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Gene expression in chromosomal Ridge domains : influence on transcription, mRNA
stability, codon usage, and evolution
Gierman, H.J.
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Gierman, H. J. (2010). Gene expression in chromosomal Ridge domains : influence on transcription, mRNA
stability, codon usage, and evolution
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Download date: 19 Jun 2017
1
Introduction: Gene Regulation by
Chromosomal Domains
.
1
Introduction: Gene Regulation by Chromosomal Domains
Hinco J. Gierman and Rogier Versteeg
Department of Human Genetics, Academic Medical Center, University of Amsterdam, P.O. Box 22700,
1100 DE Amsterdam, the Netherlands.
Published in part as: Clustering of highly expressed genes in the human genome. Encyclopedia of Life
Sciences. 2008 Apr;30:a0005931 John Wiley & Sons, Ltd: Chichester.
1.1 Introduction
Gene expression is the most fundamental of all biological processes and can be
viewed as the sum of mechanisms that transcribe DNA into RNA, into protein. As
important as the function of a protein, is the place, the time and the quantity of
expression. The cellular mechanisms that underlie these three determinants are what
make up ‘the regulation of gene expression’. Together, they control in which cells
(the place), at what point during development (the time) and how many molecules
(the quantity) of any protein is produced. This control ensures the correct expression
of all genes during development. When the regulation of these genes is disturbed,
e.g. by mutations in the DNA, diseases like cancer can arise.
Identifying and understanding the mechanisms involved in gene regulation is
essential for understanding how cancer arises. Many cellular mechanisms are known
that regulate the expression of individual genes. In this thesis we asked, whether in
addition to these well-known mechanisms, genes are also regulated at the level
of chromosomal domains called ‘Ridges’ (abbreviated from ‘Regions of IncreaseD
Gene Expression’). In this thesis, we show that Ridges increase transcription by
a domain-wide mechanism, that Ridge genes have an increased messenger RNA
(mRNA) stability and finally, that Ridge messenger RNA (mRNA) have codons that
facilitate highly efficient translation. We propose that this system provides a highway
enabling an expansion of the protein expression range in the genome and we discuss
the implications of the Ridge system for the evolution of the human genome.
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Introduction: Chromosomal domains
1.2 The Human Transcriptome Map
The expression of genes is regulated in the first instance by transcription factor
complexes. These complexes bind to regulatory sequences, usually in the promoter
region of a gene. The concentration and composition of these complexes determine
the amount of mRNA that is produced. This system of individual gene regulation in
principle allows genes to be randomly positioned throughout a genome, and this was
long assumed to be the case for most genes. However, if clustering of genes with
similar activity or related function occurs, this predicts that chromosomal regions
would either show differences in average activity, or co-expression under specific
conditions or in certain tissues.
With the emergence of high-throughput screening of mRNA levels (within this context
commonly referred to as expression profiling), it became possible to analyze the
expression levels of thousands of different genes at once. One of the techniques
used to this end was Serial Analysis of Gene Expression (SAGE) (Velculescu 1995).
In short, concatemers of 3’ fragments of mRNA molecules are cloned into bacterial
plasmids. Sequencing of 50,000 to 100,000 of these 3’ tags yielded a quantitative
expression profile of a cell or tissue. In the same year, the first complete genomic
sequence of a free living organism was published: Haemophilus influenzae Rd.
(Fleischmann 1995). The convergence of these two techniques, expression profiling
and whole genome sequencing, allowed mapping the expression of every gene
onto its chromosomal position. These so-called ‘transcriptome maps’ allowed to test
whether genes of similar activity or function show clustering. This was first done
for the budding yeast Saccharomyces cerevisiae (Velculescu 1997). The study
showed some clustering of co-expressed genes, but found no clusters of high or low
expression on any chromosome. A second study looked deeper into the clustering
of these co-expressed genes in yeast and concluded that yeast possesses small
chromosomal domains of gene expression (Cohen 2000). They found that clusters
of 2–3 genes, adjacent and non-adjacent, showed co-expression.
The sequencing of the human genome had been underway for a decade by then,
and was nearing its completion. Our lab used an early radiation hybrid map of the
human genome (Deloukas 1998) to map expression data from SAGE libraries (Caron
2001). The resulting Human Transcriptome Map (HTM) revealed an unexpected
organization in the human genome: Highly expressed genes were found to cluster in
so-called Regions of IncreaseD Gene Expression (Ridges). A more detailed mapping
using the first draft human genome sequence (Lander 2001), revealed that poorly
expressed genes also clustered in separate regions termed anti-Ridges (Versteeg
2003). Figure 1 shows a transcriptome map of the q-arm of chromosome 1. Each
black vertical bar represents a gene. The height of each bar indicates the activity of
the domain surrounding that gene. To measure the activity of a domain, the median
expression over a window of genes is calculated. A window encompasses the gene
itself and an equal number of adjacent genes on both sides. The domain activity was
calculated for all genes on each chromosome, by sliding the window one gene at a
time. In Figure 1, the typical window size of 49 genes was used. Comparable results
are obtained for window sizes ranging from 19 to 59 genes. Figure 1 shows that a
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Figure 1. Physically mapped transcriptome profile of the q-arm of human chromosome 1. Giemsa banding
is illustrated below the transcriptome map (centromere/heterochromatic region is green and marked
‘cen’). Ridges are shaded red and marked with an ‘R’, anti-Ridges are shaded blue and marked ‘AR’.
Black vertical bars represent genes and their height indicates domain activity for a moving median window
of 49 genes (MM49) in 133 pooled SAGE libraries from different tissues. Below is the chromosomal
position in megabases (UCSC Genome Build HG18). Illustration adapted from Gierman et al. (Figure 1;
Gierman 2007).
number of domains have a high expression: These are Ridges (shaded red). Equally
so, the anti-Ridges (shaded blue) clearly have a lower overall expression.
On average, Ridges and anti-Ridges consist of 80–90 genes, but these domains can
range from 30 to 500 genes in size. There are about 30 Ridges and 30 anti-Ridges
in the human genome. Although the exact number depends on the window size
and statistical threshold used, almost every chromosome has at least one Ridge
or anti-Ridge. Roughly 20–25% of all human genes reside within a Ridge and 10–
15% are in an anti-Ridge. The bulk of the human genome however, is made up
of domains of intermediate gene expression harboring the remaining 60–70% of
genes. Chromosomes are thus an assemblage of different expression domains that
form a higher-order organization of the human genome.
Many other studies have investigated gene clustering. For example, in mice domains
exist with dense or sparse transcription (Carninci 2005). However, most studies have
focused on chromosomal clustering of co-expressed genes. This has been found
to occur in various organisms like S. cerevisiae (Velculescu 1997; Cohen 2000;
Burhans 2006), Drosophila (Spellman 2002), C. elegans (Roy 2002) and Arabidopsis
(Williams 2004; Ren 2005), mice (Mijalski 2005) and humans (Bortoluzzi 1998;
Vogel 2005) (see also Lee 2003; Hurst 2004). These clusters are conserved during
evolution, indicating the importance of the chromosomal organization of these genes
(Singer 2005; Sémon 2006).
1.3 Gene Expression in Ridges: Housekeeping and Tissue-specific Genes
Ridges are enriched for highly expressed genes, but medium and poorly expressed
genes populate Ridges as well. Also, many highly expressed genes are found outside
Ridges. Genes can be categorized into tissue-specific and ubiquitously expressed
genes. Genes that are expressed throughout all tissues are called housekeeping
12
Introduction: Chromosomal domains
genes. In general, Ridge genes are broadly expressed over different tissue types
(Lercher 2002). Lercher et al. proposed that Ridges are formed by clustering of
housekeeping genes (Lercher 2002). For the calculation of Ridges, the average
expression of each gene in a collection of SAGE libraries of different tissue types is
used (Versteeg 2003). This means that genes that are ubiquitously highly expressed
will have the highest average expression. Conversely, genes that are highly
expressed in just one or a few tissues will have high tissue-specific expression but a
low average expression. This raises the question whether the only difference between
Ridge genes and other genes is the broad expression, or whether Ridge genes are
also more highly expressed. Figure 2A shows that also the maximal tissue-specific
expression of genes follows the pattern of Ridges and anti-Ridges (Versteeg 2003).
Ridge genes are thus both more highly and more broadly expressed. This probably
reflects that many housekeeping genes are both broadly and highly expressed.
Nevertheless, genes in Ridges are subject to tissue-specific regulation. Figure 2B
shows the variation in individual gene expression of genes on chromosome 9 over
62 different SAGE libraries from different tissues. Ridge domains thus appear to be
favorable for genes with a high and/or ubiquitous expression, but equally allow for
tissue-specific regulation of genes.
1.4 Ridges and anti-Ridges Differ in Organization, GC Content and Chromatin
Detailed analysis of the Human Transcriptome Map showed that many physical
parameters of the genome correlate with Ridges (Versteeg 2003). Many of these
correlations confirmed earlier observations (Bernardi 1985a). The clearest correlation
is with gene density and can be observed in Figure 1: As each vertical black bar
marks the position of a single gene, the density of bars directly indicates the gene
density. Figure 3 shows this more clearly with a direct comparison of gene density
and gene expression (panels E and F). Ridges also have shorter genes and shorter
introns (panel D) and most repeats (e.g. LINEs) are less frequent in Ridges, with
the exception of SINEs which are more abundant (panels A and B). An important
genomic feature is the genomic GC content (i.e. the ratio of G/C versus A/T bases),
which is also higher in Ridges (panel C).
The genome of warm-blooded vertebrates (i.e. birds and mammals), display a strong
variation in the GC content of large chromosomal regions, also known as isochores
(Bernardi 1985a; Costantini 2006). These regions can be hundreds of kilobases long
and in humans their GC content varies from 30% to 60%. Isochores are predominantly
the result of the accumulation of changes caused by a mutation bias (Duret 2009). The
mutation bias most likely arose to compensate for the hypermutability of methylated
cytosines, which spontaneously deaminate to thymines. C/G pairs thus frequently
mutate into T/G mispairs, and the base excision repair system has become strongly
biased towards repairing G/T mispairs in favor of the guanine to compensate for
this (Brown 1987; Brown 1988; Brown 1989). This repair system thus increases the
GC content of loci where T/G or A/G mispairs originate from A/C pairs. Conversely,
despite the bias in repair, cytosines that are methylated will disappear over time.
This is because as the cytosine is continuously mutated, eventually the T/G mispair
will be repaired in favor of the thymine, creating a T/A pair instead of the original
13
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1
Figure 2. Transcriptome maps of chromosome 9. (A) Moving median of the height of average expression
(blue) and tissue-specific expression (red) per 100,000 tags. Expression values were determined in a
collection of 57 SAGE libraries of 50,000 tags or more. Blue and red bars indicate anti-Ridge and Ridge.
Genes are sequentially ordered according to chromosomal position, but not physically spaced (window
size 49 genes). (B) Individual gene expression over 62 SAGE libraries of 50,000 or more tags (horizontal
lines). Each vertical line is a gene. The levels of expression are given by a color code, ranging from zero
(blue) to 25 (purple) or more tags/100,000 transcript tags in a library. Illustrations adapted from Figure 5
and 1G Versteeg et al. (Versteeg 2003).
G/C. This repair occurs during meiotic recombination producing the so-called ‘biased
gene conversion’ which has shaped isochores (Filipski 1987; Sueoka 1988; Wolfe
1989; Press 2006; Duret 2008 and reviewed by Duret 2009). This mutational bias
affects the GC content of all sequences in isochores, including the coding sequences
of genes (Bernardi 1985b; Cruveiller 2004). Recombination is thus thought to drive
the formation of isochores, and the non-uniform distribution of GC content is likely
formed to some extent by the different rates of recombination throughout the genome
(Fullerton 2001; Kong 2002; Montoya-Burgos 2003; Meunier 2004). Analysis of the
mouse Fxy gene has shown that GC content can increase rapidly. For the third
codon position (i.e. the wobble base for which a base pair change often encodes the
same amino acid), GC content increased from 50% to 73% in 3 million years (Perry
14
Introduction: Chromosomal domains
1999). This is an evolutionary short period of time: humans and their closest living
relative the chimpanzee, diverged 5–7 million years ago (Patterson 2006).
There is a straightforward linear correlation between the GC content and gene
expression of e.g. a window size of 49 genes (R2 = 0.51, P < 10-99; Versteeg 2003,
data not shown). The difference in GC content between Ridges and anti-Ridges,
applies to all of the genomic sequence, including the coding sequences of genes.
The GC content of most anti-Ridge mRNA lies between 40% and 50%, whilst Ridge
mRNA typically have a GC content of 50% to 65% (see also chapter 3 and 4).
But not only the composition of DNA is different in Ridges. DNA is packaged into
chromatin, which consists of histone proteins. These histones can be modified on a
multitude of residues, mostly by phosphorylation, methylation and acetylation. These
modifications influence transcription in two ways: directly, by binding transcriptional
complexes and indirectly, by changing the chromatin structure. Recent studies using
chromatin immunoprecipitation (ChIP) of histone modifications show that Ridges
are associated with active histone marks associated with transcription (Bernstein
2005; Roh 2005; Barski 2007). Importantly, Ridges were also found to have an open
chromatin structure throughout their entire domain, even where genes in Ridges
are not expressed (Gilbert 2004; Goetze 2007). Open chromatin facilitates gene
expression and could be a consequence of the increased transcription in Ridges.
However, the broad open chromatin structure of Ridges might also contribute to the
expression of genes in Ridges (Sproul 2005).
1.5 Nuclear Organization and Ridges
Just as genes are not randomly distributed over the genome, the chromatin fiber
is not randomly packaged into the nucleus. Many studies have shown that active
genes usually reside more towards the nuclear center than inactive genes (reviewed
by Cremer 2001; Lanctôt 2007). It has been suggested that the nuclear localization
of chromosomal domains contributes to the regulation of their expression. For
example, it has been shown for Drosophila that hundreds of inactive genes cluster
and interact with the lamina at the nuclear periphery (Pickersgill 2006). These
genes are characterized by inactive histone marks, which could be caused by the
histone deacetylase activity present at the nuclear lamina (Somech 2005). However,
induction of gene expression disrupts the interaction with the lamina, suggesting that
localization at the nuclear periphery is a consequence of low gene expression rather
than a cause (Pickersgill 2006). Similarly, induction of gene expression in the human
major histocompatibility complex (Volpi 2000), CFTR locus (Zink 2004), or Hox
cluster (Morey 2009), was also found to drive nuclear position. Transcription itself
might not be directly responsible for the looping and repositioning of chromosomal
regions. Rather, the increase in histone acetylation that occurs upon induction of
transcription, might contribute to the behavior of the chromatin fiber (Tumbar 1999;
Belmont 1999).
In humans, Lamina Associated Domains (LADs) with low overall gene expression
were also discovered and reported to cover 40% of the genome (Guelen 2008;
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Figure 3. Profiles showing gene expression and physical parameters for chromosome 9: (A) Inverse LINE
density, (B) SINE density, (C) GC content, (D) inverse intron length, (E) gene density (F), average gene
expression. All profiles are moving medians over the parameter values per gene for a window size 49.
Bars indicate anti-Ridge (AR) and Ridge (R). Genes are sequentially ordered according to chromosomal
position, but not physically spaced (window size 49 genes). Illustration adapted from Figure 1 Versteeg
et al. (Versteeg 2003).
Wen 2009). LADs coincide with gene poor regions (Guelen 2008), and there is a
good correspondence between LADs and anti-Ridges (data not shown). LADs are
also enriched for histone marks associated with heterochromatin (Guelen 2008).
Domains of heterochromatin have been proposed to act as organizing centers that
might help position active euchromatic domains within the nuclear center (van Driel
2004).
Goetze et al. showed for six different cell lines that a specific Ridge on chromosome 11
was always more in the nuclear interior than an anti-Ridge on the same chromosome.
Although there were clear differences in expression levels for individual genes in the
16
Introduction: Chromosomal domains
Ridge, the overall expression level of the Ridge was similar in all six cell lines. This
was also the case for the anti-Ridge. These results are in agreement with the idea
that the overall activity of a chromosomal domain drives nuclear organization. This
might explain the apparent paradox of why an inactive gene (residing in a Ridge),
can be located in the nuclear interior.
1.6 Domain-wide Regulation of Chromosomal Domains in Cancer
Ridges and anti-Ridges in general have a consistent activity throughout different
tissue types. There are however, a number of smaller specialized clusters of related
genes in the human genome, such as the Hox, globin and histone gene clusters.
These groups of genes are known to be regulated together in a coordinated fashion.
Recently, a number of studies have shown that small clusters of unrelated genes
can also show co-expression throughout different tissues. Most notably a study
on bladder carcinomas showed that clusters of up to 12 unrelated genes have a
correlated expression pattern in a subset of bladder carcinomas (Stransky 2006).
The authors demonstrate for one of these clusters that the genes are silenced by
a domain-wide increase in histone methylation. The spreading of histone marks
is a well known mechanism, but until now has only been implicated in particular
processes, such as heterochromatin formation and the inactivation of the X
chromosome. Although these clusters are smaller than Ridges, these findings show
that epigenetic regulation of gene clusters might play a more important role in the
genome than previously thought.
1.7 Specific Aims of This Thesis
The existence of Ridges raises the question what causes the high expression of
Ridge genes: individual regulation of genes by strong promoters, or an additional
domain-wide effect that up-regulates transcription? In Chapter 2 we address this
question by creating a collection of 90 clones of a human embryonal cell line with a
single randomly integrated fluorescent lentiviral reporter construct. We determined
the chromosomal integration site and fluorescence of each clone. Thus, we compared
the transcriptional activity of clones with a Ridge-integrated reporter construct versus
clones with their reporter situated in an anti-Ridge. This showed that Ridges upregulate expression 4- to 8-fold compared to anti-Ridges.
The correspondence between Ridges and the isochore structure of the human
genome, suggests that transcription of Ridge genes produces mRNAs with a
distinct nucleotide composition (i.e. higher GC content). Chapter 3 investigates the
effect of GC content on the stability of Ridge mRNAs. We find that due to their
high GC content, mRNAs from Ridges have higher folding stabilities as predicted
by their minimal free energy. Microarray analysis on human cells treated with two
transcriptional inhibitors, shows that Ridge mRNAs have 1.5–2 hour longer half-lives
than anti-Ridge mRNAs.
Chapter 4 looks into the effect of GC content on the codon usage and translation
of Ridge genes. We find that the high GC content in Ridge mRNAs causes an
increase in preferred codons and optimal translation initiation sites. We propose
17
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an evolutionary model that explains how genes can acquire extreme levels of
protein expression by translocating to Ridges. The chapters 2, 3 and 4 describe
how Ridges increase the transcription of their embedded genes, while mRNAs of
Ridge genes are in addition more stable and also have codons that facilitate highly
efficient translation. This suggests that Ridges and their physical properties enable
a ‘highway’ for gene expression in the genome. Since the range of expression levels
of cellular proteins is quite extreme, Ridges might contribute to very high protein
expression levels by superimposing the three mechanisms proposed in chapters
2–4 to achieve an exponential system of gene expression.
Chapter 5 describes the role the histone methyltransferase enhancer of zeste
homolog 2 (EZH2) in neuroblastoma. In cancer, chromosomal domains were shown
to be deregulated by chromatin modifying enzymes. This prompted us to investigate
the role of EZH2 in the pediatric cancer neuroblastoma, where it is highly expressed.
EZH2 belongs to the Polycomb group proteins and has been implicated in cancer
as an oncogene. Here we show that EZH2 is required for cell cycle progression in
neuroblastoma and is associated with a poor prognosis.
In Chapter 6 we discuss the likelihood of several well-known mechanisms as
mediators of domain-wide up-regulation of transcription by Ridges. We propose a
mechanism to explain how Ridges function and the potential impact they have on
evolution.
18
Introduction: Chromosomal domains
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