|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 800-807
PLENARY PAPER
 -Thalassemia resulting from a negative chromosomal position
effect
Virginia M. Barbour,
Cristina Tufarelli,
Jacqueline A. Sharpe,
Zoe E. Smith,
Helena Ayyub,
Cynthia A. Heinlein,
Jacqueline Sloane-Stanley,
Karel Indrak,
William G. Wood, and
Douglas R. Higgs
From the MRC Molecular Haematology Unit, Institute of Molecular
Medicine, John Radcliffe Hospital, Headington, Oxford, England, and
the Department of Clinical Haematology, Faculty Hospital,
IP Pavlova, Olomouc, the Czech Republic.
 |
Abstract |
To date, all of the chromosomal deletions that cause  -thalassemia
remove the structural genes and/or their
regulatory element (HS -40). A unique deletion occurs
in a single family that juxtaposes a region that normally lies
approximately 18-kilobase downstream of the human cluster, next to
a structurally normal -globin gene, and silences its expression.
During development, the CpG island associated with the -globin
promoter in the rearranged chromosome becomes densely methylated and
insensitive to endonucleases, demonstrating that the normal chromatin
structure around the -globin gene is perturbed by this mutation and
that the gene is inactivated by a negative chromosomal position effect.
These findings highlight the importance of the chromosomal environment
in regulating globin gene expression.
(Blood. 2000;96:800-807)
© 2000 by The American Society of Hematology.
| |
Introduction |
The human  -globin gene cluster lies in the telomeric
region of chromosome 16 (16p13.3). A 285-kb (kilobase) segment of DNA extending from the terminal (TTAGGG)n repeats of chromosome 16 has been
fully sequenced and shown to be a GC- and
Alu-rich region containing a high density of
CpG islands and genes. The cluster includes an embryonic gene ( )
and 2 fetal/adult genes (2 and 1) arranged along the
chromosome in the order in which they are expressed during development
(Figure 1). Expression of these genes is
critically dependent on a single erythroid-specific positive regulatory
element (HS -40), which lies 60 kb from the genes in the
intron of an adjacent widely expressed gene.1
View larger version (22K):
[in this window]
[in a new window]
| Fig 1.
Structure of the terminal region (approximately 300 kb)
of the human chromosome 16p.
The oval on the left represents the telomere. Previously described
genes (4-203) are shown as black boxes
above the line (transcribed toward the centromere) or below the line
(transcribed toward the telomere). The -globin regulatory element is
shown as a white box (approximate coordinates 103 500-103 850). The
embryonic () and fetal/adult () genes are
indicated. Below the chromosome, the positions of previously
characterized DNaseI hypersensitive sites (DHSs) and CpG islands
(labeled A-N) are shown. Below this is a graph of the percent of
Alu sequences per 3 kb. Dashed vertical lines represent the
5' and 3' extents of the ZF deletion. The scale is in base
pairs. Coordinate 1 is the first nucleotide in the chromosomal sequence
described in Flint et al.3
|
|
The relationship between chromosome structure and function has been
well characterized in this region.2,3 It exists as a
segment of "open," transcriptionally active chromatin containing many DNase1 hypersensitive sites, punctuated by areas
of apparently "closed" chromatin, including the most telomeric
region and a long segment of nuclease-insensitive chromatin between
coordinates approximately 180 000 to 218 000. The CpG islands (Figure
1, A-N) in the 285-kb region are unmethylated in all cell
types and tissues examined, but most of the interisland CpG
sites appear to be densely methylated (D.R.H., unpublished data,
1999). Although the subtelomeric region replicates late
in the cell cycle, the adjacent GC-rich region replicates early in all
cell types examined.4
More than 50 natural deletions from this region have been identified,
most of which were detected because they down-regulate the gene expression and cause one of the well characterized hematologic
phenotypes of -thalassemia.5 All previously described mutations of this type either delete the -globin genes or remove the
remote regulatory element (HS -40), but the mutations cause no
discernible changes in the patterns of methylation, DNase1 hypersensitivity, or replication. Here we describe an approximately 18-kb deletion that extends downstream of the cluster. Although HS
-40 and the 2 gene remain fully intact, -globin
expression from this chromosome is abolished. Changes in the pattern of
methylation and DNase1 hypersensitivity within the -globin cluster
suggest that this deletion silences the 2 gene by a negative
chromosomal position effect rather than by removing positive regulatory elements.
| |
Materials and methods |
Hematologic investigations
Full blood counts, examination of peripheral blood films,
and identification of hemoglobin H (HbH) inclusions were performed as
previously described in Weatherall et al.6
Cell culture
Epstein-Barr virus (EBV)-transformed cell lines were established
for each family member. Interspecific hybrids containing the abnormal
 ZF chromosome were established by fusing
EBV-transformed lymphocytes to mouse erythroleukemia cells as
previously described in Higgs et al.1
Characterization of the chromosomal rearrangement
The -globin genotypes were determined as
previously described in Winichagoon et al.7
Further mapping of the ZF breakpoint by Southern
blot analysis was performed with a variety of restriction enzymes
(Pst1, PvuII, StuI,
HpaI, SacI, ScaI, BglII, and
NcoI); a 0.5-kb HindIII)/PstI -globin
specific fragment representing the 3' region of the -globin
genes (to determine the 5' breakpoint); and HER50, a
1.1-kb HindIII/EcoRI fragment from approximate
coordinate 193 000 (to determine the 3' breakpoint), were used
as probes. Known DNA sequences3 around the 5' and
3' breakpoints were used to design forward 280 (5'-GGTGCTGAACCATCCCCTGTC-3') and reverse 279 (5'-CCCATTTCCTAAAAGTGTCCCTTC-3') polymerase
chain reaction (PCR) primers. These primers amplify a 928-bp breakpoint
fragment that was sequenced directly using the same primers with
incorporation of fluorescently labeled ddNTP (dideoxy
nucleoside 5'-triphosphate) followed by analysis on an ABI 377 sequencer (Applied Biosystems, Foster City, CA). The
2-globin gene and the regulatory element (HS -40) from the abnormal
ZF chromosome were amplified from DNA derived
from the interspecific hybrid (ZF8), as described below, and directly
sequenced. (The primers and DNA sequence are available on request.)
Analysis of the pattern of methylation
Patterns of methylation were analyzed in DNA from peripheral blood,
EBV-transformed lymphocytes, and interspecific hybrids. DNA was also
obtained from a sample of semen, supplementing the normal lysis buffer
with 20 mmol/L dithiothreitol (DTT). DNA was initially cut with a
restriction enzyme that is insensitive to methylation to generate
specific fragments spanning the sequences of interest. These fragments
were then digested with methylation-sensitive restriction enzymes
(EagI, HpaII, or SstII and
analyzed by Southern blot analysis. Many probes
(Bg6.6, RA2.2, PL1, and IZHVR) have been previously
described in Nicholls et al.8 In addition, we used several
previously unreported probes including 510 × 511 (coordinates
66 093-66 785), 436 × 437 (coordinates 217 684-217 891),
HR91 (approximate coordinate 232 000), HR23 (approximate coordinate
267 000), and HR22 (approximate coordinate 273 000). Methylation
analysis using bisulphite-mediated genomic sequencing was adapted from
Clark et al9 using DNA templates obtained from peripheral
blood. After DNA modification, initial amplification of the 2-globin
gene was achieved using the primers APS1
(5'-CAAAAAACAACACCATAATAAATTCTCTCT-3') and
APAS1
(5'-GGGGGTGTGGGTTGATTTTTTTTTT-3').
Second-round amplification and genomic sequencing were
performed with the primers APS2
(5'-CCATAATAAATTCTCTCTAAATCTATAA-3') and APAS2 (5'-
GGGTTGATTTTTTTTTTTGTTAGGG-3') using a Perkin Elmer Cycle
sequencing kit (Perkin Elmer, Foster City, CA).
DNaseI and endonuclease sensitivity
DNaseI hypersensitive sites were analyzed as described in Higgs et
al.1 In addition to previously described probes, the hypersensitive sites (HSs) at H and K were analyzed
with the probes 382 × 383 (coordinates 158 487-159 287) and
HR77 (approximate coordinate 218 000). Nuclei for assays of
endonuclease sensitivity were prepared as described in Higgs et
al1 and resuspended at approximately 107 nuclei
per mL in the appropriate restriction enzyme digestion buffer.
Increasing amounts (10-100 units) of enzyme were added to consecutive
aliquots and incubated at 37°C for 60 minutes. DNA was subsequently
extracted and analyzed by Southern blotting using the following probes:
0.5-kb HindIII/PstI, as described above; a probe
corresponding to the -globin regulatory element (approximate
coordinate 103 000); and RA0.6 (approximate coordinate 67 000) at the
5' end of the MPG gene.10
Studies of replication timing
Replication timing was analyzed throughout the entire terminal
region of 16p13.3 using a modification of the fluoresence in situ
hybridization (FISH)-based protocol described by Selig
et al11 as set out in Smith and Higgs.4
Briefly, interspecific somatic cell hybrid cell lines were grown to the
mid log phase. To identify S-phase cells, 0.1 mmol/L BrdU was added to
the culture 90 minutes prior to harvesting. The cells were collected by
centrifugation, swollen in hypotonic solution, and fixed prior to
making a batch of approximately 30 slides. To each slide 20-50 ng of
nick-translated, Cot-1 suppressed cosmid probe was added and hybridized
at 37°C overnight in 50% formamide, 2 × SSC,
and 1% Tween 20. The following day, the slides were
washed in 50% formamide and 2 × SSC, followed by 4 more
changes of 2 × SSC. Stringent washing consisted of 3 changes of
0.1 × SSC at 60°C. S-phase cells were detected with anti-BrdU, and cosmid signals were detected with anti-DIG-FITC antibodies. A total of 200 nuclei were scored on each slide, and the
proportion of duplicated signals was determined. For each batch of
slides, the timing of replication was also determined for known early
or late replicating control probes. The replication timing of 12 individual cosmids spanning the telomeric region of 16p3
was analyzed for a normal homologue of chromosome 16p in an
interspecific hybrid and the abnormal ZF chromosome.
Preparation and analysis of transgenic mice
The transgenic construct cDH2 (Figure
2) was made by modification of Cos12, which
contains the region from approximate coordinates 160 000-196 000 in
the vector sCOS.12 The centromeric Not 1 site was
inactivated by partially digesting with Not1, filling in with
the Klenow enzyme, and by religating. The -globin regulatory element
was then inserted into the telomeric Not 1 site of this modified
cosmid. A substantial part of the insert (approximate coordinates
160 000-185 660) linked to HS -40 was subsequently released with
ClaI. This fragment was prepared, and transgenic lines were
produced as described in Higgs et al.1 The total RNA was
isolated from embryonic blood and fetal liver (10.5-12.5 days
post coitum) and from peripheral blood of adult transgenic mice. Human
and mouse -globin messenger RNA (mRNA) was detected using
quantitative RNAse mapping as previously described.1
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
Details of the region around the ZF
breakpoints.
The positions of the Alu elements orientated toward
(Up) or away from (Down) the telomere are shown. Below
this, tandem repeats, other repeats, and DNaseI HS and CpG islands
(labeled F-L) are shown. The , , and  1
genes are transcribed toward the centromere, and the gene
16PHQG;16 is transcribed toward the telomere. The
ZF deletion is shown as a black bar below the
chromosome. Genomic mapping localized its 5' breakpoint between
an HpaI site (at coordinate 164 012) and a SacI site
(at coordinate 164 356), beyond the polyA addition site of the
2-globin gene. Pulsed field gel electrophoresis of a Not1
-specific fragment, approximately 380 kb, indicated that the
deletion extends for approximately 18 kb (data not shown). The 3'
breakpoint was localized between a BglII site
(coordinate 180 096) and a BamHI site (coordinate 182 417).
Forward 280 and reverse 279 primers were designed from the breakpoint
regions, and a 928-bp fragment spanning the breakpoint was amplified
(data not shown) in the propositus (Z.F.) and his mother (H.F.). DNA
sequence analysis demonstrated that the breakpoints lie between
coordinates 164 044-45 and 182 395-96 and arose via an illegitimate
recombination event (sequence available on request). Five previously
described deletions that remove overlapping segments of this region are
denoted a-e14; none of these silence gene
expression, although deletion a,
3.7, only removes 1 gene. Cosmids
described in the text are shown at the bottom of the figure. The black
box attached to cDH2 represents the -globin regulatory element (HS
-40).
|
|
| |
Results |
-Thalassemia resulting from a deletion extending downstream of
the -globin cluster
Hematologically normal individuals have 4 -globin genes
(/), whereas most carriers of
-thalassemia have only 2 -glob genes
(/) or 3 genes
(/).5 -Thalassemia trait was identified in 2 members (Z.F., the propositus, and
H.F., the affected mother) of a Polish family (Table
1). Provisional data demonstrated that a
segment of more than 18-kb DNA extending downstream from between the
2 and 1 genes had been deleted from the affected chromosome
(subsequently referred to as ZF), thereby
completely removing the 1 gene.13 Further analysis (Figure 2 and legend) localized the breakpoints precisely between coordinates 164 044-45 and 182 395-96 and showed that the deletion results from an illegitimate recombination event that removes 18 352-bp DNA.
The hematologic phenotype in Z.F.
(/ZF) is more severe than the
phenotype normally seen in patients with 3 functional genes
(/), which suggests that the remaining
gene on the affected chromosome
(ZF) might also be down-regulated by this
mutation. The similar phenotype in H.F.
(/ZF) supports this hypothesis
(Table 1, legend). To test this directly, the abnormal
ZF chromosome 16 was isolated and analyzed in an
interspecific hybrid after fusion of a lymphoblastoid cell line from
Z.F. with a mouse erythroleukemia (MEL) cell line. Upon induction with
HMBA and hemin, such hybrids terminally differentiate and normally
express high levels of mouse and human globin mRNAs. The ratio of
human/mouse -globin mRNA in hybrids with a normal human chromosome
16 is 28.2% ± 15.1%.14 No human -globin mRNA was
expressed from the ZF chromosome in such hybrids,
thereby confirming that the remaining gene is completely
inactive (Figure 3A).
View larger version (46K):
[in this window]
[in a new window]
| Fig 3.
Expression studies of cDH2.
Nuclease protection assays to analyze human -globin gene expression
in (A) the abnormal interspecific hybrid (ZF2) and (B) adult peripheral
blood from lines of transgenic mice carrying the cDH2 construct. Two
samples from different individuals from line 84 are shown.
|
|
The 5' breakpoint of the ZF deletion lies
335- to 337-bp (coordinates 164 044-164 045) beyond the polyA
addition site of the 2-globin gene, and all cis-acting sequences
known to regulate expression of this gene remain intact on the
ZF chromosome. To ensure that
the 2 gene was down-regulated by the
associated deletion and not as a result of any other mutation, the gene
from the affected chromosome was amplified, and its sequence (coordinates 162 595-163 878) was shown to be normal. Similarly the
sequence of the -globin regulatory element (HS -40, coordinates 103 390-103 925) from this chromosome was entirely normal. Finally, when linked to HS -40, the 2 gene from the affected chromosome was
expressed normally in stable transfectants of the MEL cells and
transgenic mice (data not shown). Thus the 18 352-bp deletion not only
removes the 1 gene but also inactivates the remaining 2-globin
gene on the ZF chromosome.
The ZF deletion does not remove a
positive cis-acting element
These data suggest that the ZF deletion either
removes a positive cis-acting element or juxtaposes negative sequences
that down-regulate expression of the remaining 2-globin gene. To
date, the only well characterized positive cis-acting sequences
controlling gene expression lie in the promoters and the
remote regulatory element (HS -40). Removal of HS -40 by natural
deletion5 or by specific "knock out"15
reduces the gene expression to less than 1% of normal,
indicating that there are no other sequences in the 16p region which,
on their own, are capable of up-regulating gene expression.
To test this further, a construct (cDH2, Figure 2) containing HS
-40 linked to a segment of DNA spanning approximate coordinates 159 000-185 660, including both genes and the entire
segment of DNA deleted in the ZF chromosome, was
analyzed in 5 independent lines of transgenic mice (Table
2 and Figure 3B). With this construct, the
patterns and levels of human -globin expression were not
significantly different from those obtained with previously analyzed
constructs completely lacking the down-stream region (Table 2, legend). As noted with other human -globin transgenes, the level of human -globin mRNA compared to endogenous mouse -globin mRNA was lower in adult than embryonic erythroid cells.14 Therefore these
findings confirm that the region within cDH2 does not contain any
additional hitherto unidentified positive elements and show that the
remaining 2 gene on the ZF chromosome
is negatively regulated by the associated deletion.
Methylation of the -globin CpG island on the silenced
ZF chromosome
We have previously shown that all CpG islands in the 16p
segment, with the exception of B (Figures 1 and
4A), were unmethylated in all tissues and
newly established cell lines that were analyzed. This applies to the
islands at the gene promoters (Figure 4B, H and I) even though the
genes are expressed in a tissue-specific manner.2,3 We examined the methylation state of islands on the ZF chromosome in spermatocytes (ZF),
peripheral blood (ZF and HF), EBV-transformed lymphocytes (ZF and HF),
and an interspecific hybrid carrying the ZF
chromosome using the methylation-sensitive restriction enzymes Eag1 (Figure 4B-D) and/or HpaII (data not shown).
View larger version (68K):
[in this window]
[in a new window]
| Fig 4.
Analysis of the pattern of methylation along the
ZF chromosome.
(A) CpG islands (A-N, see also Figure 1) were analyzed
with a combination of enzymes and probes, which were previously
described.2,53 In each panel, the left-hand lane is the
chosen digest (eg, BglII) using DNA from the peripheral blood
of an unaffected individual. The middle lane represents a double digest
incorporating a methylation-sensitive enzyme (eg,
BglII/SacII) using DNA from the peripheral blood of an
unaffected individual. The third lane represents the same double digest
(eg, BglII/SacII) using DNA from the peripheral blood
of Z.F. The presence of 2 variably sized bands using IZHVR reflects the
2 different VNTR alleles detected with this probe. (B) Analysis
of the CpG islands (H and I) associated with the -globin genes using
DNA from peripheral blood. In each case the left-hand lane is a
PstI digest, and the right-hand lane is a
PstI/EagI digest. (Only EagI cuts unmethylated
sites.) N indicates an unaffected individual; ZF, the propositus; AF,
the unaffected father; and HF, the affected mother. ND is a patient in
whom an gene was inactivated by a nondeletional form of
-thalassemia, demonstrating that such mutations do not alter the
pattern of methylation. Note that both CpG islands (H and I) are
examined in this assay. The different signal intensities of uncut DNA
(methylated) to cut DNA (unmethylated) in Z.F.
(/ZF) and H.F.
(/ZF) is explained by their different
genotypes. Only the ZF chromosome is methylated
at CpG island H. (C) Analysis of DNA from a sample of semen from Z.F.
(D) Analysis of the CpG islands (H and I) in EBV-transformed lymphocyte
lines from an unaffected individual (N), Z.F., and a MEL16 hybrid
containing only the abnormal copy of chromosome 16 (ZF2). (E)
Bisulphite modified sequence analysis of DNA from the peripheral blood
of the propositus (ZF) and his unaffected father (AF). During
bisulphite treatment of DNA, unmethylated cytosines were converted to
uracil and subsequently PCR-amplified as thymidine. Methylated
cytosines were resistant to this conversion and hence were amplified as
cytosine. Arrows indicate that whereas all cytosines were converted to
thymidines in A.F., many remain unconverted (methylated) in Z.F. Note
that only the cytosines on one allele in Z.F. are methylated.
|
|
Using Eag1, the CpG island (H) associated with the remaining
2 gene on the ZF chromosome was
unmethylated in spermatocytes (Figure 4C), but it was methylated in
peripheral blood, EBV lymphocytes, and the interspecific hybrid (Figure
4B,D). Island H was analyzed further using HpaII. Although this
island contains 16 sites (CCGG), none appeared to be cut in DNA from
peripheral blood (data not shown). Finally, the CpG dinucleotides in
the segment of H spanning the 2 promoter (coordinates
162 529-162 887) were analyzed using bisulphite-modified sequencing
(Figure 4E and legend). In DNA from the peripheral blood of the
unaffected father (AF), 1 of 47 CpGs were methylated, whereas in the
propositus (ZF), 40 of 47 CpGs were methylated (Figure 4E).
Thus it seemed likely that at least part of the mechanism by which the
2 gene had been silenced involved methylation of the CpG
dinucleotides. It was therefore of interest to know how far this effect
propagated along the chromosome. All other CpG islands within the 16p
segment were analyzed, as previously described, using
methylation-sensitive restriction enzymes (Figure 4A), but none was
inappropriately methylated. The closest CpG island (G) appeared
unmethylated and the peripheral blood of Z.F. from all CpG dinucleotides analyzed in DNA from approximately 2 kb upstream of
island H had the same pattern of methylation as a normal chromosome (data not shown).
The silenced -globin promoter shows reduced
sensitivity to endonucleases
Although the CpG islands (H and I) associated with the genes are normally unmethylated in all tissues, the corresponding DNase1 HSs are expressed in a strictly erythroid-specific
manner.16 Similarly, in vivo, these promoters are also
sensitive to specific endonucleases (eg, HinfI), which
cut within previously defined hypersensitive sites in erythroid cells
(Figure 5 and D.R.H., unpublished data,
1999). DNase1 hypersensitivity and
endonuclease sensitivity at the promoters were analyzed
in interspecific MEL hybrids containing either normal (, JY5-4)
or abnormal (ZF, ZF2, and ZF8) copies of
chromosome 16.
View larger version (57K):
[in this window]
[in a new window]
| Fig 5.
Analysis of endonuclease sensitivity in the
ZF chromosome.
DNA from nuclei of (A) EBV lymphocytes, (B) an interspecific MEL hybrid
containing the ZF chromosome (ZF2), and (C) the
erythroid cell line K562. The DNA was incubated with increasing amounts
of Hinf I ("Materials and methods") and analyzed with probes
around gene no. 6 (RA0.6, approximate coordinate
67 000), which encodes the ubiquitously expressed MPG gene,
the -globin regulatory element HS -40 (approximate coordinate
103 000), and the -globin gene. Whereas the site associated with HS
-40 is clearly present in ZF2, the sites are missing from the silenced
-globin promoter.
|
|
Whereas the normal promoters are usually sensitive to
endonucleases in these erythroid cells (Figure 5, K562, and Figure 6, JY5-4), the 2 promoter on the
ZF chromosome was insensitive (Figure 5, ZF2, and
Figure 6, ZF8). By contrast, the remote -globin regulatory element
(HS -40) was sensitive in both normal and abnormal chromosomes (Figure
5, ZF2), adding further weight to previous studies15,16
demonstrating that tissue-specific chromatin remodeling at HS -40 and
the promoters occurs independently (see "Discussion").
View larger version (38K):
[in this window]
[in a new window]
| Fig 6.
Analysis of DNase1 hypersensitive sites along the
ZF chromosome.
DNA from the nuclei of an interspecific MEL hybrid containing the
ZF chromosome (ZF8) or a normal chromosome
(JY5-4) using probes and enzymes that detect the hypersensitive sites
at the CpG islands associated with the 2-globin promoter (H) and the
surrounding CpG islands B, G, E, and K (see Figure
1). Analysis of HSs at E and H in normal hybrids
(JY5-4) and abnormal hybrids (ZF8) shows that although the constitutive
site at E is sensitive in both, the HS at the silenced promoter
is insensitive.
|
|
As for the methylation pattern described previously, it was
of interest to determine how far the abnormal pattern of reduced chromatin sensitivity extended from the ZF
breakpoints. Again it appeared that this effect was quite localized because the nearest known constitutive HSs lying upstream and downstream of the 2 promoter were sensitive as normal (Figure 6).
Silencing of the -globin gene is not associated with any change
in the pattern of replication timing
Unlike the rest of the GC-rich chromosomal segment, the region
downstream of the ZF deletion (coordinates
180 000-218 000) is devoid of both HSs and CpG islands. It therefore
seemed possible that the 2 gene in the
ZF chromosome was inactivated by juxtaposition
next to this potentially repressive chromatin environment. There is
evidence that such chromatin replicates late in the cell
cycle17,18 and that multiprotein complexes at some origins
of replication can silence gene expression.19,20 Therefore,
it was of interest to study the effect of the ZF
deletion on the normal pattern of replication along this region of 16p.
Using a FISH-based assay to score duplicated loci in S-phase, we have
recently shown that in MEL x chromosome 16 hybrids, the most telomeric region (approximately 20 kb) of 16p replicates late
in the cell cycle, whereas the adjacent GC-rich region replicates early
(Figure 7 and Smith and
Higgs4). However, we noted that the Alu-dense
region between approximate coordinates 180 000 and 218 000, where the
3' breakpoint of the ZF chromosome lies,
consistently replicates later than its flanking regions, albeit still
relatively early in S-phase.4 Furthermore, in contrast to
other segments of 16p, duplicated signals from this region, contained
within Cos12 (Figure 2), often remain closely paired in many nuclei.
This suggests that more time is taken for this region to separate
during S-phase than for other regions along the duplicated chromatids.
View larger version (20K):
[in this window]
[in a new window]
| Fig 7.
The pattern of replication along the ZF
chromosome.
(A) Replication of the human 16p region was assayed by FISH as
set out in Smith and Higgs4 using a set of
12 cosmids spanning this region. The pattern in an interspecific hybrid
containing a normal copy of chromosome 16 (closed boxes) is compared
with a hybrid containing the ZF chromosome (open
circles). In the ZF chromosome, there is no value
for Cos 12 (midpoint at approximate coordinate 177 000) because this
region is deleted. Black dashed lines indicate the mean percent doublet
scores for the early replicating control cosmids (top line) and late
replicating control cosmids (bottom line). Below, the key features of
the 16p region are shown. (B) Replication timing of control cosmids.
The mean value for all early (p48) and late (cSamD4) mouse
controls4 are represented as columns. Values for controls
measured in the hybrid containing the ZF
chromosome are shown as a black diamond with lines representing plus or
minus 1 SD.
|
|
Using the FISH-based replication assay with 12 cosmids along the 300-kb
16p telomeric region, we found that this entire segment of the
ZF chromosome, including the silenced gene (corresponding to cGG1 in Figure 2), remains
early replicating with a profile that is almost indistinguishable from
a normal chromosome (Figure 7). This suggests that replication of the
ZF chromosome initiates at the same origins at
the same phase of the cell cycle as a normal chromosome and that
silencing of gene expression in this case is not associated with any
switch in the timing of replication.
The 3' breakpoint lies in an Alu dense region and
disrupts a widely expressed gene
The ZF deletion was further evaluated in the
light of previously described structural and functional data relating
to the region surrounding its breakpoints. We recently suggested that there may be some heterogeneity in chromatin structure across the
285-kb 16p segment.3 In particular, in the cell lines
tested, we did not detect either the HSs or CpG islands in the most
telomeric region (coordinates 1 to approximately 36 000) or in a
region downstream of the cluster (approximate coordinates
180 000-218 000). Both regions contain many repetitive DNA elements.
The downstream region contains the highest density of Alu
elements in the entire segment (Figure 1). Although neither of the
ZF breakpoints lie within the Alu
elements, the deletion juxtaposes the intact 2 gene next to
an almost continuous block of Alu repeats (Figure 2).
There are several expressed sequence tags (ESTs) in
the downstream region thought to represent transcripts originating from the CpG islands K and/or L (Figures 1 and 2). Reverse
transcriptase-PCR (RT-PCR) and Northern blot analysis indicated that
this region contains a gene (16pHQG;16) that is widely
expressed in a variety of tissues (heart, brain, lung, liver, muscle,
kidney, and pancreas) and cell lines (EBV-transformed B lymphocytes,
K562, HEL, HepG2, and HT29). Its alternatively spliced RNA transcripts
have been further characterized and shown to encode a putative
RNA-binding protein expressed from at least 10 exons spanning
coordinates 178 856-219 261 (C.T., unpublished data,
1999). The introns of this gene consist almost entirely
of Alu elements. The gene is transcribed in the opposite
orientation to the -globin genes, with a polyA tract attached to a
mRNA from the last exon (coordinates 178 856-179 220). Therefore, in
addition to removing the 1 gene, the
ZF deletion disrupts the 16pHQG;16 gene,
removing its last 3 known exons (Figure 2).
| |
Discussion |
We have shown that an 18.3-kb deletion from the 3' end of the
human -globin cluster stably silences -globin gene expression from its otherwise natural chromosomal environment. Unlike all previously described deletions causing -thalassaemia, this mutation does not remove any positive cis-acting elements. Therefore it appears
to silence gene expression by what is referred to as a
negative chromosomal position effect, a term that covers a variety of
mechanistically different phenomena in mammals,21,22
Drosophila,22,23 plants,24 and
yeast.25 Although such silencing mechanisms are thought to
underlie several human genetic diseases and altered mouse
phenotypes,21,26 there has been no detailed molecular characterization of negative position effects resulting from natural chromosomal rearrangements.
To date the best evidence that the mammalian "chromosomal
environment" can influence gene expression comes from many
observations demonstrating that genes on identical DNA segments may be
expressed with variable patterns and at different levels when randomly
integrated into the mouse genome. At the cellular level this often
produces a variegated effect on gene expression. Some epigenetic
characteristics (eg, methylation and DNaseI sensitivity), by which the
chromosomal environment is currently defined, have been analyzed in
such lines of transgenic mice with different
conclusions.27-34 This suggests that there may be many ways
in which chromosome position can affect gene expression.
The simplest explanation of our data would be that the gene
is silenced by its juxtaposition to the Alu-rich downstream region. It is known that stable, heritable gene silencing can occur
when a euchromatic gene is juxtaposed to a region of heterochromatin. The region downstream of -globin (approximate coordinates
180 000-218 000) may represent a relatively "heterochromatic"
segment of DNA within the euchromatic 16p13.33 environment.
The evidence for this is necessarily indirect. First, it contains a
very high density of methylated repeat sequences (Alu and MER
families), and it has been proposed that these features alone can be
sufficient to create a hypoacetylated repressive chromatin
environment.35,36 Second, in contrast to much of the
surrounding chromosome, we did not detect DNaseI HSs or CpG islands in
this region (Figure 1). Third, studies of replication timing using FISH
analysis have shown that during the S-phase, the region within Cos12
(Figure 2, approximate coordinates 160 000-185 660) always replicates
and separates later than its flanking regions.4
Although the silencing effect described here may be due to a single
cis-element within the downstream region, preliminary sequence analysis
did not identify significant homology to any previously described
silencer elements. An alternative explanation is that multiple closely
spaced Alu repeats act as a nucleation site for the formation
of a stable repressive chromatin structure that extends, albeit a
relatively short distance, to incorporate the juxtaposed gene. The silencing phenomenon reported here may therefore be related
to the previously described effects originating at the
euchromatin/heterochromatin boundaries and polycomb response elements
in Drosophila.
An unexpected finding was that a widely expressed gene encoding a
putative RNA binding protein extends through this Alu dense region, transcribed in the opposite direction with respect to the
genes (C.T., unpublished data, 1999). It is
possible that this gene represents an example of a human
"heterochromatic gene" of the type described in
Drosophila (eg, rolled and light), which are similarly
embedded within regions containing high densities of repeats and
transposons.37-39
Abnormal transcripts from this gene, extending across
the breakpoint, may contribute to silencing and/or methylation of the juxtaposed 2-globin gene in the ZF chromosome,
as recently proposed for regulation of some imprinted genes (eg,
lgf2r, UBEA3, and LIT1)40-42 and
the Xist gene, which is involved in X
inactivation.43 Provisional experiments using RT-PCR
suggest that such antisense transcripts are produced from the
ZF chromosome (C.T., unpublished data,
1999) although the role of such transcripts in
silencing is not yet clear. One hypothesis suggests that they directly
interfere with transcription of the gene on the opposite DNA
strand.44 Alternatively, antisense RNA might locally
inactivate the chromosome by interacting with its chromatin in a
similar way to the RNA product of Xist on the inactive X
chromosome.45,46
Methylation is frequently associated with silencing of gene expression.
For example, CpG islands normally become methylated during
X-inactivation and genomic imprinting and may become abnormally methylated in tumorigenesis. Analysis of the CpG island associated with
the promoter on the ZF chromosome showed that
although it is unmethylated in spermatocytes, it too becomes densely
methylated during development.
Although methylation may play an important role in silencing it is not
clear whether it is required to establish silencing or maintain
silencing initiated by changes in chromatin structure. Recently,
potentially important links between the processes of DNA methylation
and histone deacetylation have been established. The protein
MeCP2 specifically binds methylated DNA and has also been shown to interact with histone deacetylase and mammalian Sin3,
providing a plausible connection between DNA methylation and the
formation of a repressive chromatin environment.36,47,48 Similarly, the protein MBD2, which also binds methylated DNA, can
recruit the repressive NuRD complex, which contains the histone deacetylanes HDAC1 and HDAC2. At present, the order of events leading
to hypoacetylation, methylation, and repression has not been established.
The ZF mutation adds to our understanding of the
relationship between gene regulation and its chromosomal
environment. Despite their common ancestry, we have previously
suggested that the - and  -globin genes are regulated in a
different manner, which may be related to their different chromosome
environments.2,4,16 These genes may provide models for
other genes located in contrasting regions of the genome.49
The region of chromosome 11 containing the -globin locus control
region (-LCR), which regulates expression of the entire -cluster,
influences long-range and local chromatin structure, the timing of
replication, and the accumulated levels of the -globin gene
expression.50,51 When linked to -LCR, the transgenes are
protected from position effects.
By contrast the -globin regulatory element, although absolutely
required for gene expression, has no discernible effect on
chromatin structure, methylation, or replication
timing.4,15,16 Transgenes linked to this element are always
expressed but appear sensitive to their position of integration.
Although HS -40 enhances the accumulated levels of -globin mRNA
expression, other cis-acting feature(s) must create the permissive
chromosomal environment that allows this interaction to occur. All
known positive cis-acting sequences are present on the
ZF chromosome, and the regulatory element is
active, as judged by the presence of the HS, yet the gene
is silenced. Clearly the HS -40/ interaction is
insufficient to overcome the ZF position effect.
Although we have not ruled out the presence of as yet unidentified
specific elements, the normal permissive environment possibly results
from an inherent, albeit unevenly distributed, feature of the 16p
GC-rich chromosomal segment. The rearrangement described here may
simply have changed a normally permissive microenvironment containing
the gene to a nonpermissive one.
In a normal chromosome the genes are protected from the
influence of this repressive environment that only lies approximately 12 kb away. Furthermore, some previously described deletions move the
genes to within approximately 6 kb of this region (Figure 2) and yet have no effect on expression of the remaining gene(s). One possibility is that the genes are normally
protected by a cis-acting element(s) which remains intact in these
deletions but is removed in the ZF chromosome.
The region of interest contains several Alu elements, a
variable number tandem repeat (3' hypervariable
region) and a DNaseI hypersensitive site (approximate coordinates
178 500-179 000) at the end of the 16PHQG;16 gene. It has
been suggested that boundary elements commonly flank chromosomal
domains, protecting genes within them from position effects induced by
surrounding heterochromatin or unrelated positive regulatory
elements.52 However, the level and pattern expression of
the cDH2 construct, which spans the entire region deleted in the
ZF chromosome, does not provide evidence for such
a boundary because the genes in this construct were not
consistently protected from position effects in transgenic mice.
In summary, juxtaposition of a sequence that normally lies 18 kb
downstream of the - globin complex next to the 2 gene
silences its expression, thereby providing an entirely new mechanism
that may cause -thalassemia. Further work is required to determine the relative contributions of repressive chromatin
environment, antisense transcripts, and the removal of a
potential boundary element in causing this negative chromosomal
position effect.
| |
Acknowledgments |
We are grateful to Professor D. J. Weatherall for his continued support
and encouragement. We are also grateful to the late Professor
T. H. J. Huisman, who originally brought our attention to the
ZF deletion. We thank David Garrick, Jonathan
Flint, and Veronica Buckle for their comments on the manuscript and Liz
Rose and Milly Graver for preparation of the manuscript.
| |
Footnotes |
Submitted January 19, 2000; accepted March 27, 2000.
Reprints: D. R. Higgs, the MRC Molecular Haematology Unit,
Institute of Molecular Medicine, John Radcliffe Hospital, Headington,
Oxford, England; e-mail: drhiggs{at}molbiol.ox.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Higgs DR, Wood WG, Jarman AP, et al.
A major positive regulatory region located far upstream of the human -globin gene locus.
Genes Dev.
1990;4:1588-1601[Abstract/Free Full Text].
2.
Vyas P, Vickers MA, Simmons DL, Ayyub H, Craddock CF, Higgs DR.
Cis-acting sequences regulating expression of the human globin cluster lie within constitutively open chromatin.
Cell.
1992;69:781-793[Medline]
[Order article via Infotrieve].
3.
Flint J, Thomas K, Micklem G, et al.
The relationship between chromosome structure and function at a human telomeric region.
Nature Genet.
1997;15:252-257[Medline]
[Order article via Infotrieve].
4.
Smith ZE, Higgs DR.
The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression.
Hum Mol Genet.
1999;8:1373-1386[Abstract/Free Full Text].
5.
Higgs DR.
-Thalassaemia. In:
Higgs DR,Weatherall DJ, eds.
Baillière's Clinical Haematology. International Practice and Research: The Haemoglobinopathies. London: Baillière Tindall; 1993:117-150.
6.
Weatherall DJ, Clegg JB.
The Thalassaemia Syndromes. Oxford: Blackwell Scientific Publications; 1981:744-769.
7.
Winichagoon P, Higgs DR, Goodbourn SEY, Clegg JB, Weatherall DJ, Wasi P.
The molecular basis of -thalassaemia in Thailand.
EMBO J.
1984;3:1813-1818[Medline]
[Order article via Infotrieve].
8.
Nicholls RD, Fischel-Ghodsian N, Higgs DR.
Recombination at the human -globin gene cluster: sequence features and topological constraints.
Cell.
1987;49:369-378[Medline]
[Order article via Infotrieve].
9.
Clark SJ, Harrison J, Paul CL, Frommer M.
High sensitivity mapping of methylated cytosines.
Nucl Acids Res.
1994;11:2990-2997.
10.
Vickers MA, Vyas P, Harris PC, Simmons DL, Higgs DR.
Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere.
Proc Natl Acad Sci U S A.
1993;90:3437[Abstract/Free Full Text].
11.
Selig S, Okumura K, Ward DC, Cedar H.
Delineation of DNA replication time zones by fluorescence in situ hybridization.
EMBO J.
1992;11:1217-1225[Medline]
[Order article via Infotrieve].
12.
Wahl GM, Lewis KA, Ruiz JC, Rothenberg B, Zhao J, Evans GA.
Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer.
Proc Natl Acad Sci U S A.
1987;84:2160-2164[Abstract/Free Full Text].
13.
Indrak K, Gu Y-C, Novotny J, Huisman THJ.
A new -thalassemia-2 deletion resulting in microcytosis and hypochromia and in vitro chain imbalance in the heterozygote.
Am J Hematol.
1993;43:144-145[Medline]
[Order article via Infotrieve].
14.
Higgs DR, Sharpe JA, Wood WG.
Understanding globin gene expression: a step towards effective gene therapy.
Sem Hematol.
1998;35:93-104[Medline]
[Order article via Infotrieve].
15.
Bernet A, Sabatier S, Picketts DJ, et al.
Targeted inactivation of the major positive regulatory element (HS -40) of the human -globin gene locus.
Blood.
1994;86:1202-1211[Abstract/Free Full Text].
16.
Craddock CF, Vyas P, Sharpe JA, Ayyub H, Wood WG, Higgs DR.
Contrasting effects of and globin regulatory elements on chromatin structure may be related to their different chromosomal environments.
EMBO J.
1995;14:1718-1726[Medline]
[Order article via Infotrieve].
17.
Wolffe A.
Chromatin: Structure and Function. London: Academic Press; 1995.
18.
Holmquist GP.
Role of replication time in the control of tissue-specific gene expression.
Am J Hum Genet.
1987;40:151-173[Medline]
[Order article via Infotrieve].
19.
Fox CA, Loo S, Dillin A, Rine J.
The origin recognition complex has essential functions in transcriptional silencing and chromosomal replication.
Genes Dev.
1995;9:911-924[Abstract/Free Full Text].
20.
Lustig AJ.
Mechanisms of silencing in Saccharomyces cerevisiae.
Curr Opin Genet Dev.
1998;8:233-239[Medline]
[Order article via Infotrieve].
21.
Kleinjan D-J, van Heyningen V.
Position effect in human genetic disease.
Hum Mol Genet.
1998;7:1611-1618[Abstract/Free Full Text].
22.
Weiler KS, Wakimoto BT.
Heterochromatin and gene expression in Drosophila.
Annu Rev Genet.
1995;29:577-605[Medline]
[Order article via Infotrieve].
23.
Wakimoto BT.
Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila.
Cell.
1998;93:321-324[Medline]
[Order article via Infotrieve].
24.
Matzke AJ, Matzke MA.
Position effects and epigenetic silencing of plant transgenes.
Curr Opin Plant Biol.
1998;1:142-148[Medline]
[Order article via Infotrieve].
25.
Sherman JM, Pillus L.
An uncertain silence.
Trends Genet.
1997;13:308-313[Medline]
[Order article via Infotrieve].
26.
Bedell MA, Jenkins NA, Copeland NG.
Good genes in bad neighbourhoods.
Nature Genet.
1996;12:229-232[Medline]
[Order article via Infotrieve].
27.
Garrick D, Fiering S, Martin DIK, Whitelaw E.
Repeat-induced gene silencing in mammals.
Nature Genet.
1998;18:56-59[Medline]
[Order article via Infotrieve].
28.
Guy L-G, Kothary R, Wall L.
Position effects in mice carrying a lacZ transgene in cis with the -globin LCR can be explained by a graded model.
Nucl Acids Res.
1997;25:4400-4407[Abstract/Free Full Text].
29.
Elliott JI, Festenstein R, Tolaini M, Kioussis D.
Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element.
EMBO J.
1995;14:575-584[Medline]
[Order article via Infotrieve].
30.
Garrick D, Sutherland H, Robertson G, Whitelaw E.
Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status.
Nucl Acids Res.
1996;24:4902-4909[Abstract/Free Full Text].
31.
Walters MC, Magis W, Fiering S, et al.
Transcriptional enhancers act in cis to suppress position-effect variegation.
Genes Dev.
1996;10:185-195[Abstract/Free Full Text].
32.
Festenstein R, Tolaini M, Corbella P, et al.
Locus control region function and heterochromatin-induced position effect variegation.
Science.
1996;271:1123-1125[Abstract].
33.
Milot E, Strouboulis J, Trimborn T, et al.
Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.
Cell.
1996;87:105-114[Medline]
[Order article via Infotrieve].
34.
Huber MC, Krüger G, Bonifer C.
Genomic position effects lead to an inefficient reorganization of nucleosomes in the 5'-regulatory region of the chicken lysozyme locus in transgenic mice.
Nucl Acids Res.
1996;24:1443-1452[Abstract/Free Full Text].
35.
Walsh CP, Bestor TH.
Cytosine methylation and mammalian development.
Genes Dev.
1999;13:26-34[Abstract/Free Full Text].
36.
Razin A.
CpG methylation, chromatin structure and gene silencing: a three-way connection.
EMBO J.
1998;17:4905-4908[Medline]
[Order article via Infotrieve].
37.
Devlin RH, Bingham B, Wakimoto BT.
The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster.
Genetics.
1990;125:129-140[Abstract].
38.
Eberl DF, Duyf BJ, Hilliker AJ.
The role of heterochromatin in the expression of a heterochromatic gene, the rolled locus of Drosophila melanogaster.
Genetics.
1993;134:277-292[Abstract].
39.
Cook KR, Karpen GH.
A rosy future for heterochromatin.
Proc Natl Acad Sci U S A.
1994;91:5219-5221[Free Full Text].
40.
Wutz A, Smrzka OW, Schweifer N, Schellander K, Wagner EF, Barlow DP.
Imprinted expression of the lgf2rgene depends on an intronic CpG island.
Nature.
1997;389:745-749[Medline]
[Order article via Infotrieve].
41.
Rougeulle C, Cardoso C, Fontes M, Colleaux L, Lalande M.
An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript.
Nature Genet.
1998;19:15-16[Medline]
[Order article via Infotrieve].
42.
Mitsuya K, Meguro M, Lee MP, et al.
LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids.
Hum Mol Genet.
1999;8:1209-1217[Abstract/Free Full Text].
43.
Lee JT, Davidow LS, Warshawsky D.
Tsix, a gene antisense to Xist at the X-inactivation centre.
Nature Genet.
1999;21:400-404[Medline]
[Order article via Infotrieve].
44.
Tilghman SM.
The sins of the fathers and mothers: genomic imprinting in mammalian development.
Cell.
1999;96:185-193[Medline]
[Order article via Infotrieve].
45.
Reik W, Constancia M.
Making sense or antisense?
Nature.
1997;389:669-671[Medline]
[Order article via Infotrieve].
46.
Heard E, Lovell-Badge R, Avner P.
Anti-Xistentialism.
Nature Genet.
1999;21:343-344[Medline]
[Order article via Infotrieve].
47.
Kass SU, Landsberger N, Wolffe AP.
DNA methylation directs a time-dependent repression of transcription initiation.
Curr Biol.
1997;7:157-165[Medline]
[Order article via Infotrieve].
48.
Nan X, Ng HH, Johnson CA, et al.
Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
Nature.
1998;393:386-389[Medline]
[Order article via Infotrieve].
49.
Craig JM, Bickmore WA.
Chromosome bands: flavours to savour.
BioEssays.
1993;15:349-354[Medline]
[Order article via Infotrieve].
50.
Grosveld F, Blom van Assendelft G, Greaves DR, Kollias G.
Position-independent, high-level expression of the human -globin gene in transgenic mice.
Cell.
1987;51:975-985[Medline]
[Order article via Infotrieve].
51.
Forrester WC, Epner E, Driscoll MC, et al.
A deletion of the human -globin locus activation region causes a major alteration in chromatin structure and replication across the entire -globin locus.
Genes Dev.
1990;4:1637-1649[Abstract/Free Full Text].
52.
Udvardy A.
Dividing the empire: boundary chromatin elements delimit the territory of enhancers.
EMBO J.
1999;18:1-8[Medline]
[Order article via Infotrieve].
53.
Nicholls RD, Higgs DR, Clegg JB, Weatherall DJ.
o-thalassemia due to recombination between the 1-globin gene and an AluI repeat.
Blood.
1985;65:1434-1438[Abstract/Free Full Text].
54.
Higgs DR, Vickers MA, Wilkie AOM, Pretorius I-M, Jarman AP, Weatherall DJ.
A review of the molecular genetics of the human -globin gene cluster.
Blood.
1989;73:1081-1104[Free Full Text].
55.
Gourdon G, Sharpe JA, Wells D, Wood WG, Higgs DR.
Analysis of a 70 kb segment of DNA containing the human and globin genes linked to their regulatory element (HS [EN]40) in transgenic mice.
Nucl Acids Res.
1994;22:4139-4147[Abstract/Free Full Text].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Garrick, M. De Gobbi, V. Samara, M. Rugless, M. Holland, H. Ayyub, K. Lower, J. Sloane-Stanley, N. Gray, C. Koch, et al.
The role of the polycomb complex in silencing {alpha}-globin gene expression in nonerythroid cells
Blood,
November 1, 2008;
112(9):
3889 - 3899.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Feuk, C. R. Marshall, R. F. Wintle, and S. W. Scherer
Structural variants: changing the landscape of chromosomes and design of disease studies.
Hum. Mol. Genet.,
April 15, 2006;
15(suppl_1):
R57 - R66.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Tufarelli
The silence RNA keeps: cis mechanisms of RNA mediated epigenetic silencing in mammals
Phil Trans R Soc B,
January 29, 2006;
361(1465):
67 - 79.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Ahituv, S. Prabhakar, F. Poulin, E. M. Rubin, and O. Couronne
Mapping cis-regulatory domains in the human genome using multi-species conservation of synteny
Hum. Mol. Genet.,
October 15, 2005;
14(20):
3057 - 3063.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, D. W. Emery, H. Han, J. Sun, M. Yu, and G. Stamatoyannopoulos
Differences of globin transgene expression in stably transfected cell lines and transgenic mice
Blood,
April 15, 2005;
105(8):
3346 - 3352.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Anguita, C. A. Johnson, W. G. Wood, B. M. Turner, and D. R. Higgs
Identification of a conserved erythroid specific domain of histone acetylation across the alpha -globin gene cluster
PNAS,
September 26, 2001;
(2001)
201413098.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Anguita, C. A. Johnson, W. G. Wood, B. M. Turner, and D. R. Higgs
Identification of a conserved erythroid specific domain of histone acetylation across the alpha -globin gene cluster
PNAS,
October 9, 2001;
98(21):
12114 - 12119.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction