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A-T-Rich Scaffold Attachment Regions Flank the Hematopoietic Serine Protease
Genes Clustered on Chromosome 14q11.2
By Robin D. Hanson and Timothy J. Ley
We have analyzed approximately 70 kb of the chromosome
14q11.2 hematopoietic serine protease gene cluster for the
presence of nuclear scaffold attachment regions (SARs). At
least 12 potential attachment sites were identified.SARs are
present on both sides of the CGL-IICSP-B and CGL-21
CCP-X genes and upstream from the cathepsin G (CG) gene.
We have further characterized the SARs immediatelyflanking
the cytotoxic lymphocyte-specificCGL-1ICSP-B gene. These
5‘ and 3’ SARs are highly A-T-rich, contain multiple attachment sites. and are associated with the scaffolds of nuclei
derived from both lymphoid and erythroid cell lines. These
SARs contain multiple consensus elements frequently associated with A-T-rich sequences, includingthe vertebrate topoisomerase II (topo II) consensus sequence, the A-box and
T-box elements, and the yeast autonomous replicating sequence (ARS). The potential role for the nuclear scaffold in
the transcriptional regulation of CGL-1/CSP-B expression is
discussed.
o 1992by The American Society of Hematology.
A
ment regions (SARs), also called matrix association regions
(MARS), typically contain multiple topoisomerase I1 (topo
11) binding sites: and top0 I1 is a major component of the
nuclear scaffold.” Finally, juxtaposition of regulatory DNA
sequences with the nuclear scaffold may increase the
efficiency of transcription factor utilization, perhaps by
reducing the effective volume in which a factor must find its
cognate DNA binding sites.
We recently described the cloning of an hematopoietic
serine protease gene cluster.” The three known genes in
this locus are expressed in distinct lineages of hematopoietic cells, and at distinct stages of development. The
CGL-1/CSP-B gene is expressed at high levels in activated
cytotoxic T lymphocytes,lymphokine-activated killer (LAK)
cells, and natural killer (NK) cell^.''^'^ CGL-2/CCP-X is
expressed at lower levels in NK and LAK cells.’4Cathepsin
G (CG) is expressed exclusively in myeloid and monocytic
precursor^.'^-'^ Expression of CGL-1 /CSP-B and CG is
mutually exclusive in all of the cell lines and tissues that we
have examined thus far.I4 We are interested in understanding the differential regulation of these linked genes, and we
reasoned that one mechanism that could contribute to the
mutually exclusive pattern of CGL-1 versus CG expression
might be organization of these genes into separate chromatin domains. In this model, transcriptional activity in one
domain might depend on the adjacent domain being closed.
To begin to define the chromatin domains within the
serine protease locus, we have identified, mapped, and
characterized the in vitro scaffold attachment sites within
this region. In vivo, the proximal SARs on either side of the
CGL-1/CSP-B gene are associated with the scaffold in both
lymphoid and erythroid cell lines. Sequencing of the CGL1/CSP-B SARs demonstrated extensive A-T richness. In
particular, the 5’ S A R contains at least 2.4 kb of DNA,
which is 70% A-T-rich overall; this region appears to be
associated with the nuclear scaffold along its entire length.
Our results suggest that each serine protease gene may be
contained within a separate domain, and that these domains may somehow be involved with the differential
expression of the genes within this cluster.
EUKARYOTIC GENE can be regulated by cis-acting
DNA sequences close to it, as well as sequences
located many kilobases away. For some genes, an additional
level of regulation may be provided by the physical state of
the gene’s DNA. The properties of torsional stress and
DNA accessibility are known to differ for some genes in
different tissues, and may reflect differences in transcriptional activity.’,’ For example, all of the DNA in the human
P-globin gene cluster is “sensitive,” or accessible, to DNAse
I in vivo in erythroid cells. The same sequences are
inaccessible to DNAse I in cells derived from nonerythroid
tissues.’ We do not yet know whether changes in the
physical state of DNA are responsible f o r - o r caused
by-transcriptional activity.
The nuclear scaffold, or nuclear matrix, is a poorly
characterized network of proteins that tightly binds specific
DNA sequences that have been evolutionarily con~erved?~
The loops of chromatin observed by electron microscopy of
metaphase chromosomes may correspond to DNA domains
anchored at each end by the nuclear ~caffold.~~’
Three
possible roles for the nuclear scaffold in gene regulation
have been suggested.8 First, the scaffold may serve to
sequester a gene or a gene cluster from surrounding
chromatin, perhaps blocking interactions with adjacent
regulatory sequences, or dampening the spread of torsional
stress among domains. Second, it may be a site for the
unwinding of DNA during transcription, or it may facilitate
the establishment of an open or closed chromatin conformation by controlling superhelicity. Indeed, scaffold attachFrom the Division of HematologylOncology, Departments of Medicine and Genetics, Jewish Hospital at Washington University Medical
Center, St Louis, MO.
Submitted April 8, 1991; accepted September 24, 1991.
Supported by National Institutes of Health Grants No. DK 38682
and CA 49712, and by the Washington University-MonsantoAgreement.
Address reprint requests to Timothy J. Ley, MD, Division of
Hematology /Oncology, Depa rtments of Medicine and Genetics, Jewish Hospital at Washington University Medical Center, 216 S Kingshighway Blvd, St Louis, MO 63110.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971I9217903-0023$3.00/0
610
MATERIALS AND METHODS
Cell culture and isolation of nuclei. Cell lines were maintained in
Iscoves modified Eagle medium with 5% fetal calf serum (GIBCO,
Grand Island, NY) and 5% controlled process serum replacement-4 (Sigma, St Louis, MO). PEER cell activations were
Blood, Vol79, No 3 (February 1). 1992: pp610-618
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SERINE PROTEASE GENE SARs
performed with 12-0-tetradecanoylphorbol-13-acetate(TPA;
Sigma) and N6-2-0-dibutyryladenosine3',5'-cyclic monophosphate
(bt,cAMP; Sigma) at final concentrations of 1.6 x
mol/L and
0.5 mg/mL, respectively.
Nuclei were prepared by washing 5 x lo7cells with 1x Hebs (137
mmol/L NaCI, 5 mmol/L KCI, 0.7 mmol/L Na,HPO,, 6 mmol/L
dextrose, 20 mmol/L HEPES [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; pH 7.051) and resuspending in RSB (10
mmol/L TRIS, pH 7.5, 10 mmol/L NaCI, 5 mmol/L MgCI,, 0.1
mmol/L phenylmethylsulfonyl fluoride [PMSF]). After a 15minute incubation on ice, cell membranes were broken by three to
five forceful ejections through a 22-gauge needle. Nuclei were
pelleted, washed with isolation buffer (3.75 mmol/L TRIS, pH 7.4,
0.05 mmol/L spermine, 0.125 mmol/L spermidine, 20 mmol/L
KCI, 0.01% digitonin, 1% thiodiglycol, 0.5 mmol/L PMSF, 0.5
+g/mL leupeptin, 0.7 kg/mL pepstatin, 0.5 mmol/L EDTA-KOH,
pH 7.4) and resuspended in isolation buffer minus EDTA, Nuclei
were either used immediatelyor stored at -20°C in 50% glycerol.
Preparation of nuclear scaffolds. Nuclear scaffolds were prepared essentially as described." Briefly, 10 OD,,, units of nuclei
were mixed with an equal volume of isolation buffer containing 0.5
mmol/L CuSO, and incubated for 10 minutes at 37°C. Seven
milliliters of lithium salt buffer (5 mmol/L HEPES, pH 7.4, 0.25
mmol/L spermidine, 2 mmol/L EDTA-KOH, pH 7.4, 2 mmol/L
KCI, 0.01% digitonin, 15 mmol/L lithium 3,5-diiodosalicylate)was
slowly added, followed by a 10-minute incubation at room temperature. Nuclear scaffolds were pelleted by centrifugation for 20
minutes at 2,400 x gat 4"C, washed twice with digestion buffer (20
mmol/L TRIS, pH 7.4, 0.05 mmol/L spermine, 0.125 mmol/L
spermidine, 20 mmol/L KCI, 70 mmol/L NaCI, 10 mmol/L MgCl,,
0.01% digitonin, 0.1 mmol/L PMSF, 0.5 kg/mL leupeptin, 0.7
pg/mL pepstatin), and resuspended in 2 mL of digestion buffer.
I n vitro SAR assay. One hundred microliters of the nuclear
scaffold preparation was mixed with 5 pg of sonicated Escherichia
coli DNA, 50 to 100 U BamHI and/or EcoRI, and 1 to 4 ng of
end-labeled DNA (5 X 1Ohcpm/+g)and incubated for 2 to 4 hours
at 37°C. The reaction was pelleted by centrifugationfor 10 minutes
at 3,100 x g at 4"C, the supernatant saved, and the pellet washed
once more with digestion buffer and resuspended in l x TE (10
mmol/L TRIS pH 7.5, 1mmol/L EDTA). The pellet and supernatant fractions were adjusted to 160 mmol/L NaCI, 0.5% sodium
dodecyl sulfate (SDS), and 0.2 mg/mL proteinase K and incubated
for 16 hours at 50°C. DNA was phenol/chloroform-extracted,
ethanol-precipitated, and electrophoresed on agarose or polyacrylamide gels.
In vivo SAR assay. Five hundred microliters of nuclear scaffold
preparation was incubated with restriction enzymes as above, but
without exogenous competitor or probe DNA. Genomic DNA was
purified from pellet and supernatant fractions as described above.
Approximately equal amounts of DNA from each fraction were
electrophoresed, transferred to nitrocellulose membranes, and
analyzed by Southern blot hybridization as previously described"
using random primer-labeled probes.I8
61 1
. ,.,.$
,.,
. , . ,.,
LI_I
CGL-I/CSP+?
CGL-PKCP-X
I
CG
I C6
32
Fig 1. Identificationof potential SARs in the serine protease gene
cluster. The cosmids C2.10 and C6.32 were digested with various
restriction endonucleases, end-labeled, and incubated with nuclear
scaffolds in the presence of competitor DNA. Regions of DNA that
bind preferentially to the scaffold are indicated by asterisks, with
brackets indicating the limits of the smallest DNA fragment that
defines each SAR. The relative positions and transcriptionalorientations of the three hematopoietic serine protease genes are shown in
reference to the two cosmlds used in this study. Note that multiple
SARs are detected on either side of CGL-l/CSP-B and CGL-P/CCP-X
and upstreamfrom CG.
CGL-2/CCP-X and upstream of CG bind the PEER
scaffold in vitro. In contrast, most o r all of the transcribed
regions of each gene d o not contain SARs. W e cannot rule
out the existence of a SAR in the 5' transcribed portion of
the CGL-2/CCP-X gene, since a fragment containing this
region, as well as upstream sequences, bound to the
scaffold. Figure 2 contains an example of the SAR mapping
experiments that are summarized in Fig 1. The cosmid
C2.10, which extends from 5' of CGL-1/CSP-B to 3' of
CGL-2/CCP-X, was end-labeled after digestion with either
BamHI or EcoRI. The resulting fragments were either
incubated directly with the scaffold, o r were bound to the
scaffold following secondary restriction enzyme cleavage
with EcoRI or BamHI. The scaffold and associated D N A
fragments were pelleted by centrifugation. D N A was purified from the pellet and supernatant and electrophoresed
on an agarose gel along with a sample of the input DNA. As
shown in Fig 2A, some D N A fragments are recovered in the
pellet, while others are found exclusively in the supernatant. Some fragments are portioned almost completely into
the pellet fraction, suggesting a very high affinity for the
scaffold. For example, the 1.9-kb BamHIIEcoRI fragment
is recovered entirely in the pellet fraction. A summary of
the results of this experiment is shown in Fig 2B. The D N A
fragments that clearly associate with the PEER-cellderived nuclear scaffold are indicated.
Mapping of potential SA& 5' and 3' to the CGL-1ICSP-B
gene. Since the region containing the transcribed portion
of the CGL-1/CSP-B gene did not bind the scaffold, we
next focused on the SARs immediately flanking this gene.
The 2.4-kb EcoRI fragment 5' from CGL-l/CSP-B and the
7.6-kb EcoRI fragment extending 3' from CSP-B were
RESULTS
subcloned and further analyzed. The strategy used to
Identification of potential SA& in the serine protease gene
further define the scaffold attachment points within these
cluster. D N A sequences associated with the nuclear scaftwo fragments is outlined in Fig 3C. The 5' SAR fragment
fold in vivo have been shown to preferentially bind to
was end-labeled at its EcoRI sites. A portion of this D N A
lithium diiodosalicylate (LIS)-extracted nuclei in ~ i t r 0 . I ~ was then secondarily cleaved with either BamHI o r PstI. A
We therefore analyzed the serine protease locus by identifymixture containing all of the indicated D N A fragments was
ing end-labeled cosmid D N A restriction fragments that
then incubated with a PEER scaffold as before, and
bind to LIS-extracted nuclei from the PEER T-cell line. A
analyzed as shown in Fig 3A. While the 3.0-kb vector D N A
summary of these results is presented in Fig 1. Multiple
is not recovered in the scaffold pellet, all of the 5'
D N A fragments on either side of CGL-l/CSP-B and
CGL-1/CSP-B fragments are recovered in approximately
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HANSON AND LEY
612
A
I PS
I PS
I PS
IPS
=
-
1l.k
-
86.60.8.0(v)
4.8
3.1- 000
2.3- o 0
1.9 0 0
1.6-0 0
-
- -
0.5
-
0.5
*mu1
BomHl
'BomHI
1
Eco RI
-- -
*EcoRI
t
8omHI
CCP-x
CGL-U
17
,
.
&&
I12
1
12.1
Bound
67
equal proportions. This indicates that multiple scaffold
attachment sites must occur along the length of the 2.4-kb
fragment. The 3' CGL-l/CSP-B fragment was end-labeled
following digestion with BumHI and EcoRI. The 6.4-kb
fragment and the vector DNA were recovered almost
Fig 2. Mapping of potential
SARs near the CGL-1/CSP-B and
CGL-2/CCP-X genes. (A) Cosmid
C2.10 was end-labeled at either
the BsmHl or EcoRl sites. Labeled DNA fragments were incubated with LIS-extracted nuclei
from PEER cells in the presence
of either BamHl or EcoRl or both.
Nuclei were pelleted, and DNA
was purified from pellet (P) and
supernatant (S)fractions. DNA
was electrophoresed on an agarose gel, dried, and autoradiographed. The sizes of individual
fragments (in kb) are indicated;
(v) denotes vector DNA fragments derived from the cosmid.
Lanes labeled "I" contain a sample of the input DNA mixture.
Note that several DNAfragments
associate preferentially with the
scaffold and are recovered in the
pellet fraction. (B) Restriction enzyme map of the serine protease
locus contained in cosmid C2.10.
DNA fragments clearly partioning with the nuclear scaffold or
supernatant are indicated as
bound or free, respectively. B,
BamHI. E, EcoRI. Sizes are in kb.
entirely in the supernatant, whereas the 1.2-kb fragment
was found almost exclusively in the pellet.
The 5' and 3' SAR-containing fragments were isolated
from plasmid DNA, digested with Ah1 or HueIII, and then
end-labeled. Phage @X174 DNA was also digested with
B
A
I PS
I P
-
(vector) (3.0)
2.4
1.9
1.4
1.0
6.4
-
-
(vectod(2.7)
0.5 Fig 3. Mapping of SARs 5' and 3' of the CGL-l/
CSP-B gene. Plasmids containing the 2.4-kb EcoRl
fragment 5' of CGL-1/CSP-B and the 7.6-kb EcoRl
fragment 3' of CGL-1/CSP-B were end-labeled at the
EcoRl sites. (A) Aliquots of the 5' SAR DNA were
secondarily digested with nothing, BamHI, or Pstl,
and then were mixed together and incubated with
LIS-extracted PEER nuclei. The input (I)and pellet (P)
DNAs are shown. Note that all of the subfragments
derived from the 5' SAR are recovered in the pellet.
(B) The labeled 3' SAR DNA was secondarily digested
with BamHl and then incubated with LIS-extracted
PEER nuclei. Input (I),pellet (P), and supernatant (S)
DNA fractions are shown. Note that only the 1.2-kb
BamHIIEcoRI fragment is recovered in the pellet. (C)
The relative locations and sizes of the 5' and 3' SAR
DNA fragments are shown. E, EcoRI. B, BamHI. P,
Pstl.
-
1.2-
C
--
H
0.5
I
1.0
I
{ 2.4
7
I .9
1.4
4 6.4
H1.2
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613
SERINE PROTEASE GENE SARs
HaeIII and end-labeled as a control. As shown in Fig 4A,
the QX174 DNA is entirely recovered in the supernatant
following incubation with a PEER-cell-derived scaffold, as
expected. In contrast, many of the AluI and HaeIII fragments from the 5' S A R are found in the pellet. The 128and 76-bp Ah1 and the 181-bp HaeIII fragments are from
nonoverlapping regions and do not bind appreciably to the
scaffold. These fragments therefore define distinct regions
within the 5' SAR, which by themselves are unable to bind
the scaffold. The results of a similar experiment using A M
and HaeIII fragments from the 3' S A R are shown in Fig 4B.
Significant amounts of the 704-bp AluI and 947-bp HaeIII
fragments are recovered in the pellet, while the smaller
fragments are recovered mostly or entirely in the supernatant.
5' and 3' S& are attached to the nuclear scaffold in vivo.
Binding of a particular DNA sequence to a nuclear scaffold
in vitro does not necessarily reflect scaffold association of
that sequence in vivo. For example, DNA sequences 3' to
the chicken pA-globingene bind equally well in vitro to
nuclear scaffolds prepared from either brain or erythroid
cells; however, these regions are associated only with
erythroid scaffolds in vivo." We therefore investigated the
in vivo status of the CGL-l/CSP-B 5' and 3' SARs in
different nuclear environments in vivo.
The PEER cell line does not transcribe the CGL-I/
CSP-B gene constitutively, but it does express the gene at
high levels following 48 hours of stimulation with TPA and
bt2cAMP." Nuclei from untreated or TPA + bt,cAMPtreated PEER cells were extracted with LIS, digested with
BamHI and EcoRI, and separated into pellet and supernatant fractions by centrifugation. Genomic DNA was puri-
A
I1
P S"I P s"1 P
7-
1350
1080
870
--
-
600-
31Q281/271
234
I94
-
--
118-
s'
fied from each fraction, electrophoresed, transferred to
nitrocellulose, and analyzed by Southern blot hybridization,
as shown in Fig 5A. The 5' and 3' SARs identified with the
in vitro assay are associated with the nuclear scaffold in vivo
in both untreated and activated PEER cells. This blot was
first hybridized with the 5' 2.4-kb EcoRI fragment. Several
partial digestion products are detected, possibly due to
inaccessibility of the restriction enzyme sites as a result of
scaffold attachment, or perhaps to more general inhibitors
of restriction enzymes in these crude preparations. The
2.4-kb fragment results from the lack of cleavage at the
internal BamHI site (see Fig 3C). This fragment, together
with the 1.9- and 0.5-kb complete digestion products, is
recovered predominately in the pellet fraction from either
untreated or stimulated PEER cells. The blot was next
stripped and rehybridized with the 3' 1.2-kb BamHIIEcoRI
fragment. The 1.2-kb 3' SAR is also detected primarily in
the pellet from both cell types. Finally, the blot was again
stripped and hybridized with a 1.0-kb EcoRI fragment from
within the CSP-B gene. This fragment did not bind the
PEER scaffold in vitro, and it is likewise recovered almost
entirely in the supernatant in the in vivo assay.
To determine whether the 5' and 3' SARs are associated
with the scaffold exclusively in lymphocytes, an identical
experiment was next performed using uninduced K562
erythroleukemia cells, as shown in Fig 5B. Uninduced K562
cells do not express detectable amounts of CGL-1, CGL-2,
or CG mRNA.I4 Again, the 5' and 3' SAR fragments are
completely recovered in the pellet fraction, whereas the
internal 1.0-kb fragment is found entirely in the supernatant. Therefore, the SARs flanking the CSP-B gene are
matrix-associated in vivo, but matrix binding does not
B lI
P
s'
'I
P
s'
-
587
458
434-
-
29%
267
227
--
I74
-
102
-
80
-
I
I
3'SAR
*Alul
1
1
3'SAR
.Haem
Fig 4. Fine mapping of SARs 5' and 3' ofthe CGL-l/CSP-B gene. The 2.4-kbEcoRl fragment contalningthe 5' SAR and the 1.2-kbBamHI/€coRI
fragment containing the 3' SAR were gel-purified, digested with Alul or HeeIII, and end-labeled. As a control, 9x174 RF DNA was digested with
Heelll and end-labeled. DNA fragments were incubated with LIS-extractedPEER nuclei, and input (I),pellet (P), and supernatant (S)
fractions were
electrophoresedon a polyacrylamidegel and autoradiographed. (A) Analysis of 9x174 and 5' SAR fragments. Sizes of the 6x174 Huelll fragments
are indicated (in bp) on the left. No 6x174 fragments bind to the scaffold, as expected, while multiple regions within the 5' SAR bind. (B) Analysis
of 3' SAR fragments. Sizes (in bp) of Heelll digested fragments of pUC9 are indicated on the left. Note that several specific fragments from both
the 5' and 3' SARs bind to the scaffold.
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614
HANSON AND LEY
PEER
A
"1ps'
4.423-
2.0-
0.612 3 4
5'SAR
12
34
3' SAR
12 3 4
CGL- I ICSP-B
gene
K562
B
P S
P S
P S
-
2.3201 2
1 2
5'SAR
3'S AR
1 2
CGL- I/CSP -8
gene
appear to be lineage-specific in these two hematopoietic
cell lines.
The sequence of the 5' and 3' SARs is highly A-T-rich.
We next sequenced the 5' 2.4-kb EcoRI fragment and the 3'
1.2-kb BumHIIEcoRI fragment (Fig 6). The 5' SAR contains 69% A + T, while the 3' SAR is 68% A + T. In
addition to overall A-T richness, several sequence motifs
have been found in association with SARS."".~ These
motifs are all expected to occur at a very high frequency in
Fig 5. In vivo analysis of CGL-l/CSP-B 5' and 3'
SARs in PEER and K562 cells. Nuclei were extracted
with LIS and incubated with EcoRl and BsmHI. Following centrifugation, genomic DNA was purified from
the pellets (P) and supernatants (S).Equal amounts
of DNA were electrophoresed and analyzed by Southern blot hybridization. (A) Analysis of untreated (-)
and TPA + bt,cAMP-treated (+) PEER cells. Blots
were consecutively hybridized with the 2.4-kb EcoRI
fragment located upstream from CGL-1/CSP-B (5'
SAR), the 1.2-kb BamHIIEcoRI fragment 3' of CGL-/
CSP-B (3' SAR), and the 1.0-kb EcoRI fragment contained within the CGL-1ICSP-B gene. (6) Analysis of
K562 cells. Blots were hybridized as in (A). Note that
DNA fragments from the 5' and 3' SARs are recovered preferentially in the pellets (matrix-attached),
and that the CLG-1/CSP-B gene itself is found in the
supernatant (non-matrix-attached) fractions.
70% A-T-rich regions by virtue of their A-T richness; the
significance of these consensus motifs in this context is as
yet unknown. Regardless, we searched the 5' and 3' SAR
regions for these motifs, allowing one mismatch in the
consensus: the A-box motif (AATAAAT/CAAA)9is found
four times in the 5' SAR at positions 146,215,663, and 839,
and once in the 3' SAR at position 124. The T-box motif
(TTATTTTTTT or TTlTATTATT) is found in the 5'
SAR at positions 212,264,1428,2016, and 2018, and in the
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
615
SERINE PROTEASE GENE SARs
.
.
.
.
.
.
.
.
.
.
. ,M
SAR at positions 867, 961, 1627, and 1891, and twice in the
.
.
.
.
.
.
.
.
.
.
.
210
3' SAR at positions 399 and 1013.
u\IcouuuLLn~TTIoTrTu,U~-,,~~nucffi,~-,~~"n"~,-~,wCU,~~
A search for direct and inverted repeats within and
.
.
.
.
.
.
.
.
.
.
. 1 6 0
between the 5' and 3' SARs identified four motifs. The
.
.
.
.
.
.
.
.
.
.
.
e
n
sequence II?TTAAAA is found twice in the 5' SAR (at
.
.
.
.
.
.
.
.
.
.
, 6 0 0
positions
1316 to 1323 and 1919 to 1926) and twice in the 3'
LIaTu*LnuiircII"DI~~~-~rA~r~~IcIl~"CU~--~-UnU
SAR (at positions 190 to 197 and 236 to 243). The sequence
.
.
.
.
.
.
.
.
.
.
.
RI
~ u c I I ~ c c o u L c c u ~ ~ ~ ~ ~ ~ u ~ ~ ~ ~ ~ c u - ~ ~ ~ ~ ~ u ~ ~ ~ m ~ ~ - ~ ~ - ~ ~ ~ - ~ " - - u ~
TTA'ITGTAT occurs twice in the 5' SAR (at positions 829
.
.
.
.
.
.
.
.
.
.
.
w
c I I u ~ u c T f f i v * * I c L L ~ ~ r ~ ~ " ~ ~ m ~ ~ r ~ to
~ c821
i ~ and
~ m 1446
~ ~ ~ to
~ ~
~ - ~ and
~ ~ Once
~ n ~ in
~ -the 3' SAR (at
1454)
.
.
.
.
.
.
.
.
.
positions 248 to 256). The sequence 'I?TTAATTT is
TLurcurmlllTITuiE-~,-m~~U,-,~u","CU,~n--~
repeated three times in the 5' SAR (at positions 998 to
. . . . . . . . . . . 1m
* u L o \ ~ I u u n w ~ - m w ~ ~ I u u n ~ - U ~ ~ - ~ - ~ - ~ ~ ~ - ~ ~ " - ~ ~ * ~ - ~ u ~ ~
1006,1439 to 1431, and 1913 to 1921) and is present once in
.
.
.
.
.
.
.
.
.
.
.
1240
T n w T U ~ r u c I I x t l w * r , ~ , ~ m ~ , ~ - - ~ ~ - m " ~
the 3' SAR (at positions 1078 to 1086). Finally, the
.
.
.
.
.
.
.
.
.
.
. 1 3 w
the~5' mSAR
~ ~ ~ T c c I u u E y v . u u u u ~ ~ ~ u ~ m sequence
~ ~ ~ ~ATTT?TTAA
~ u ~ ~ ~ is
~ found
~ ~ three
r ~ times
~ ~ ~in m
~ ~
.
.
.
.
.
.
.
.
.
.
.
<YO
(at positions 1434 to 1426, 1871 to 1879, and 2067 to 2075)
- T - r r i T U C Y U C C L r ~ - - , ~ ~ ~ ~ ~ ~ W r U ~ ~ ~ i ~ " ~ ~ ~ ~ - n and once in the 3' SAR (at positions 1185 to 1193). The
. . . . . . . . . . . 1~60
T*CWrT~nCr~iU"-,,~,~,~,~n,~~,~,~,~-i~~~,-,""~-,-~"~"~"cII"~
significance of these repeated A-T-rich motifs is unknown.
.
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Figure 7A shows an A-T richness plot of the 5' flanking
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sequence of the CGL-1/CSP-B gene, including the 5 ' SA.'
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Fig 6. DNA sequence of the CGL-l/CSP-B 5' and 3' SARs. The 5'
2.4-kb €coRI and 3' 1.2-kb &mHI/€coRI fragments were sequenced
on both strands using modified T7 DNA polymerase (Sequenase 2,
USB) and sequence-specific oligonucleotide primers, using the sequencing strategy shown in the diagram. The sequences shown carry
the Genbank accession members M62717 (5' SAR) and M62716 (3'
SAR).
3' SAR at positions 194 and 410. The vertebrate top0 I1
consensus cleavage sequence (A/GnC/TnnCnnGC/TnGG/
TXIC/T~C/T)'~
is found once in the 5' SAR at position
1405, and once in the 3' SAR, at position 646. Finally, the
yeast autonomous replicating sequence (ARS) consensus
(A/II?TTATA/GTITA/T)*l is found four times in the 5'
Fig 7. A-T richness plot of DNA sequences 5' and 3' of CGL-1/
CSP-B. The Beckman Microgenie sequence analysis program was
used t o plot the percentage of A T in 5' CGL-l/CSP-B sequences
extending from positions -3599 t o +1 relative t o the transcriptional
start-site (A, indicated as 1-3600 on the x-axis) dnd 3' CGL-l/CSP-B
sequences beginning approximately 4.8 kb down tream of the poly-A
addition site (B, indicated as 1-1200 on the x-axis At each nucleotide
position, the percentage of A + T i n the surround ng 35 nucleotides is
plotted. The 5' SAR described here corresponds t positions 1t o 2429
of (A). The black blocks designate binding of sp , ific DNA fragments
to the scaffold, and the open blocks designate bonassociated DNA
fragments. Note the overall A-T richness of thest regions; however,
not all A-T-rich regions bind t o the scaffold.
+
T3
.
~
~
~
~
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
616
HANSON AND LEY
S A R corresponds to positions 1 to 2429. This region is very
A-T-rich. In contrast, the transcribed portion of the CGL1/CSP-B gene (not shown) contains only a few A-T-rich
stretches. The region from positions 2430 to 3020 (corresponding to position - 1170 to -580 relative to the transcription start site) has an A-T profile similar to that of the 5'
SAR, but it does not bind scaffold preparations in vitro. The
locations of the AluI and Hue111 5' SAR subfragments
identified in Fig 4 are also shown. Notably, the three
fragments that fail to bind the scaffold are also located in
A-T-rich regions. A-T richness alone is therefore not
sufficient for scaffold attachment; sequence context must
also play a role.
An A-T richness plot of the 3' SAR is shown in Fig 7B.
This sequence displays "periodicity" of A-T-rich DNA
stretches from positions 530 to 1197. A-T-rich stretches of
50 to 100 bp are separated by short regions that are not
A-T-rich. The A-T-rich stretch from positions 1066 to 1135
appears to be important for scaffold binding, since it
represents the overlap between the 188-bpHaeIII fragment
(positions 947 to 1135) and the 161-bp Mu1 fragment
(positions 1066 to 1227), which both associate with the
scaffold in vitro. However, fragments containing one or two
A-T-rich stretches on either side of the 1066 to 1135 region
do not bind to the scaffold.
DISCUSSION
Recent studies from several laboratories have suggested
a role for scaffold attachment sites in the organization of
chromatin and the regulation of gene e x p r e s s i ~ n ?Given
~-~~
the disparate development- and lineage-specificpatterns of
expression of the three linked serine protease genes found
on chromosome 14 band q11.2, we were interested in a
possible role for the nuclear scaffold in the organization
and regulation of this locus. Nuclear scaffolds prepared
from LIS-extracted nuclei can bind SAR-containing DNA
fragments with great specificity in vitro. We initially surveyed the serine protease gene cluster for in vitro SAR
activity using DNA fragments obtained from overlapping
cosmids. A number of scaffold attachment sites were
detected on either side of CGL-1/CSP-B and CGL-2/
CCP-X and upstream from CG.
The locations of SARs surrounding each serine protease
gene are similar. A SAR is located 1.2 kb 5' to the
CGL-1/CSP-B transcriptional start site, no more than 2.4
kb 5' to the CGL-2/CCP-X start site, and no more than 4.3
kb 5' to the CG transcriptional start site. The first SAR 3' to
CGL-1/CSP-B is located 4.8 kb downstream of the final
exon, while the first SAR downstream of CGL-2/CCP-X is
between 1.8 and 4.9 kb 3' to the last exon. Thus, if the SARs
identified in vitro are all scaffold-associated in vivo, then
each serine protease gene may be contained within a
separate chromatin loop of no more than 7 to 8 kb in length.
The size of chromatin loops in Drosophila melanogaster may
be inversely proportional to the level of expression of genes
within the loop.*Highly expressed genes such as Adh, Sgs-4,
andftz are contained in loops of 4 to 13 kb, while genes with
less transcriptional activity (such as those in the rosy locus)
are found on loops of at least 50 kb. The two well-studied
serine protease genes, CGL-1/CSP-B and CG, are both
expressed at very high levels in specific lineage^'^ and are
tightly flanked by SARs; these observations may therefore
corroborate the proposed relationship between small loop
size and high transcriptional activity.
Further analysis of the SARs proximal to the CGL-1/
CSP-B gene showed that both the 5' and 3' SARs are
actually composed of multiple scaffold attachment sites. At
least four distinct regions within the 5' SAR have scaffoldbinding activity, and at least three regions of the 3' SAR
have activity. Similarly, the mouse K immunoglobulin gene
has been shown to contain multiple, overlapping binding
~ites.~"."
The presence of multiple binding sites within an
attachment region may permit transcription or replication
to occur without disrupting the DNA-scaffold association?"
The apparent affinity of CGL-l/CSP-B S A R subfragments generated by AluI or HaeIII digestion appears to
correlate in part with fragment length. For both the 5' and
3' SARs, no subfragments smaller than 130 bp bound to the
scaffold, three of four subfragments 130 to 190 bp in length
bound weakly to the scaffold, but all subfragments longer
than 400 bp were associated strongly with the scaffold. We
also noted that none of the AluI or Hue111 subfragments
was completely depleted from the supernatant (scaffoldunattached) fraction. In contrast, the complete 2.4-kb 5'
SAR and 1.2-kb 3' S A R fragments were recovered entirely
in the pellet fraction in the in vitro binding assays. Each
individual scaffold attachment site may therefore contribute to the overall affinity of the SAR for the scaffold, as
previously suggested.*
Chromatin upstream from the CGL-1/CSP-B gene is in a
"closed" or inaccessible conformation in untreated PEER
cells, but it becomes DNAse I hypersensitive after TPA
bt,cAh4P treatment?"Therefore, we wished to know whether
the scaffold association of the 5' and 3' SARs was altered
with PEER cell stimulation. No change was observed; both
SARs were clearly scaffold-attached before and after stimulation. Similarly, the 5' and 3' CGL-1 SARs were both
matrix-associated in K562 cells, a line in which none of the
genes in this cluster is expres~ed.'~
Although development
and lineage-specific differences in SAR attachment have
previously been reported for the D melanogasterAdh gene'
and the chicken P-globin gene" in fresh tissues, a number of
SARs associated with genes that are expressed in specific
tissues show constitutive matrix attachment in all cell lines
analyzed. Examples include the human apolipoprotein B
gene? and the mouse Ig heavy-~hain~~
and K light-~hain'~
genes. Therefore, the constitutive scaffold attachment of
the CGL-1/CSP-B 5' and 3' SARs in untreated versus
stimulated PEER cells, as well as in K562 cells, is probably
to be expected. To further define the tissue and lineage
specificity of matrix association for this group of S A R s , a
more detailed examination of freshly obtained tissues will
need to be performed.
The sequences of the CGL-1/CSP-B 5' and 3' SARs are
highly A-T-rich, a feature that is common to all SARs.
However, A-T richness alone does not explain all SAR
+
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
617
SERINE PROTEASE GENE SARs
activity in this region. For example, the sequence extending
from positions -1170 to -580 relative to the transcriptional start site (positions 2430 to 3020 in Fig 7A) is as
A-T-rich as the 5‘ SAR (positions 1 to 2430), but a 1.8-kb
EcoRI fragment containing this region does not bind to the
scaffold in vitro. However, this region could still conceivably
bind to the scaffold when it is linked with the 5’ SAR.
The 5’ SAR lies adjacent to CGL-1/CSP-B 5‘ regulatory
sequences; specifically, we have shown that sequences from
positions -1170 to +31 relative to the transcriptional start
site are necessary and sufficient for high-level, inducible
CGL-1/CSP-B expression in PEER cellsZoand in the T
lymphocytes of transgenic m i ~ e . 3The
~ juxtaposition of
SARs with the regulatory sequences of other genes, including the drosophilatldh, Sgs-4, andftz genes, and the mouse
Ig heavy-chain and K light-chain genes, has previously been
d e s ~ r i b e d . ’ ,We
~ ~ , have
~ ~ previously demonstrated that the
activation of CGL-1/CSP-B transcription in PEER cells is
accompanied by a change in chromatin conformation, as
evidenced by the appearance of a DNAse I hypersensitive
site just upstream from the gene.” Therefore, we speculate
that one mechanism of CGL-1/CSP-B regulation may
involve promoter accessibilityto transcription factors. SARs
are candidates for such chromatin conformational control.
For example, the functional domains of the chicken lysozyme gene36and the human apolipoprotein B gene:’ as
defined by DNAse sensitivity, are also immediately flanked
by scaffold attachment sites.
SARs may play a role in establishing and maintaining
chromatin “domains.” The 5’ chicken lysozyme gene SAR
confers high-level, integration site-independent, copy number-dependent expression on linked lysozyme gene regulatory sequences.26 Similarly, the 3’ chicken @-globin enhancer provides integration site-independent, copy number-
dependent expression of linked chicken @-globingenes in
transgenic mice3’; this region also behaves as a powerful,
erythroid-specific SAR in vivo.1y.3833y
Flanking SARs also
provide integration site-independent expression for the ftz
gene.‘3 Finally, the S A R adjacent to the mouse Ig K gene
enhancer increases expression of the intact K gene fourfold
in stably transfected plasmacytoma cells.” All of these
experiments suggest that SARs may somehow isolate transferred genes from negative regulatory sequences that may
be located in DNA that flanks an integration site. Regulatory elements in the transfected gene fragment may then
respond to “normal” regulatory influences, regardless of
differences among particular integration sites.
We recently generated a number of transgenic mice using
CGL-l/CSP-B sequences from positions -1170 to +31 to
drive expression of the human growth hormone gene; this
fusion gene lacks the CGt-l/CSP-B SARs. While the
majority of mice had appropriate tissue-specific expression
of the transgene, the level of expression per transgene copy
was consistently less than that of the endogenous mouse
homologue, CCPI. In addition, no correlation between
level of expression and transgene copy number was observed, indicating that levels of transgene expression were
influenced by sequences flanking the site of integration in
the mouse genome.35These experiments therefore provide
a model for testing potential functions of the SARcontaining regions. We will examine the effects of the
CGL-1/CSP-B 5’ and 3’ SARs on chromatin organization
and gene expression in stably transfected cell lines and
transgenic mice.
ACKNOWLEDGMENT
The authors thank Diana Coleman for her expert editorial
assistance.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1992 79: 610-618
A-T-rich scaffold attachment regions flank the hematopoietic serine
protease genes clustered on chromosome 14q11.2
RD Hanson and TJ Ley
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