Thioredoxin-2 (Trx-2). Jag har upptäckt att flugor som saknar Trx-2 är
livsdugliga, men att de lever kortare än vildtypsflugor, medan flugor med extra
mycket Trx-2 har en ökad tålighet mot oxidativ stress. Slående nog är en total
avsaknad av alla tre thioredoxiner inte förenat med letalitet, tvärtemot vad man
skulle kunnat vänta sig. Alla tre thioredoxiner finns i de olika Drosophilider som
undersökts och den ovanliga genorganisation som delas av TrxT och dhd är
generellt konserverad.
Gen-namnet Painting of fourth härstammar från upptäckten att POF
binder till (”målar”) Drosophilas kromosom 4. Jag visar i min avhandling att
POFs bindning till den fjärde kromosomen är bevarad i olika Drosophila-arter och
att POF kolokaliserar med både ett protein och en histon-modifiering, som är
förknippade med doskompensation, i arter där POF också binder till hanens Xkromosom. POF uttrycks överallt i både honor och hanar, men i väldigt höga
nivåer i hanens testiklar. Jag visar här att POF finns i cellkärnan hos primära
spermatocyter, men också i kärnan på mognande spermatider, och att avsaknad av
POF in hanens könsceller orsakar en global nedreglering av gener som ligger på
kromosom 4. Kombinationen av mina POF- resultat tyder på att POF har en viktig
funktion i det första kända fallet av genreglering av en hel autosomal kromosom.
43
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
I would like to thank all the people who have contributed in some way to this thesis, or
been important to me and the journey that has brought me to this point in time:
Janne, my wonderful supervisor. You are a brilliant scientist AND you have a lovely
family and a social life! You are of a rare kind. ;-) You have done a fantastic job through
all my PhD-student years, giving me a kick when I needed it, supporting me whenever I
was low and letting me spread my wings just at the right time. Thank you also for all your
good advice regarding this thesis. I don’t think I could have had a better supervisor than
you!
Per, my fellow trekker (or is it trekkie?). It was as small step for humanity, but a great step
for GroupJL when you joined us. ;-) It has been great to have someone to share my movie
and music taste with! You are one of the most amusing people I know, and a fine scientist.
Anna-Mia, my lab-pal, my office-buddy, my IKSU-mate. From the moment you set your
foot in our office I knew you would fit right in. You are a true friend and I have
appreciated our friendship very much! And I will definitely be dropping by some time in
the future to check out your beautiful house. ;-)
Karin, our fly-wizard. Thank you for your excellent fly work and great party spirit. Work
would have been both less fun and more cumbersome without you.
Project students and past members of group JL: Thomas, Mikael, Maria, Carolina, Elin,
Theresa and the latest addition to group JL, Lina. You all contributed to the positive
atmosphere in group JL. I would like to especially thank Theresa for performing the major
part of that huge fertility-experiment. You did a fantastic job! (And thank you Lina for
solving my apartment renting problem!)
Thanks to Per, Simon and Åsa for constructive criticism and reading of this thesis.
Anssi, thank you for sharing your never ending knowledge and personal views on science,
religion and everything else and thank you for all the invaluable help I have received
during the years!
Liz and Åsa for constructive criticism and encouragement during our post-Floortalkcommittee meetings. And thank you for the fact that you were there as “safety nets” if I
ever would have needed it.
All the UCMPers: Our fellow fly groups RHP (thank you Caroline for making me feel
welcome into ‘the Clan’) and DH, the Worm group and the two X-Ray groups (Anders O
and Malin LP, thanks for nice company in the office). Thanks for making UCMP such an
incredibly fun and friendly place to work! I want to especially thank Maria Westling for
your invaluable help with everything during my years at UCMP.
44
ACKNOWLEDGEMENTS
Everybody at the old genetics department: Anna, Bettan, Gunilla (tack för superbra
sevice med flugmaten), Helena, Jesper, Kerstin, Magnus, Marcus, Marianne, Mikael,
Sa, Stefan (tack för att jag fick dela rum med dig när jag skrev avhandlingen och för att du
ställer upp som toastmaster på festen) and everbody else. Thanks for all the incredibly fun
genetics parties and nice company during lunches and coffee breaks in the ‘good old days’!
Jenny, Åsa och Elisabeth mina kompisar från biologprogrammet, ni gjorde
grundutbildningen väldigt trevlig! Vi hade nog alla fyra en ganska omvälvande tid då. ;-)
Tack särskilt till Jenny och Jörgen för att jag fått hyra er lägenhet, det har varit super!
Anki, jag vet inte om det var helt optimalt för betygen och studiemoralen att vi hängde
ihop som ler och långhalm på gymnasiet, men det var desto bättre för själen. Tack för att
jag fått lära känna Bertram och Elliot! Du kommer alltid vara mina allra bästa vän.
ACC-folket, tack för alla trevliga tillställningar, av alla de slag. Jag vet, jag borde ha
skrivit min avhandling i LaTeX. ;-) Nya och gamla vänner i Fjollträsk, tack vare er känns
det inte så läskigt att flytta söderut.
Ulrika Östh, min klassföreståndare på högstadiet. Du har en viktig del i att jag alls har
kommit så här långt. Minns du den där fåniga uppsatsen jag skrev? Sometimes life is
stranger than fiction.
Daniel, jag lärde mig mycket om mig själv och världen under vår tid tillsammans. Jag är
väldigt glad att vi förblivit goda vänner och jag kommer sakna våra trevliga sci fi-kvällar.
Är du säker på att du inte ska flytta söderut du med? ;-)
Karin och Bertil, ni har verkligen fått mig att känna mig som en i familjen. Tack för all
hjälp! Jag ser fram emot många besök av er och oss, i Stockholm, Umeå och Bakviken.
Mamma och Pappa, tack för att ni alltid ställer upp för mig och att ni stöttar mig och låter
mig vara den jag är! Hille och Jonas, vi hörs av alldeles för sällan. Nu när jag flyttar lite
närmre hoppas jag vi kan hälsa på varandra oftare. Jag älskar er allihop! Tack oxå till
övriga släkten för att ni bidragit till att göra min uppväxt lycklig och ljus!
Och framförallt min egen personliga isbjörn. Jag vet att du var lite ledsen över att du inte
kunde finnas där för mig och hjälpa mig med alla små vardagsbestyr som jag inte hade tid
med när jag skrev avhandlingen. Men vet du, det räckte med att du fanns där i tanken (och
några telefonsignaler bort). Din kärlek får mig att tro på min egen förmåga. Nu är snart
mer än tre års väntan äntligen över... Jag tokälskar dig! :-)
Live Long and Prosper!
45
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