|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3600-3604
RED CELLS
Independent formation of DnaseI hypersensitive sites in the
murine  -globin locus control region
M. A. Bender,
Michelle G. Mehaffey,
Agnes Telling,
Bruce Hug,
Timothy J. Ley,
Mark Groudine, and
Steven Fiering
From the Fred Hutchinson Cancer Research Center, Seattle, WA;
Departments of Pediatrics and Radiation Oncology, University of
Washington, Seattle, WA; Departments of Internal Medicine and Genetics,
Washington University, St Louis, MO; and Department of Microbiology,
Dartmouth Medical School, Hanover, NH.
 |
Abstract |
Mammalian  -globin loci are composed of multiple orthologous genes
whose expression is erythroid specific and developmentally regulated.
The expression of these genes both from the endogenous locus and from
transgenes is strongly influenced by a linked 15-kilobase region of
clustered DNaseI hypersensitive sites (HSs) known as the locus control
region (LCR). The LCR encompasses 5 major HSs, each of which is highly
homologous among humans, mice, and other mammals. To analyze the
function of individual HSs in the endogenous murine -globin LCR, we
have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In
this report, we demonstrate that deletion of the conserved region of
5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion
site but has no influence on the formation of the remaining HSs in the
LCR. Therefore, in the endogenous murine locus, there is no dominant or
initiating site whose formation must precede the formation of the other
HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of
-globin regulation.
(Blood. 2000;95:3600-3604)
© 2000 by The American Society of Hematology.
| |
Introduction |
The regulation of the -like globin genes is of
significant interest because it is a paradigm for a complex multigenic
and developmentally regulated locus1 and because of the
prevalence of mutations causing -thalassemia and sickle cell anemia.
The identification of deletions upstream of the embryonic  -globin gene that left all of the -like globin genes intact but that eliminated expression of all of the genes implied the existence of
cis-acting regulatory elements in this region.2-4 A series of erythroid-specific DNaseI hypersensitive sites (HSs) were identified in this region,5,6 which subsequently was named the locus control region (LCR). Six 5'HSs have been described in the human and mouse, and homologues to at least 4 have been detected to date in
other mammals.7-9 In the mouse, 5'HS 5 forms very
weakly, and 5'HS 6 is prominent, while in humans the opposite
pattern is observed.
A great number of experiments have been performed in attempts to
determine how the LCR influences expression of genes of the -globin
locus located 6 to 50 kilobases (kb) away. The majority of these
experiments have used human -globin transgenes in cell lines or
transgenic mice. Interpretation of the transgenic studies have been
complicated by variations in integration site, copy number, size of
constructs, and the exact deletions made. However, in aggregate,
analyses of large transgenes containing sequences from the human
-globin locus have revealed that loci with wild-type LCRs express at
the vast majority of integration sites, but not all integration sites,
at levels that are within a twofold to fourfold range relative to
the number of integrated copies.10-13 In contrast, when
5'HS 1, 2, 3, or 4 is mutated, there is greater variability among
lines, and expression levels are often distinctly reduced.14-17
The sequence the arrangement of gene and regulatory elementsand
chromatin structure of the -globin locus are highly conserved among
mammals. This conservation is present within the LCR as well as within
the coding regions. Within the LCR, the highest homology is found near
the sequences at which the HSs actually form, in what are termed the
cores of the HSs. The regions immediately flanking the cores of the HSs
have less homology, while regions between HSs have no substantial
homology. Sequence and structural conservation extends upstream of the
LCR and downstream of the coding regions and includes several
orthologous olfactory receptor genes that flank the human and mouse
loci.9 The high degree of homology observed between species
suggests conservation of function. To gain insights into the regulation
of mammalian -globin loci, we have generated mice with targeted
deletions of the individual HSs of the endogenous murine
LCR8,18,19 (also in M.A.B., unpublished data). In contrast
to the analysis of human -globin locus transgenes, where one must
always consider integration site effects when interpreting results, the
HR approach permits analysis of the contribution of the LCR to the
function of the endogenous mouse -globin locus in its native
genomic, cellular, and developmental state.
To determine how any single HS influences the formation of other HSs in
the native murine locus, we have performed a DNaseI HS formation
analysis of mice with each of the individual HS deletions. We show that
while the formation of each site is abolished when its core and
flanking sequences are deleted, the deletion of any individual HS does
not noticeably influence the formation of the remaining HSs. Thus, no
single HS is required for the formation of the other HSs in the
endogenous locus. These results are most compatible with a model in
which multiple elements composing the LCR act independently to alter
chromatin structure and to contribute to the level of expression of the
linked -like globin genes.
| |
Materials and methods |
Production of mutant mice
Mutant mice were produced by performing homologous recombination in
embryonic stem (ES) cells and deriving chimeric mice that transmitted
the ES cell genome through the germline. The details of production of
the 5'HS 2, 5'HS 3, and 5'HS 5/6 deletion mice were
reported previously.8,18,19 The descriptions
of production and transcriptional characterization of the 5'HS 1 and
5'HS 4 deletion mice are in preparation. These latter deletions remove nucleotides  4675 to 6999 and 19 849 to
22 561 relative to the Ey cap site,
respectively. All selectable markers used were flanked by recombinase
target sites for either Flp or Cre
site-specific recombinases and were deleted by expression of the
recombinases specific to either the Flp (FRT) or the Cre (loxP) site.
Correct structure of the targeted alleles was confirmed by Southern blotting.
DNase I hypersensitivity assay
Nuclei were isolated from spleens of mice after phenylhydrazine
treatment to induce anemia, at which time more than 50 percent of cells
are erythroid. The phenylhydrazine treatment, DNaseI digestion, and
Southern blotting were done as described.20 Probes used
span the following bases relative to the Ey cap site (1/+1): probe 1 (2867 to 3469), probe 2 (16 779 to
17 656), probe 3 (20 508 to 222), and probe 4 (27 981 to 28 567) (Figure 1).
View larger version (12K):
[in this window]
[in a new window]
| Fig 1.
Map of the murine -globin locus.
The top line diagrams the entire locus, including the LCR and the
expressed genes. The bottom line is an expansion of the LCR region of
the top line showing the restriction fragments and location of probes
used in this study and the sites of various deletions. The deleted
sequences are specified with negative numbers representing the number
of bases from each side of the deletion to the cap site of the Ey
messenger RNA, which is designated as 1/+1.
|
|
| |
Results |
Formation of 5'HS 1, 2, and 3
To determine how deletion of a single 5'HS affects the
formation of the other 5'HSs, we analyzed the formation of
5'HS 1 through 6 in wild type and HS-knockout mice. Formation of
5'HS 1, 2, and 3 was assayed by digesting nuclei with DNaseI
followed by restriction with HpaI and probing Southern blots with probe
1 (Figure 1). Figure 2 presents the data on
formation of 5'HS 1, 2, and 3 in mice with deletions of
5'HS 1, 2, 3, 4, or 5/6 and the wild-type control. Figure 2A
shows the formation of 5'HS 1, 2, and 3 in the wild-type mice.
Figure 2B shows that deletion of 5'HS 1 results in the failure of
5'HS 1 to form; however, 5'HS 2 and 5'HS 3 still form. Figure 2C shows that deletion of the region comprising 5'HS 2 does not impair the formation of 5'HS 1 and 5'HS 3, even
though 5'HS 2 does not form. Figure 2D shows that 5'HS 1 and 5'HS 2 form in the absence of the sequences composing
5'HS 3, even though 5'HS 3 does not form. Similarly,
5'HS 1, 2, and 3 form normally in mice with deletions of
5'HS 4 (Figure 2E) or 5'HS 5/6 (Figure 2F), respectively.
To confirm the presence and placement of 5'HS 1, 2, and 3, HSs
were mapped with the use of probes to the opposite end of the parent
HpaI fragment (data not shown). The kinetics of DNaseI digestion varies
among different digestion series; thus, quantitative comparisons of
sensitivity among experiments cannot be made.
View larger version (64K):
[in this window]
[in a new window]
| Fig 2.
Formation of 5'HS 1, 5'HS 2, and 5'HS 3 in mice with homozygous single-site deletions assayed by HpaI digestion
and probe 1.
(A) HS formation in wild-type mice, showing the expected sites and the
parent band. M denotes the marker lane; the DNaseI concentration is 0 in the first lane and increases as shown by the shaded triangle. Below
the autoradiograph is a restriction map of expected sizes for the HS
and parent band. (B) Similar to (A), with the assays done on mice with
5'HS 1 deleted and the site of the deletion shown on the gel and
in the map below. (C) Assay done on mice with 5'HS 2 deleted,
with the map below. (D) Assay done on mice with 5'HS 3 deleted,
with the map below. (E) Assay done on mice with 5'HS 4 deleted.
The map is identical to that in (A). (F) Assay done on mice with
5'HS 5/6 deleted. The map is identical to that in (A).
|
|
Formation of 5'HS 4, 5, and 6
Formation of 5'HS 4 and minor bands 5'HS 4.1 and 4.2 were analyzed with the use of HpaI-digested blots and probe 3. Unlike the major HS bands 5'HS 1, 2, 3, 4, and 6, which always form
strong sites in erythroid tissues, 5'HS 4.1 and 4.2 are more
variable in intensity. There is formation of 5'HS 4 in wild-type
mice and in mice with individual deletions of 5'HS 1, 2, or 3 (Figure 3); 5'HS 4 also forms
normally in mice with deletion of 5'HS 5/6, as previously
published8 (data not shown).
View larger version (83K):
[in this window]
[in a new window]
| Fig 3.
Formation of 5'HS 4 and minor sites 5'HS 4.1 and 5'HS 4.2 in mice with single-site deletions assayed by HpaI
digestion and probed with probe 3.
The map appearing in (A) is appropriate for all parts of this figure,
and is explained in the legend describing (A). (A) HS
formation in wild-type mice with a restriction map below the film
showing the expected sizes for the HS and parent band. M denotes the
marker lane; the DNaseI concentration is 0 in the first lane and
increases as shown by the shaded triangle. (B) Similar to (A), with the
assay done on mice with 5'HS 1 deleted. (C) Similar to (A), with
the assay done on mice with 5'HS 2 deleted. (D) Similar to A,
with the assay done on mice with 5'HS 3 deleted.
|
|
HSs in the region of 5'HS 4 were mapped in the 5'HS 4 deletion mice with the use of an SphI digest and probe 4 (Figure
4). HpaI was not used because the targeted
deletion of 5'HS 4 alters the HpaI restriction map. Analysis of
the wild-type samples on the left of Figure 4 reveals the presence of
5'HS 4, 4.1, and 4.2. In this assay, 5'HS 3 is difficult to
discern since the gel did not clearly resolve the 14-kb 5'HS 3 sub-band from the 16.2-kb parent band. The targeted deletion of
5'HS 4 removes the region encompassing 5'HS 4, but leaves
the region of 5'HS 4.1 and 4.2 intact. HS mapping of the
5'HS 4 deletion mice reveals that 5'HS 4.1 and 4.2 form,
but that 5'HS 4 is not present (Figure 4). Two additional minor bands are visible but do not map to the region of the
5'HS 4 deletion. The band just below the parent band is 5'HS 3, which is resolved more easily from the parent band owing to the 2.8-kb 5'HS 4 deletion and which is also more easily
visualized in the absence of 5'HS 4. The smaller of the 2 bands,
5'HS 3.5, is seen sporadically in these assays and is more
prominent in the absence of 5'HS 4 since the fragment is more
frequently available to be cut by DNaseI at 5'HS 3.5. In both
series, the major sites, 5'HS 3 and 5'HS 4, first appear at
lower DNaseI concentration than the minor sites (5'HS 4.1, 4.2, and 3.5), suggesting their greater accessibility to DNaseI. As with the
mapping of 5'HS 1, 2, and 3, these results were confirmed by
mapping sites from the opposite end of the parent fragment.
View larger version (54K):
[in this window]
[in a new window]
| Fig 4.
Formation of 5'HS 3, 5'HS 4, and minor
associated sites in mice with the 5'HS 4 region deleted.
The restriction digest is with SphI, and the probe is probe 4 from
Figure 1. M denotes the marker lane; the DNaseI concentration is 0 in
the first lane; and increases as shown by the shaded triangle. The
portion of the film to the left of the marker shows an assay done on
wild-type mice, and below this is the restriction map of expected
bands. The portion of the autoradiograph to the right of the marker
shows an assay done on mice with 5'HS 4 deleted, and below this
is the restriction map of expected bands.
|
|
Previously, we showed that deletion of the region that spans 5'HS
5 and 6 results in no formation of HSs at the site of the deletion,
while 5'HS 4, 4.1, and 4.2 form normally.8 We extend these findings to show that deletion of 5'HS 5/6 does not affect formation of 5'HS 1, 2, or 3 (Figure 2F). Thus, our analyses
reveal that deletion of any LCR 5'HSs (5'HS 1, 2, 3, 4, or
5/6) does not affect the formation of the other LCR HSs.
| |
Discussion |
A great deal of experimental effort has been devoted to
understanding the role of the LCR in regulating the -globin locus; however, many of the fundamental questions remain unresolved. We have
taken the approach of investigating the LCR by using homologous recombination in ES cells and deriving mouse lines with individual deletions of each of the major HSs in the murine LCR8,18,19 (also in M.A.B., unpublished data). None of these deletions in the
endogenous murine -globin LCR disrupts the normal pattern of
-like-globin switching. Deletion of HS 2, HS 3, or HS 4 reduces expression of -globin in adults by roughly 30%, 40%, and 20% respectively, whereas deletion of 5'HS 1 or 5'HS 5/6
results in more minor reductions in -globin gene expression. In this
report, we have investigated the formation of HSs in adult mice
carrying individual HS deletions. Our results, in combination with
previous studies, lead to 3 major conclusions regarding the murine LCR HSs: (1) Although each HS contributes to the overall expression of the
locus, no individual HS is necessary for the expression of a specific
gene or for expression at any specific stage of development. (2)
Deletion of the DNA region that forms an HS results in the elimination
of that HS. (3) Deletion of a given HS has little or no effect on
formation of the other HSs. Our data support the notion that the HSs
form and act independently and, in an additive or synergistic manner,
contribute to overall LCR activity.
On the basis of results obtained with single-copy transgenes containing
a single 5' HS, it was suggested that 5'HS 3 possessed the
dominant chromatin opening activity in the LCR; thus, the chromatin
overlaying 5'HS 3 would be altered initially, and subsequently an
open chromatin state could spread to the rest of the LCR and -globin
locus.21 Although, compared with the other HSs in the LCR,
HS 3 may have a greater chromatin opening activity when linked by
itself to globin genes in transgenic mice, it is clearly not required
for establishment of normal chromatin structure of the other HSs in the
endogenous mouse LCR. Similarly, during differentiation of a
multipotent murine hematopoietic cell line, the endogenous 5'HS2
formed prior to the other HSs,22 suggesting that formation of 5'HS2 might be required to precede the formation of the other HSs. We now demonstrate that this is not the case.
A current model for LCR function postulates that the HSs and associated
bound proteins form a single large "holocomplex" that is required
for expression of the -globin locus.21,23 The observations that HSs act additively or synergistically to stimulate expression, both in the endogenous locus and in transgenes, have been
cited in support of this model. Clearly, however, these data are
equally compatible with an additive effect of the HSs on modifying chromatin, directing subnuclear localization, fostering tracking of
factors from the LCR to the genes, or other plausible models of LCR
function. We have shown that in the endogenous murine locus, HSs form
independently, and no particular HS is necessary for near-normal
expression of any of the genes. Moreover, synthetic LCRs, which include
various combinations of HS sites and intervening DNA, generally confer
high-level expression in cell lines and in mice.21,24-26 In
fact, HS 2, 3, or 4 stimulates expression of linked globin transgenes
when it is the only HS present.23,27,28 Thus, if the
postulated holocomplex does exist and is required for LCR function,
then no specific HS is required to participate in such a complex, and
formation of such a holocomplex would not be dependent on the presence
or arrangement of any of the individual LCR HSs or associated binding
proteins. Such a complex, with little or no structural constraints,
would be unique among present examples of multiprotein complexes
regulating transcription in higher eukaryotes.29 Ultimately, the holocomplex model can be proved or disproved only by
biochemical and structural analyses of the protein-bound LCR in a
normal erythroid cell.
Our failure to observe any influence of the deletion of 1 HS on the
formation of others in the endogenous murine locus contrasts with the
more variable results obtained with human -globin locus transgenes
in mice. Comparison of results between
homologous-recombination-mediated HS deletion at the endogenous locus
and the deletion of HSs in human transgenes is difficult owing to
several factors, including position effects due to variable integration
sites of the transgenes, differences in the sizes of the deletions, and
cross-species differences.
One unavoidable variable in the transgenic studies is that each of the
transgenes is integrated at a different position in the genome, and
position effects on expression of transgenes are well documented.
Although the full LCR significantly suppresses position effects, this
suppression is incomplete; the same large, apparently unrearranged,
single-copy and wild-type transgenes consistently show twofold to
fourfold variability in expression level in different mouse lines. In
addition, a 150-kb YAC transgene containing the human -globin locus
and an intact LCR demonstrates position effect variegation in mice: the
transgene is not expressed in 58% of the red cells in these
animals.30 Thus, LCRs containing transgenes are sensitive
to position effects. Moreover, deletion of any of the HSs further
sensitizes the transgene to position effects.14 Depending
on the integration site, position effects may influence the chromatin
structure of the transgene, which may be manifested in part as variable
HS formation. Examination of each construct at a larger number of
integration sites or examination of multiple constructs at a single
integration site will be required to address this issue.
In general, studies of transgenes with relatively large HS deletions
encompassing the HS core and flanking regions revealed minor effects on
transcription, similar to the effects of our studies of targeted
deletions in the endogenous LCR. However, chromatin structure has not
been evaluated in transgenic mice with these large HS deletions. In
contrast, small deletions of the HS cores have been reported to result
in dramatic decreases in transcription and in significant diminution of
HS formation.17,31 As discussed previously,32
these core deletions may remove binding sites for activators and leave
intact sites for the binding of repressors that may nucleate formation
of a chromatin structure incompatible with transcription. It is
possible that such core deletions in the endogenous mouse locus may
reveal suppressive effects on the formation of the remaining
HSs in the mouse locus; these experiments are currently underway.
It is also possible that species differences may account for the
different results obtained in the analysis of the human -globin locus in the transgenes compared with studies of the murine locus. Although we cannot completely rule out any cross-species effects, the
high degree of sequence and structural homology between the mouse and
human loci, and the fact that the human locus functions in a transgenic
mouse, further support the idea that the loci are regulated similarly.
Studies of the endogenous human -globin locus in mouse
erythroleukemia somatic cell hybrids carrying the entire human
chromosome 11 revealed that deletion of human 5' HS 2 through
5' HS 5 eliminates -globin gene expression without affecting
the formation of the remaining site, 5'HS 1.33
Similarly, deletion of 5'HS 2 through 5'HS 4 leads to
complete loss of expression but normal formation of 5'HS 1 and 5. The HSs at either the murine or the human endogenous -globin loci
form independently of each other. Thus, the endogenous human locus in a
murine environment has a chromatin phenotype similar to that observed
in our knockout mouse studies.
Further insight into the differences in results obtained in studies of
LCR function at ectopic genomic sites and those at the endogenous locus
may derive from our recent demonstration that 5'HS 2 confers
stability of transgene expression at repressive genomic sites by
influencing the subnuclear localization of the transgene
locus.34 By extrapolation, we suggest that the deletion of
individual or multiple HSs from -globin locus transgenes increases the probability that the transgene associates with heterochromatic nuclear compartments, resulting in the lack of transcription factor accessibility, the loss of HS formation and the failure to activate transcription. In this scenario, transgenes with mutated LCRs would be
expressed only at genomic sites that are normally in a subnuclear
compartment permissive for expression in erythroid cells, whereas these
mutated transgenes would not be expressed at genomic sites that are
normally heterochromatized in erythroid cells. In contrast, multiple,
redundant elements in the endogenous globin locus prevent its
localization in such repressive subnuclear compartments, resulting in
the maintenance of the locus in an "open" DNaseI sensitive
conformation upon deletion of the LCR. Clearly, elucidation
of the molecular mechanism(s) by which the LCR influences the
-globin locus will require additional experimentation.
| |
Acknowledgments |
We are grateful to Jessica Halow, Joan Hamilton, and Jennie Close for
expert technical assistance.
| |
Footnotes |
Submitted November 19, 1999; accepted February 1, 2000.
Supported by the National Institutes of Health grants DK54071 (S.N.F.,
M.G.), DK44746 (M.G.), and P30 HD28834 through the University of
Washington Child Health Research Center (M.A.B.); and by a
Burroughs-Wellcome Fund Career Development Award (S.N.F.). M.A.B. is a Howard Hughes Medical Institute Physician
Postdoctoral Fellow and a J. S. McDonnell Foundation Scholar.
Reprints: Steven Fiering, 6W Borwell, DHMC, Lebanon, NH, 03756;
e-mail, fiering{at}dartmouth.edu; or Mark Groudine, FHCRC, 1100 Fairview
Ave N, Seattle, WA 98109; e-mail, markg{at}fhcrc.org.
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.
Stamatoyannopoulos G, Nienhuis A.
Hemoglobin switching. In:
Stamatoyannopoulos G, ed.
The Molecular Basis of Blood Diseases. Philadelphia, PA: WB Saunders; 1994:107.
2.
Curtin P, Pirastu M, Kan YW, Gobert-Jones JA, Stephens AD, Lehmann H.
A distant deletion affects -globin gene function in an atypical   -thalassemia.
J Clin Invest.
1985;76:1554.
3.
Taramelli R, Kioussis D, Vanin E, et al.
thalassemias 1 and 2 are the result of a 100 kb deletion in the human -globin cluster.
Nucleic Acids Res.
1986;14:7017[Abstract/Free Full Text].
4.
Driscoll C, Dobkin CS, Alter BP.
thalassemia due to a de novo mutation deleting the 5' -globin locus activating region hypersensitive sites.
Proc Natl Acad Sci U S A.
1989;86:7470[Abstract/Free Full Text].
5.
Tuan D, Solomon W, Li Q, London IM.
The -like-globin gene domain in human erythroid cells.
Proc Natl Acad Sci U S A.
1985;82:6384[Abstract/Free Full Text].
6.
Forrester WC, Thompson C, Elder JT, Groudine M.
A developmentally stable chromatin structure in the human -globin gene cluster.
Proc Natl Acad Sci U S A.
1986;83:1359[Abstract/Free Full Text].
7.
Hardison R, Slightom JL, Gumucio DL, Goodman M, Stojanovic N, Miller W.
Locus control regions of mammalian -globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.
Gene.
1997;205:73[Medline]
[Order article via Infotrieve].
8.
Bender M, Reik A, Close J, et al.
Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse -globin locus control region.
Blood.
1998;92:4394[Abstract/Free Full Text].
9.
Bulger M, von Doorninck J, Saitoh N, et al.
Conservation of sequence and structure flanking the mouse and human -globin loci: the -globin genes are embedded within an array of odorant receptor genes.
Proc Natl Acad Sci U S A.
1999;96:5129[Abstract/Free Full Text].
10.
Strouboulis J, Dillon N, Grosveld F.
Developmental regulation of a complete 70-kb human -globin locus in transgenic mice.
Genes Dev.
1992;6:1857[Abstract/Free Full Text].
11.
Porcu S, Kitamura M, Witkowska E, et al.
The human -globin locus introduced by YAC transfer exhibits a specific and reproducible pattern of developmental regulation in transgenic mice.
Blood.
1997;90:4602[Abstract/Free Full Text].
12.
Peterson KR, Clegg CH, Huxley C, et al.
Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human -globin locus display proper developmental control of human globin genes.
Proc Natl Acad Sci U S A.
1993;90:7593[Abstract/Free Full Text].
13.
Kaufman RM, Pham CT, Ley TJ.
Transgenic analysis of a 110-kb human -globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome.
Blood.
1999;94:3178[Abstract/Free Full Text].
14.
Milot E, Stroubolis J, Trimborn T, et al.
Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.
Cell.
1996;87:105[Medline]
[Order article via Infotrieve].
15.
Peterson KR, Clegg CH, Navas PA, Norton EJ, Kimbrough TG, Stamatoyannopoulos G.
Effect of deletion of 5'HS3 or 5'HS2 of the human -globin LCR on the developmental regulation of globin gene expression in -YAC transgenic mice.
Proc Natl Acad Sci U S A.
1996;93:6605[Abstract/Free Full Text].
16.
Bungert J, Dave U, Lim K-C, et al.
Synergistic regulation of human -globin gene switching by locus control region elements HS3 and HS4.
Genes Dev.
1995;9:3083[Abstract/Free Full Text].
17.
Bungert J, Tanimoto K, Patel S, Liu Q, Fear M, Engel J.
Hypersensitive site 2 specifies a unique function within the human -globin locus control region to stimulate globin gene transcription.
Mol Cel Biol.
1999;19:3062[Abstract/Free Full Text].
18.
Fiering S, Epner E, Robinson K, et al.
Targeted deletion of 5'HS2 of the murine -globin locus reveals that it is not essential for proper regulation of the -globin locus.
Genes Dev.
1995;9:2203[Abstract/Free Full Text].
19.
Hug B, Wesselschmidt RL, Fiering S, Bender MA, Groudine M, Ley TJ.
Analysis of mice containing a targeted deletion of the -globin locus control region 5'HS-3.
Mol Cell Biol.
1996;16:2906[Abstract].
20.
Reitman M, Lee H, Westphal H, Felsenfeld G.
An enhancer/locus control region is not sufficient to open chromatin.
Mol Cel Biol.
1993;13:3990[Abstract/Free Full Text].
21.
Ellis J, Tan-un K, Harper A, et al.
A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human -globin locus control region.
EMBO J.
1996;15:562[Medline]
[Order article via Infotrieve].
22.
Jimenez G, Griffiths SD, Ford AM, Greaves MF, Enver T.
Activation of the -globin locus control region precedes commitment to the erythroid lineage.
Proc Natl Acad Sci U S A.
1992;89:10,618[Abstract/Free Full Text].
23.
Fraser P, Pruzina S, Antoniou M, Grosveld F.
Each hypersensitive site of the human -globin locus control region confers a different development pattern of expression on the globin genes.
Genes Dev.
1993;7:106[Abstract/Free Full Text].
24.
Forrester WC, Novak U, Gelinas R, Groudine M.
Molecular analysis of the human -globin locus activation region.
Proc Natl Acad Sci U S A.
1989;86:5439[Abstract/Free Full Text].
25.
Moon AM, Ley TJ.
Functional properties of the -globin locus control region in K562 erythroleukemia cells.
Blood.
1991;77:2272[Abstract/Free Full Text].
26.
Blom van Assendelft G, Hanscombe O, Grosveld F, Greaves DR.
The -globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner.
Cell.
1989;56:969[Medline]
[Order article via Infotrieve].
27.
Curtin PT, Liu D, Liu W, Chang JC, Kan YW.
Human -globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.
Proc Natl Acad Sci U S A.
1989;86:7082[Abstract/Free Full Text].
28.
Ryan TM, Behringer RR, Martin NC, Townes TM, Palmiter RD, Brinster RL.
A single erythroid-specific DNase I super-hypersensitive site activates high levels of human -globin gene expression in transgenic mice.
Genes Dev.
1989;3:314[Abstract/Free Full Text].
29.
Groschedl R.
Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination.
Curr Opin Cell Biol.
1995;7:362[Medline]
[Order article via Infotrieve].
30.
Alami R, Greally JM, Tanimoto K, et al.
-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.
Hum Mol Genet.
2000;9:631[Abstract/Free Full Text].
31.
Li G, Lim K, Engel J, Bungert J.
Individual LCR hypersensitive sites cooperate to generate an open chromatin domain spanning the human -globin locus.
Genes Cells.
1998;3:415[Abstract].
32.
Bulger M, Groudine M.
Looping vs. linking: toward a model for long-distance gene activation.
Genes Dev.
1999;13:2465[Free Full Text].
33.
Reik A, Telling A, Zitnik G, Cimbora D, Epner E, Groudine M.
The locus control region is necessary for gene expression in the human -globin locus but not the maintenance of an open chromatin structure in erythroid cells.
Mol Cell Biol.
1998;18:5992[Abstract/Free Full Text].
34.
Francastel C, Walters M, Groudine M, Martin D.
A functional enhancer suppresses silencing of a transgene and prevents its localization close to centromeric heterochromatin.
Cell.
1999;99:259[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
J. Ross, S. Bottardi, V. Bourgoin, A. Wollenschlaeger, E. Drobetsky, M. Trudel, and E. Milot
Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters
Nucleic Acids Res.,
September 1, 2009;
37(16):
5295 - 5308.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Sawado, J. Halow, H. Im, T. Ragoczy, E. H. Bresnick, M. A. Bender, and M. Groudine
H3 K79 dimethylation marks developmental activation of the {beta}-globin gene but is reduced upon LCR-mediated high-level transcription
Blood,
July 15, 2008;
112(2):
406 - 414.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Ma, S. Fiering, C. Black, X. Liu, Z. Yuan, V. A. Memoli, D. J. Robbins, H. A. Bentley, G. J. Tsongalis, E. Demidenko, et al.
Transgenic cyclin E triggers dysplasia and multiple pulmonary adenocarcinomas
PNAS,
March 6, 2007;
104(10):
4089 - 4094.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Hu, M. Bulger, M. A. Bender, J. Fields, M. Groudine, and S. Fiering
Deletion of the core region of 5' HS2 of the mouse {beta}-globin locus control region reveals a distinct effect in comparison with human {beta}-globin transgenes
Blood,
January 15, 2006;
107(2):
821 - 826.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Fang, J. Sun, P. Xiang, M. Yu, P. A. Navas, K. R. Peterson, G. Stamatoyannopoulos, and Q. Li
Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5'HS3 Deletion Mutations: Implication for LCR Chromatin Architecture
Mol. Cell. Biol.,
August 15, 2005;
25(16):
7033 - 7041.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Martowicz, J. A. Grass, M. E. Boyer, H. Guend, and E. H. Bresnick
Dynamic GATA Factor Interplay at a Multicomponent Regulatory Region of the GATA-2 Locus
J. Biol. Chem.,
January 21, 2005;
280(3):
1724 - 1732.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. D. Marsden and R. E. K. Fournier
Chromosomal Elements Regulate Gene Activity and Chromatin Structure of the Human Serpin Gene Cluster at 14q32.1
Mol. Cell. Biol.,
May 15, 2003;
23(10):
3516 - 3526.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Jackson, J. C. McDowell, and A. Dean
{beta}-Globin locus control region HS2 and HS3 interact structurally and functionally
Nucleic Acids Res.,
February 15, 2003;
31(4):
1180 - 1190.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Thornton, C. Zhang, M. A. Kowalska, and M. Poncz
Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent, high-level megakaryocyte expression
Blood,
November 15, 2002;
100(10):
3588 - 3596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, K. R. Peterson, X. Fang, and G. Stamatoyannopoulos
Locus control regions
Blood,
October 16, 2002;
100(9):
3077 - 3086.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Bender, J. N. Roach, J. Halow, J. Close, R. Alami, E. E. Bouhassira, M. Groudine, and S. N. Fiering
Targeted deletion of 5'HS1 and 5'HS4 of the {beta}-globin locus control region reveals additive activity of the DNaseI hypersensitive sites
Blood,
October 1, 2001;
98(7):
2022 - 2027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Farrell, A. Grinberg, S. P. Huang, D. Chen, J. G. Pichel, H. Westphal, and G. Felsenfeld
A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus
PNAS,
December 19, 2000;
97(26):
14554 - 14559.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Goodwin, J. M. McInerney, M. A. Glander, O. Pomerantz, and C. H. Lowrey
In Vivo Formation of a Human beta -Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1
J. Biol. Chem.,
July 13, 2001;
276(29):
26883 - 26892.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction