By
From the Laboratory of Cell and Viral Regulation, Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
To investigate regulation of human immunoglobulin heavy chain expression, we have cloned
DNA downstream from the two human C genes, corresponding to the position in the mouse
IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype
switching. Within 25 kb downstream of both the human immunoglobulin C
1 and C
2 genes
we identified several segments of DNA which display B lymphoid-specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences
downstream from each of the two human C
genes are nearly identical to each other. These
enhancers are also homologous to three regions which lie in similar positions downstream from
the murine C
gene and form the murine LCR. The strongest enhancers in both mouse and
human have been designated HS12. Within a 135-bp core homology region, the human HS12
enhancers are ~90% identical to the murine homolog and include several motifs previously
demonstrated to be important for function of the murine enhancer; additional segments of high
sequence conservation suggest the possibility of previously unrecognized functional motifs. On
the other hand, certain functional elements in the murine enhancer, including a B cell-specific
activator protein site, do not appear to be conserved in human HS12. The human homologs of
the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but
for at least two of the functional motifs in the murine HS4 (a
B site and an octamer motif ) the
human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes
a region of high sequence conservation with mouse, suggesting the possibility of another novel
functional element.
The regulation of human immunoglobulin heavy chain
gene expression is incompletely understood, despite
clinically significant conditions in which specific isotypes
are inappropriately up- or downregulated, e.g., allergies
due to inappropriate IgE response, and various forms of
immunodeficiency associated with low IgA expression.
Clearly, cytokines and interactions between B and T cells
play a role in regulating isotype switching, and cis elements in the IgH gene locus which mediate these effects have
been documented in the murine and human promoters of
the sterile transcripts associated with each heavy chain constant region gene (1, 2). However, in the mouse an additional control region which contributes to regulation of
isotype switching has been reported to lie downstream from C The existence of a regulatory region downstream from
murine C
More recently, Madisen and Groudine (12) analyzed B
cell-specific DNase I hypersensitivity downstream from
C Analyses of the regulatory regions downstream from murine C Apart from the motifs mediating upregulation of the
3 A role for the 3 Because the enhancer regions lying downstream from
the mouse C Several laboratories have attempted to characterize sequences lying downstream from the two human C As an initial step toward defining the role of global control regions in the activation of the human IgH genes, our
laboratory has sought to determine: (a) are enhancer complexes located downstream from the human C Cloning of 3
, and the corresponding region of the human
heavy chain locus has not yet been investigated.
was originally inferred when it was found that
plasmacytomas which had undergone spontaneous deletions of the only heavy chain enhancer then known, which
lies in the intron between JH and Cµ, nevertheless remained capable of high level immunoglobulin secretion (3). Conversely, a myeloma subclone which retained the
intronic enhancer but lost a segment of DNA downstream
from the murine C
gene was found to have markedly reduced its heavy chain gene expression (7). A systematic
search in the homologous region of the rat heavy chain locus revealed an enhancer (8), and a homologous mouse enhancer designated 3
E was found soon after (9, 10) positioned ~16 kb downstream from C
. The mouse and rat
3
E segments lie in opposite orientations and are flanked by inverted repeats (9). In addition to the 3
E, Matthias and Baltimore also reported a weak enhancer in mouse
lying only 4 kb downstream from C
(Fig. 1 and reference 11).
Fig. 1.
Comparison of IgsH
loci of mouse and human. Line
A shows a map of the murine
IgH locus, from which the region downstream from C is expanded in line B. The murine
enhancers designated C
3
E (11)
and 3
E (9) are shown as vertical ovals, along with the DNase I
hypersensitivity site designations
(12). We have distinguished the
two copies of HS3 sequence as
HS3A and HS3B; these are included in a large palindrome (arrows) that flanks HS12, according
to the sequence analysis of
Chauveau and Cogné (13). Line
C shows the human IgH locus,
illustrating the
-
-
-
duplication units (brackets) and the possibility of two regions homologous to the murine LCR.
[View Larger Version of this Image (13K GIF file)]
and identified four hypersensitive sites. HS1 and HS2
fall in the previously described 3
E, whereas HS3 and
HS4 lie further downstream and identify two new regions
with somewhat weaker enhancer activity in transient transfection assays. The HS3 sequence is almost identical to that
of the enhancer described by Matthias and Baltimore but has an inverted orientation. This reflects the fact that the
sequence surrounding the HS12-3
E is present in the
mouse in a long inverted repeat which includes HS3 sequences at both ends (Fig. 1 and reference 13). When constructs containing HS3, HS12, and HS4 linked to a reporter gene were transfected into a B cell line, subsequently isolated stable transfectants were found to express the reporter gene in a position-independent manner. This suggested that the three enhancer sequences (HS12, HS3, and
HS4) acted together as a locus control region (LCR)1.
LCRs, first defined in the globin locus (14), activate large domains of chromatin in vivo (100 kb in the human
globin locus), and, as components of DNA constructs in
transgenic mice, support gene expression proportional to
the number of integrated copies. In contrast, integrated
gene constructs lacking LCR sequences are variably expressed, depending on the positions of integration. LCRs
typically contain several DNase I hypersensitive sites, which often represent DNA with enhancer activity. In addition to
the LCRs found in the
globin and mouse IgH loci,
LCRs have also been described as associated with macrophage-specific lysozyme, CD2, and
/
TCR loci (15).
have identified several motifs which bind specific
transcription factors to mediate different aspects of regulation of enhancer function. The 3
E has been found to activate transcription strongly in plasmacytomas, but only
weakly in earlier B lymphoid cells. Part of this developmental change is attributable to a motif known as E5, which
matches the "E-box" consensus binding site (CANNTG) characteristic for members of the basic helix-loop-helix
family of transcription factors. The contribution of the E5
site to enhancer activity is inhibited in early stages of development by the dominant negative nuclear regulator Id3,
which is expressed in early B lineage cells but downregulated in plasma cells (16). At least four other motifs in the
3
E have been reported to contribute to enhancer activity
specifically in plasmacytomas, motifs whose contribution in
B cells is inhibited by BSAP (the B cell-specific activator
protein), which disappears as B cells mature to plasma cells.
These sites include
P (17), the octamer motif (ATGCAAAT; reference 18), a
B-like site (16), and a G-rich
sequence (19). In B cells, BSAP prevents the binding of the
transcriptional activator NF-
P to the
P site, and causes
the octamer, G-rich, and
B-like motifs to exert an active
repressive influence on transcription (17, 19).
E during maturation to plasma cells, a response element
in the enhancer for activation induced by B cell receptor
cross-linking has been traced to partially overlapping sites
for the ETS family member Elf-1 and for members of the
AP-1 transcription factor family (22). Two other motifs in
the enhancer have been proposed to contribute to its regulation, but are less well documented: the µE1 and the µB
motifs, which were first noted in the rat 3
E and which
are partially conserved in mouse. The HS3 and HS4 enhancer regions of mouse have been studied in less detail,
but the HS4 enhancer apparently contains functional Oct-1
and BSAP binding sites (23).
E in isotype switching was revealed by
experiments in which this region was replaced by a neomycin resistance gene through homologous recombination in
embryonic stem cells which were then used to reconstitute
the B cell population in RAG-2 knockout mice. The resulting B cells showed normal V(D)J recombination but
marked deficiencies in switching to IgG2a, IgG2b, IgG3,
and IgE in vitro, whereas expression of IgM and IgG1 was normal (24). This observation suggests that the enhancer
exerts isotype-specific effects on switch recombination,
possibly through its regulation of germline transcription of
the different isotypes before switch recombination.
gene have been found to be important for
heavy chain gene expression and isotype switching, there
has been considerable interest in determining how homologous regions might regulate immunoglobulin gene expression in humans. The human heavy chain locus includes
two
-
-
-
segments (25, 26), apparently the product of
a large duplication in the primate lineage (27). Isotypes
from the upstream duplication, comprising the
3-
1-
-
1 constant region genes, are generally expressed at a much
higher level than those of the downstream
2-
4-
-
2 duplication. The existence of two C
genes in humans suggests the possibility that two 3
enhancer complexes may
regulate the locus, one downstream from each C
gene;
differences in these complexes could contribute to the differential regulation of the two duplications. Moreover, individuals who have a
-
1-
deletion on one chromosome show reduced expression of the downstream
2 and
4 genes on that chromosome, indicating that this deletion
may have removed a region which exerts distal control
over at least some of the human heavy chain genes (28). Finally, the possibility that there are two 3
enhancer complexes makes the human IgH locus an attractive candidate for study because there could be interactions between two
adjacent LCRs, a situation which has not been described in
any other system.
genes,
but technical difficulties have impeded this work. Gene
walking downstream from the C
genes has been difficult,
apparently because of a segment of 20-bp tandem repeats
which lies almost immediately downstream of the most 3
exon of both C
genes. These tandem repeats, described independently by three laboratories (29), include the
sequence GATC recognized by the isoschizomer restriction enzymes Sau3A and MboI. Since commercial human
DNA libraries in
phage have been constructed using genomic DNA fragments generated by partial MboI/Sau3A
digestion, the repeated Sau3A sites downstream of the C
genes make it unlikely that library clones isolated by hybridization to C
probes will contain DNA downstream
from the repeats.
genes; and
(b) how do any such human enhancers correspond to the
regulatory sequences described downstream from the murine C
gene? Towards this end we have successfully applied several strategies other than gene walking from C
to
obtain DNA clones extending downstream from the human
C
genes, enabling us, by functional analysis and sequencing, to characterize enhancer regions 3
of each C
gene
homologous to those of the mouse HS12, HS3, and HS4.
Regions from Human Genomic DNA.
To obtain DNA between C
1 and the previously reported
pseudogene which lacks associated S
sequences, we initially sought clones containing the pseudogene, which should hybridize to a C
probe but not to an S
probe. We screened a commercial library of partial MboI-digested human placental DNA in the
FixII phage vector (Stratagene, La Jolla, CA) with a C
probe:
probe f (Fig. 2), a 7-kb HindIII fragment, was isolated from
a pBR322 plasmid clone originally derived from
phage
CH·Ig·H·g-11(32) (Health Science Research Resources Bank,
Osaka, Japan; e-mail: hsrrb{at}nihs.go.jp). C
+ plaques were replated and duplicate plaque-lift filters were hybridized with the
C
probe and an S
probe (1,100-bp KpnI to PstI fragment containing human S
2; reference 33). Southern blots of BamHI-digested DNA from eight candidate clones (C
+, S
) were
hybridized with a 32P-labeled oligonucleotide Pseudogam-1 (see
Table 1 for sequences of oligonucleotides) specific for the
hinge region at 48°C in hybridization buffer (1 M NaCl, 0.1 M
sodium phosphate, pH 7.0, 10% dextran sulfate, 10 mM EDTA,
1% SDS), followed by washing at 48°C in 1× SSC, 10 mM
EDTA, 0.1% SDS. This analysis identified three overlapping
+ clones (
-25,
-2, and
-38) extending over an ~30-kb region containing the
gene (Fig. 2).
Fig. 2.
Regulatory loci
downstream of human C1 and
C
2. Lines A and C, based on
this study, show an expanded
map of the region downstream of C
1 and C
2, respectively, as
well as available DNA clones, which are shown above (
1) or
below (
2) the line: phage
clones are marked with diagrammatic phage heads, while the
subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3
are drawn with hatched lines;
and a BAC clone is drawn as a
double line, containing a deletion (dashed box). Vertical ovals
mark DNase I sites demonstrating enhancer activity and named
according to the homologous
murine HS sites. A series of small
triangles identifies the 20-bp repeats located downstream from
human C
genes. X marks the
position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is
also shown (oval). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS
site, but opposite from the orientation of HS12 in the
2 locus (line C). The thick black lines under the maps of lines A and C (single lower case letters) represent hybridization probes used in this study.
[View Larger Version of this Image (24K GIF file)]
DNase I Hypersensitive Site Analysis.
Nuclei were prepared
and digested with DNase I according to a previously described
protocol (36). K562 and HS Sultan cells (both obtained from
American Type Culture Collection) were grown to densities of
5-8 × 105 cells/ml. For each experiment, 3-6 × 108 cells were
harvested, lysed by addition of NP-40, centrifuged through a 1.7 M
sucrose cushion, and resuspended in 5 ml; 450-µl aliquots of suspended nuclei were treated with serially diluted DNase I (Boehringer-Mannheim, Indianapolis, IN) to give final DNase I concentrations of 0-8 µg/ml. Nuclei were digested with DNase I for 3 min at 25°C. For one experiment HS Sultan nuclei were digested
with the restriction endonuclease SspI (New England Biolabs,
Beverly, MA) for 15 min at 37°C. DNase I (or SspI) digestion was terminated by adding 50 µl 1% SDS, 100 mM EDTA. DNA
samples were deproteinized for 5-48 h at 37°C using proteinase
K (Boehringer-Mannheim) at a final concentration of 100 µg/ml.
DNA was purified by phenol/chloroform extraction and ethanol
precipitation, resuspended in 50-100 µl deionized water, and
DNA concentrations were measured using a Fluorometer (TKO
100; Hoeffer, San Francisco, CA). 5-µg DNA samples were digested for 5-24 h in 50 µl of appropriate restriction enzyme buffer with BglII, EcoRI, or HindIII (New England Biolabs). To assess the SspI sensitivity of the globin locus in HS Sultan nuclei, a 1,511-bp human
globin probe was amplified from total human genomic DNA using primers
5PR-A and
3PR-B. Restriction-digested samples, together with 32P-labeled size markers,
were electrophoresed, blotted, and hybridized with the probes
indicated in the figure legends. After washing the membranes, radioactive images were obtained with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Enhancer Assays.
To analyze DNA fragments for enhancer
activity, we used the luciferase reporter plasmid pGL3 (Promega
Corp., Madison, WI), modified so that the SV40 promoter between BglII and HindIII sites was replaced by a 76-bp V promoter (37) containing an octamer motif and TATA box. This
plasmid, named pGL3-V
, served as an enhancerless, promoter-only control. Fragments to be assayed for enhancer activity were
blunt-ended with Klenow fragments of DNA polymerase and ligated with MluI linkers, or amplified with primers incorporating
an MluI (or KpnI) site; the fragments were then cloned into the
MluI (or KpnI) site in the polylinker upstream of the promoter in
pGL3-V
. Plasmid DNAs for transfection were twice purified on
a CsCl gradient.
Sequence Analysis. A PCR-based methodology employing P33-labeled ddNTPs was used for sequencing reactions (ThermoSequenase kit; Amersham Life Science, Arlington Heights, IL). All samples were amplified for 50 cycles (95°C for 30 s; 60°C for 30 s; 72°C for 60 s). Sequencing reactions were electrophoresed on 6% gels, dried, autoradiographed, and read manually. All reported sequences were read on both A and B strands, except for the sequences of the HS12 59-bp repeats, which, despite several attempts employing various strategies, could be read only on one strand.
Because attempts to clone the human 3 C
regions using strategies
based on gene walking from C
or cross-species hybridization had failed in other laboratories, we used an alternative
strategy based on the fact that a C
-like pseudogene (
)
has been described downstream of human C
1 (32, 38).
We reasoned that we could clone DNA downstream of
C
1 by walking upstream from
. The similarity in the
restriction maps reported downstream of the two C
genes based on Southern blotting (26) furthermore suggested that
these regions would be highly similar to each other. Therefore, probes derived by walking upstream from
should
also hybridize to clones containing DNA from downstream
of C
2.
By screening a phage library for clones that hybridized to
C but not S
, and then screening those clones with a
-specific oligonucleotide (see Materials and Methods), we
obtained three overlapping clones spanning ~30 kb (Fig.
2). To obtain clones covering the corresponding region
from the
2 locus, the library was then rescreened with
probes b and e (Fig. 2). Clones deriving from downstream
of C
2 were selected on the basis of restriction site differences between the
1 and
2 which had been established by analysis of genomic Southern blots and comparisons
with our cloned DNA from the
1 duplication. Three
clones deriving from the
2 locus and spanning ~30 kb
were obtained (Fig. 2).
To estimate the distance between our two phage contigs
and the corresponding C genes, we performed genomic
Southern blot experiments using a panel of restriction enzymes with known sites in the C
loci and in our contigs.
For example, co-migrating BglII bands of ~35 kb were
found to hybridize to both probe a (from the C
membrane exon
m) and probe b (Fig. 2); these experiments suggested a gap of ~20 kb between each contig and the
corresponding membrane exon of C
(data not shown).
For the
2 locus, the gap was bridged by a BAC clone (see
Materials and Methods). The corresponding regions from
the
1 locus have resisted direct cloning and have been obtained by PCR using primers designed from the sequence of the
2 locus, as described in Materials and Methods.
To map potential
regulatory sequences downstream of the C genes, we
used fragments from the cloned DNA downstream of the C
2 gene to search for DNase I hypersensitivity sites; in
cells where such enhancers or promoters are active, they
generally are hypersensitive to endonucleases, apparently
because binding of transcription factors disrupts the nuclease protection afforded by nucleosomes. Intact nuclei
from the human myeloma HS Sultan were incubated
briefly with various concentrations of DNase I; DNA samples purified from these treated nuclei were then analyzed using several Southern blot strategies in order to localize
the positions of DNase I cleavage. The analyses were complicated by the fact that all of the probes we used hybridized to both the
1 and
2 loci, but various restriction map
differences between the two loci allowed us to position all
the hypersensitivity sites with respect to restriction sites
mapped from our clones.
Fig. 3 A demonstrates analyses of DNA from the promyelocyte K562 line, representative of a cell not expressing
immunoglobulin genes, and the myeloma HS Sultan line.
The DNase I-treated DNA was digested with BglII, which
cuts ~1 kb upstream of the membrane exon of both C
genes; the blots were hybridized with a probe corresponding to this exon. Since the next downstream BglII site is
>20 kb away, this strategy can display hypersensitivity sites over this wide distance from the
membrane exon. The
blot demonstrates at least seven hypersensitivity sites, which
were subsequently assigned to the
1 or
2 locus by other
blotting experiments and were named according to sequence similarity to the homologous murine regions as described below. The sites we have tentatively designated
1X and
2X do not correspond to any reported murine enhancer sequence. None of these sites were visible in the
DNA from K562, in which the enhancer region is expected to be inactive.
The BglII blot of HS Sultan DNA fails to resolve the
two HS4 sites because they are too far away from the BglII
sites. The two HS4 sites were resolved by an alternative
Southern blot strategy employing EcoRI digests of the
DNA from DNase I-digested nuclei (Fig 3 B). To determine which of the two resulting bands represented HS4
from the 1 versus
2 loci, we exploited the observation
from our sequence analysis that recognition sites for the restriction enzyme SspI lie in each HS4 site. Since many regulatory regions accessible to DNase I are also accessible to
restriction endonucleases, we digested HS Sultan nuclei
with SspI and localized the cleavage sites by isolating the
DNA, digesting with EcoRI, and hybridizing Southern
blots with probe b
as shown in Fig. 3 B. From the map positions of the
1 and
2 SspI sites we assigned the SspI
hypersensitivity bands as shown in Fig. 3. A control experiment on the same DNA isolated from SspI-digested HS
Sultan nuclei demonstrated that SspI recognition sites in
the
globin locus were not cut, indicating that the SspI
sites associated with HS4 were indeed hypersensitive in HS
Sultan cells (data not shown; see Materials and Methods).
Fig. 3 C illustrates an experiment which allowed assignment of several HS sites to the 2 locus rather than
1.
DNA samples from DNase I-treated HS Sultan nuclei
were digested with HindIII and hybridized with a probe
for the HS12 site derived from the
2 locus. Although this
probe hybridizes to both the
1 and
2 loci, in this DNA
the
1 band is ~23 kb, so large that any fragments from
this locus that were generated by HindIII and DNase I and which hybridized to the probe would be larger than the
12-kb band representing the
2 locus. Thus all HS bands
<12 kb derive from
2. This blot therefore defines the position of the HS12, X, and HS3 sites in the
2 locus. By
implication, the other HS sites in the BglII blot of Fig. 3 A
must derive from
1. (It should be noted that a common
allele in other DNA samples shows an additional polymorphic HindIII site which cuts within the 23 kb corresponding to the HS Sultan band; this allele would have confounded the above strategy, but was absent in HS Sultan.)
To analyze enhancer activity in the
cloned DNA downstream of the C2 locus, fragments
containing one or more DNase I hypersensitive sites were
subcloned into a luciferase reporter gene driven by a V
promoter as described in Materials and Methods. Luciferase activity in each sample was normalized to the
-galactosidase activity of the promoter-only control plasmid, and expressed as fold-increase over luciferase activity of that control plasmid.
The luciferase assays (Fig. 4) revealed strong enhancer
activity in the 5-kb SmaI-HindIII fragment (SM5) which
was found to contain sequence homologous to murine
HS12 (see below). Within this fragment, enhancer activity
seemed to be confined to the 1.3-kb EcoRI-HindIII fragment (EH1.3) containing the HS12 site. This segment was
further cut into an upstream 0.3-kb EcoRI-PstI fragment
(EP300), a 0.3-kb PstI-PstI fragment (P300), and a 0.6-kb PstI-HindIII fragment (PH600). Of these, only the P300
fragment, which contained HS12, showed enhancer activity. However, it should be noted that the enhancer activity
of P300 was less than that measured for a larger PCR-generated fragment A2HS12. Thus it is possible that additional
elements that do not show intrinsic enhancer activity when
isolated in constructs can nevertheless augment the activity
of the core HS12 enhancer lying in the P300 fragment. Furthermore, the EH1.3 fragment, but not the slightly
shorter A2HS12 fragment, showed significantly less enhancer activity in the A orientation, suggesting the possibility of inhibitory sequences located near one of the ends of
EH1.3.
Two fragments containing the 2 HS3 site showed no
significant enhancer activity in the luciferase construct, but
a plasmid in which the HS3-containing 0.7-kb BamHI-SmaI fragment was dimerized showed consistent low activity (Fig. 4, SM0.7-X2). The homologous murine enhancer
was reported to show greater activity with a c-myc promoter than with an immunoglobulin V
promoter (12),
but we found no significant increase in enhancer activity
when the V
promoter was replaced by a human c-myc
promoter (data not shown; construct described in Materials
and Methods).
Two restriction fragments, B5.5 and SpB0.9, containing
the 2 HS4 site demonstrated significant stimulation of luciferase activity, but only when cloned in the B (inverted)
orientation (Fig. 4 A). When these fragments were oriented so that the DNA strand continuous with the transcribed strand in the luciferase gene corresponded to the
transcribed strand of the immunoglobulin gene in genomic
DNA (the A orientation), luciferase activity was no greater
than that from the promoter-only plasmid. Unexpectedly, a smaller fragment (the 468-bp PCR-generated fragment
designated A2-HS4, designed to span the sequence showing the strongest homology to the murine HS4 site) was
found to give a strong activation of luciferase activity. The
stronger activation of luciferase conferred by the short A2-HS4
PCR fragment compared with that of the longer SpB0.9 fragment (in the same A orientation) suggests that the latter may
contain an inhibitory sequence that is either position- or orientation-dependent. This possibility is currently being explored.
Because of the lack of genomic clones spanning the HS3,
HS12, and HS4 regions of 1 locus, fragments corresponding to each of these sites were obtained by PCR and analyzed; and, to facilitate
1 versus
2 locus comparisons, the
same primer pairs were used to generate corresponding
fragments from the
2 locus. As shown in Fig. 4, the activity of each HS site fragment from
1 was similar to that
from the
2 locus (compare SM0.7-X2
2 versus HS3-X2
1; A2HS12
2 versus A1HS12T
1; and A2-HS4
2
versus A1-HS4
1). The PCR amplification of the HS12
site yielded two fragments, designated A1HS12T (top) and
A1HS12B (bottom), which differ in size by <0.1 kb and
may represent alleles (see below). Both fragments showed
substantial enhancer activity (Fig. 4 B).
Since the mouse 3 enhancers show different activation
patterns during B cell differentiation, with HS4 being activated at the pre-B cell stage, while HS12 and HS3 are only
active in mature B cells, we examined the activity of the
human 3
2 enhancers in a range of human and mouse B
cell lines (Table 2). The pattern of activation for the human
HS12 enhancer is similar to that of mouse HS12; i.e., human HS12 is inactive in the human pro-B cell line FLEB-14 and the mouse pre-B cell line 18-81, but functions in
the human mature B cell line Raji, as well as three plasmacytomas (HS Sultan, human; S194, mouse; and MOPC
315, mouse). HS3 is also inactive in mouse 18-81 pre-B
cell line, but shows modest activity in most of the more
mature lines tested. HS3 shows surprisingly strong activity
in the mouse S194 myeloma, indicating that unknown factors varying between cell lines at similar stages of differentiation can modulate the activity of this enhancer. Finally,
the HS4 enhancer shows strong activity in the human pro-B
cell line FLEB14, and is also variably active in all of the more
mature cell lines (except 18-81) in which this enhancer was
assayed in the B orientation.
|
The nucleotide sequence of all DNase
I hypersensitivity sites was determined, revealing ~99% sequence identity between the human 1 and
2 elements,
and similarity between the human and mouse enhancers
ranging from 74 to 90%.
HS12, the strongest enhancer, showed 90% sequence
identity to the homologous murine enhancer over a 135-bp core homology (Fig. 5 A). In the 2 locus, four tandem
repeats with a 59-bp consensus sequence lie immediately
upstream of the HS12 core. However, this sequence has
been inverted in Fig. 5 A, to facilitate comparison with the
homologous
1 sequence in opposite orientation. In the corresponding region of
1 (which, due to the inversion,
lies downstream of the core homology region) a 115-bp
deletion removes the second and third repeats; the
1HS12B
region shows an additional deletion of 70 bp. The core homology region includes several of the functional motifs
identified in the murine enhancer: the AP1-Ets site reported to confer responsiveness to B cell receptor cross-linking (22, 39); an exact octamer sequence (ATGCAAAT);
and a µE5 site (except in the
1HS12B sequence, in which
the µE5 is missing owing to the 70-bp deletion). The sequences of these three motifs from the human HS12 are
identical to their murine homologs except for a single base
change in the AP1 site which causes the human sequence
to exactly match the consensus AP1 site, where the murine motif has one mismatch. The murine element designated
µE1 (39), which has never been thoroughly documented
even in the murine enhancer, is poorly conserved in the
human homologs. Although the murine binding site for
NF-
B lies outside the 135-bp region of strongest sequence similarity, a reasonable match to consensus for this
element is found in a position roughly homologous to the
murine
B site in the
1HS12T and
2HS12 sequences,
but is part of the 70 bp deleted in
1HS12B. One of the
mouse BSAP sites (BSAP2) is not conserved, but most residues in a second mouse BSAP binding site (BSAP1) are
maintained in the human
2 HS12 enhancer. The murine
P site, which binds to an ETS-related transcription factor
which augments enhancer activity (17), is not conserved in
the human sequences.
The DNase I hypersensitive sites lying <3 kb downstream from the membrane exons of C1 and C
2,
roughly in the position of the weak enhancer reported by
Matthias and Baltimore (11) and here designated HS3A,
were found to contain sequences which are 74% identical
to the murine HS3 over a 200 bp core segment. The two
human HS3 segments are identical in the 326 bp shown
(Fig. 5 B), and lie in the same orientation as HS3A. We
have assumed that the correct orientation of the murine
HS3A sequence is that described by Chauveau and Cogné
(13). This orientation is opposite to that of the murine
HS3B, which lies downstream from HS12 in the mouse, as
described by Madisen and Groudine (12). Of the enhancer/HS sites downstream of murine C
, HS3 is the
least well investigated for functional motifs, in part because
of its weak enhancer activity. Independent sequence analysis of the murine HS3A and HS3B regions detected several
similarities to octamer motifs, AP1 sites, and consensus E
box motifs (CANNTG). The AP1 site identified in Fig. 4
is a precise match to the consensus AP1 binding motif
TGANTCA (40) in the two human and two mouse HS3
sequences, and the murine sequence has been shown to
bind to c-jun and c-fos in vitro (Neurath, M., personal
communication). Similarly, several of the E box consensus
motifs in the murine sequence have been shown to bind in
vitro to proteins of the HLH family (Neurath, M., personal
communication); some of these motifs are conserved in the
two human HS3 sequences. The significance of the conserved motifs remains uncertain in the absence of a functional analysis of HS3 sequences.
The DNase I hypersensitivity sites furthest downstream
from human C1 and C
2 in Fig. 3, which we have designated HS4, are 76% similar to the murine HS4 site over a
core 145-bp sequence which spans the three functional
motifs demonstrated in murine HS4 (23); see Fig. 5 C. The
NF-
B motif and the downstream octamer motif in Fig. 5
C are both precisely conserved; in the murine HS4 these
motifs both contribute to functional enhancer activity (23).
In contrast, the BSAP site which upregulates murine HS4 enhancer activity in B cells but downregulates it in pre-B
cells is completely absent from the human HS4 sequences.
Lying between HS3 and HS12 in both the 1 and
2
loci are DNase I hypersensitive sites which are not associated with any of the known enhancer elements, but that do
map to the position of a 61-bp segment of 70% mouse-
human homology. These conserved regions are provisionally designated X sites in part because of their unknown
function, and in part because both the human and mouse
segments contain XbaI restriction enzyme sites. In mouse this sequence is duplicated as part of the large inverted repeat centered on HS12, so that one copy lies between
HS3A and HS12 while a second copy lies between HS12
and HS3B. A segment of (GA)n repeats is found near the X
site in the direction of HS12 at an interval of 80 bp for
both mouse X sites (13) and an interval of ~70 bp for the
human (data not shown). Within the 61-bp conserved segment, the most highly conserved sequence is a consensus
heat shock element (HSE; 41, 42). An HSE could potentially bind heat shock transcription factors (HSTFs), which
are known to activate several heat shock response genes
(HSP70, HSP90) in response to cellular stress such as heating (43). Fragments containing an X site do not appear
to dramatically affect enhancer activity in HS Sultan, but
may contribute to regulation through mechanisms not captured in our transient transfection assays.
The enhancers clustered 3 of C
in the mouse IgH
locus can activate the upstream genes, functioning as an
LCR. In human IgH locus, arrays of enhancers homologous to those 3
of mouse C
are located at two positions
within the human IgH locus, 3
of each C
gene. On the
basis of sequence homology and conservation of restriction
sites between the two human enhancer arrays, it is apparent
that these enhancers lie near the 3
ends of the two duplication units which encompass the
3-
1-
-
1 and
2-
4-
-
2 gene clusters (25), indicating that the 3
enhancer arrays were present in an approximation of the human arrangement preceding the duplication event that gave
rise to the present human IgH locus structure. Moreover,
the arrangement downstream of both human C
genes is
5
-HS3-HS12-HS4-3
, in contrast to the large palindromic
structure downstream from mouse C
that contains a 5
-HS3A-HS12-HS3B-HS4-3
arrangement (13). Therefore, although an arrangement containing an HS3 enhancer
proximal to the C
membrane exon and an HS4 enhancer
farther downstream would seem to have been present in
the common ancestor of rodents and primates, the mouse
HS3A-HS12-HS3B palindrome probably arose after the
primate-rodent divergence. Finally, there is a major structural difference between the 3
1 and 3
2 enhancer arrays; namely, that a DNA segment containing HS12 is inverted between the two loci. Using probes containing the
135-bp human HS12 core, it should now be possible to examine DNA from a number of primates for inversion of
3
2 HS12 relative to 3
1 HS12; such data may indicate
when in evolution the inversion event occurred, and
which orientation was present initially in the locus. What
caused this inversion? Interestingly, the single mouse HS12
lies in opposite orientation from the rat HS12, and both are
flanked by inverted repeats (13) which are known to mediate inversions in other genomic contexts, e.g., in the iduronate-2-sulfatase gene causing Hunter syndrome (46) and
in the factor VIII gene (47). Limited Southern blot experiments have not provided evidence for inverted repeats
flanking the human HS12 sequences (data not shown).
Some hints about the mechanism of the inversion may be
found when the inversion breakpoints are identified and
sequenced, work currently in progress in our laboratory.
The 135-bp HS12 core
homology sequence is likely to contain essential motifs important for the strong, late, B cell-specific enhancer activity
characteristic of HS12 in mice and humans. Although the
function of transcription factor binding sites within the human HS12 core has not yet been demonstrated experimentally, this segment contains sequences nearly identical to the
murine AP1, ETS, Oct, and, in 1HS12T and
2HS12, µE5
motifs, all of which are functional in the mouse HS12 enhancer. However, the high degree of sequence conservation in the HS12 core homology extends beyond the transcription factor-binding sites identified in the mouse enhancer,
indicating that there may be additional conserved motifs
that have not been characterized in either mice or humans.
Despite the fact that a number of other transcription factor motifs whose function has been demonstrated in the
mouse HS12 lie outside the HS12 135-bp core homology
and are absent in one or more of the human 1HS12T,
1HS12B, and
2HS12 enhancers that we have studied,
these enhancers all show roughly equivalent activities. This
result suggests that elements missing from these enhancers are not essential for enhancer function in HS Sultan. These
inconsistently conserved elements include human sequences
corresponding to µE5 and NF-
B sites (absent from
1HS12B) and the BSAP2 site (absent from both
1 alleles).
On the other hand, a 1.3-kb
2 EcoRI-HindIII fragment
containing the
2 HS12 core plus considerable flanking sequence shows a dependence of enhancer activity on orientation of the fragment (Table 2), suggesting that uncharacterized elements beyond the HS12 core may have some
inhibitory function.
Outside the human HS12 core are GC-rich 59-bp repeat
units which by themselves do not have enhancer activity in
the HS Sultan myeloma (EP300, Fig. 4 A), and are not
conserved between mice and humans (Fig. 5 A). However,
it is possible that these repeats contribute to enhancer activity because they are present in the A2HS12 PCR-generated fragment, which gives significantly higher enhancer
activity than we observe in the p300 fragment containing the core homology. Deletions of the 59-bp repeats have
given rise to apparent allelic polymorphisms, as evidenced
by 1HS12T (deletion of the second and third repeats
found in
2HS12),
1HS12 (deletion extending from 28 bp 5
of the first repeat through the third repeat), and other
alleles (Harindranath, N., unpublished results).
The other two enhancer components
of the mouse 3 LCR, HS3 and HS4, are weaker enhancers than HS12, but nonetheless are essential for locus control activity (12). Although these elements are less well
characterized than the HS12 enhancer, the existing data indicate general human-mouse similarity of the HS3 and
HS4 elements, with some notable differences.
In the mouse system, the HS3A element assayed in CAT
reporter gene constructs driven by c-fos or thymidine kinase promoters (11) showed weak enhancer activity, although
the nearly identical HS3B enhancer showed substantial activity in certain constructs with other promoters tested by
another laboratory (12). These disparate results resemble
our data on the human 1 and
2 HS3 elements in that
single copies and dimers of human HS3 generally gave very
low enhancer activity, except in the mouse S194 myeloma in which the same constructs gave substantial enhancer activity comparable to that of HS12 (Table 1). Taken together, these data suggest that HS3, though typically the
weakest of the 3
enhancers, contains uncharacterized
motifs that in some cells and/or in combination with certain promoters, can mediate a strong enhancer function.
HS4 is the most downstream 3 enhancer in both mice
and humans, and shows activity intermediate between that
of HS3 and HS12. The HS4 enhancer data in the mouse
(12, 48), as well as our data on the human
1 and
2 HS4
elements, demonstrate that HS4 is active from the early
stages of the B cell lineage onward (Table 2), and thus is
qualitatively different from HS3 and HS12. In mouse HS4,
there is a binding site for the BSAP, which is expressed in
the early B cell lineage. However, in the human
1 and
2
HS4 enhancers, the BSAP site is deleted, indicating that
BSAP binding is not an essential feature for HS4 activation
in human pre-B cells. The human HS4 is inactive in the
18-81 mouse pre-B cell line, which was reported to support the activity of mouse HS4 (12). The significance of
this difference is not clear; it could be related to the BSAP
site deletion in the human HS4, or perhaps to other differences between the mouse and human HS4 sequences.
The DNase I X sites may represent novel control elements that function together with the HS3, HS12, and HS4 enhancers to activate the IgH locus. Although the significance of the conserved HSE motif is unclear, binding of HSTF protein to an HSE has been shown to be critical for maintaining the DNase I hypersensitivity of the yeast HSC82 gene promoter (49). Heat shock activation of the Drosophila HSP70 gene promoter results from binding of HSTF to HSE sites after accessibility of the HSEs has been established by binding of the GAGA protein to adjacent (GA)n repeats (50). This demonstrated interaction between HSEs and (GA)n motifs suggests that the location of a (GA)n repeat region 70-80 bp away from the X site HSE may be of some significance. Furthermore, in the context of IgH gene regulation, it is of interest to note that HSE motifs have been shown to respond to IL-2 and IL-4 (51).
Potential Locus Control Region.In the mouse, it has been
demonstrated that when HS3, HS12, and HS4 are linked
together in a construct containing the c-myc gene and stably transfected into the Raji human B cell line, the c-myc
gene is transcribed independent of integration site (12). This observation suggests that the HS1234 combination
confers LCR activity, although LCRs have more typically
been described based on position-independent transcription of mouse transgenes rather than genes transfected into
a cell line. Because the regions 3 of the human C
1 and
C
2 genes contain similar HS3, HS12, and HS4 elements
that function as enhancers, it is reasonable to hypothesize that these elements also function together in the human
system as LCRs. The different arrangement of 3
enhancers in mice and humans (HS3A-HS12-HS3B-HS4 versus
HS3-HS12-HS4) may cause some functional differences in
these control regions. Moreover the distance between 3
enhancers also differs between mice and humans, with the mouse
enhancer complex spanning a 30-kb region (13), whereas
both the human 3
1 and 3
2 enhancers span ~15 kb.
Our finding that arrays of enhancers homologous to
those in the mouse 3 LCR lie downstream of both human C
genes raises the possibility that differences in the
activation of each human
-
-
-
duplication unit result
from differences between the putative 3
1 and 3
2
LCRs. Even though sequence comparison shows that there
is near identity between homologous enhancer elements in the
1 versus
2 locus (Fig. 5), transcription and expression of
the upstream heavy chain duplication unit (
3-
1-
-
1) is greatly elevated relative to the downstream unit (
2-
4-
-
2; reference 2). This difference could result from the fact
that the 3
2 HS12 element is inverted relative to the 3
1
HS12, and is also at a greater distance from HS3 than in the
1 locus, possibly reducing synergistic interactions between
HS3 and HS12. Alternatively, it may be that only the 3
1
enhancers are activated early in B cell development, possibly falling under the influence of the upstream Eµ enhancer, which itself can function as an LCR (52, 53). In
this model, the Eµ and 3
1 enhancers together would activate a large domain encompassing the first duplication
unit, whereas the second duplication unit and the 3
2 enhancers would fall outside this combined domain. Thus activation of the second duplication unit would depend solely
on the 3
2 enhancers, and expression of genes in this unit
might therefore be reduced. Clarification of the basis for
this difference will have to await experiments that involve
specific deletion of either the 3
1 or 3
2 enhancers, as
well as studies identifying matrix attachment sites and chromatin insulator elements that define domains within the
human IgH region (52).
In the work presented here, we have laid the foundation for experimental studies on the activation of the human IgH gene transcription, as well as the regulation of isotype switching. In addition, knowledge of the action of these enhancers on distant constant region genes should contribute to a general understanding of the mechanisms underlying activation of large gene clusters.
Address correspondence to Dr. Frederick C. Mills, Division of Hematologic Products, FDA/CBER/HFM-541, Bldg. 29A, RM 2B09, 29 Lincoln Dr., MSC 4555, Bethesda, MD 20892-4555. Phone: 301-827-1808; FAX: 301-480-3256; E-mail: millsf{at}fdacb.cber.fda.gov
Received for publication 12 May 1997 and in revised form 16 July 1997.
Note added in proof. While this manuscript was under review, related investigations by two other laboratories came to our attention. Chen, C., and B.K. Birshtein (1997. J. Immunol. 159:1310-1318.) have described the HS12 enhancers from theWe thank Drs. Rose Mage, Louis Staudt, and Steven Bauer for discussions and useful comments on this manuscript. We also thank Dr. Markus Neurath for communicating unpublished results on functional elements in the mouse HS3 enhancers.
1. | Coffman, R.L., D.A. Lebman, and P. Rothman. 1993. The mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol. 54: 229-270 [Medline]. |
2. |
Sideras, P.,
L. Nilsson,
K.B. Islam,
I.Z. Quintana,
L. Freihof,
G.J. Rosen,
L. Hammarstrom, and
C.I. Smith.
1992.
Transcription of unrearranged Ig H chain genes in human B cell
malignancies. Biased expression of genes encoded within the
first duplication unit of the Ig H chain locus.
J. Immunol.
149:
244
|
3. | Klein, S., F. Sablitzky, and A. Radbruch. 1984. Deletion of the IgH enhancer does not reduce immunoglobulin production of a hybridoma IgD class switch variant. EMBO (Eur. Mol. Biol. Organ.) J. 3: 2473-2476 [Abstract]. |
4. | Eckhardt, L.A., and B.K. Birshtein. 1985. Independent immunoglobulin class-switch events occurring in a single myeloma cell line. Mol. Cell. Biol. 5: 856-868 [Medline]. |
5. | Wabl, M., and P.D. Burrows. 1984. Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer in cis. Proc. Natl. Acad. Sci. USA. 81: 2452-2455 [Abstract]. |
6. | Aguilera, R.J., T.J. Hope, and H. Sakano. 1985. Characterization of immunoglobulin enhancer deletions in murine plasmacytomas. EMBO (Eur. Mol. Biol. Organ.) J. 4: 3689-3693 [Abstract]. |
7. |
Gregor, P.D., and
S.L. Morrison.
1986.
Myeloma mutant
with a novel 3![]() |
8. |
Pettersson, S.,
G.P. Cook,
M. Bruggemann,
G.T. Williams, and
M.S. Neuberger.
1990.
A second B cell-specific enhancer
3![]() |
9. |
Dariavach, P.,
G.T. Williams,
K. Campbell,
S. Pettersson, and
M.S. Neuberger.
1991.
The mouse IgH 3![]() |
10. |
Lieberson, R.,
S.L. Giannini,
B.K. Birshtein, and
L.A. Eckhart.
1991.
An enhancer at the 3![]() |
11. |
Matthias, P., and
D. Baltimore.
1993.
The immunoglobulin
heavy chain locus contains another B-cell-specific 3![]() |
12. | Madisen, L., and M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells. Genes Dev. 8: 2212-2226 [Abstract]. |
13. |
Chauveau, C., and
M. Cogné.
1996.
Palindromic structure of
the IgH 3![]() |
14. |
Talbot, D.,
P. Collis,
M. Antoniou,
M. Vidal,
F. Grosveld, and
D.R. Greaves.
1989.
A dominant control region from
the human ![]() |
15. | Ernst, P., and S.T. Smale. 1995. Combinatorial regulation of transcription. I. General aspects of transcriptional control. Immunity. 2: 311-319 [Medline]. |
16. |
Meyer, K.B.,
M. Skoberg,
C. Margenfeld,
J. Ireland, and
S. Petterson.
1995.
Repression of the immunoglobulin heavy
chain 3![]() |
17. |
Neurath, M.F.,
E.E. Max, and
W. Strober.
1995.
Pax5
(BSAP) regulates the murine immunoglobulin 3![]() ![]() ![]() |
18. | Ernst, P., and S.T. Smale. 1995. Combinatorial regulation of transcription II: the immunoglobulin µ heavy chain gene. Immunity. 2: 427-438 [Medline]. |
19. |
Singh, M., and
B.K. Birshtein.
1993.
NF-HB (BSAP) is a repressor of the murine immunoglobulin heavy-chain 3![]() ![]() |
20. |
Neurath, M.F.,
W. Strober, and
Y. Wakatsuki.
1994.
The
murine Ig3![]() ![]() ![]() |
21. |
Singh, M., and
B.K. Birshtein.
1996.
Concerted repression of
an immunglobulin heavy-chain enhancer, 3![]() ![]() |
22. |
Grant, P.A.,
C.B. Thompson, and
S. Pettersson.
1995.
IgM
receptor-mediated transactivation of the IgH 3![]() |
23. |
Michaelson, J.S.,
M. Singh,
C.M. Snapper,
W.C. Sha,
D. Baltimore, and
B.K. Birshtein.
1996.
Regulation of 3![]() ![]() |
24. |
Cogné, M.,
R. Lansford,
A. Bottaro,
J. Zhang,
J. Gorman,
F. Young,
H.-L. Cheng, and
F.W. Alt.
1994.
A class switch
control region at the 3![]() |
25. |
Flanagan, J.G., and
T.H. Rabbitts.
1982.
Arrangement of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing ![]() ![]() ![]() |
26. | Hofker, M.H., M.A. Walter, and D.W. Cox. 1989. Complete physical map of the human immunoglobulin heavy chain constant region gene complex. Proc. Natl. Acad. Sci. USA. 86: 5567-5571 [Abstract]. |
27. | Kawamura, S., and S. Ueda. 1992. Immunoglobulin CH gene family in hominoids and its evolutionary history. Genomics. 13: 194-200 [Medline]. |
28. | Hammarstrom, L., A.O. Carbonara, M. DeMarchi, G. LeFranc, M.-P. Lefranc, and C.I.E. Smith. 1987. Generation of the antibody repertoire in individuals with multiple immunoglobulin heavy chain constant region gene deletions. Scand. J. Immunol. 25: 189-194 [Medline]. |
29. |
Gualandi, G.,
D. Frezza,
A. Scotto,
d'Abusco,
E. Bianchi,
S. Gargano,
S. Giorgi,
A. Fruscalzo, and
E. Calef.
1995.
Integration of an Epstein-Barr virus episome 3![]() ![]() |
30. |
Chen, C., and
B.K. Birshtein.
1995.
A region of 20-bp repeats lies 3![]() |
31. |
Kang, H.K., and
D.W. Cox.
1996.
Tandem repeats 3![]() |
32. | Takahasi, N., S. Ueda, M. Obata, T. Nikaido, S. Nakai, and T. Honjo. 1982. Structure of human immunoglobulin gamma genes: implications for evolution of a gene family. Cell. 29: 671-679 [Medline]. |
33. |
Mills, F.C.,
M.P. Mitchell,
N. Harindranath, and
E.E. Max.
1995.
Human Ig S![]() |
34. | Rao, V.N., K. Huebner, M. Isobe, A. Ar-Rushdi, C.M. Croce, and E.S.P. Reddy. 1989. elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints. Science (Wash. DC). 244: 66-70 [Medline]. |
35. | Bensmana, M., and M.-P. Lefranc. 1990. Gene segments encoding membrane domains of the human immunoglobulin gamma 3 and alpha chains. Immunogenetics. 32: 321-330 [Medline]. |
36. | Siebenlist, U., L. Hennighausen, J. Battey, and P. Leder. 1984. Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in Burkitt lymphoma. Cell. 37: 381-391 [Medline]. |
37. |
Bergman, Y.,
D. Rice,
R. Grosschedl, and
D. Baltimore.
1984.
Two regulatory elements for immunoglobulin ![]() |
38. | Bensmana, M., H.S. Huck, G. Lefranc, and M.-P. Lefranc. 1988. The human immunoglobulin pseudo-gamma IGHGP gene shows no major structural defect. Nucleic Acids Res. 16: 3108 [Medline]. |
39. |
Grant, P.A.,
V. Arulampalam,
L. Arhlumd-Richter, and
S. Pettersson.
1992.
Identification of Ets-like lymphoid specific
elements within the immunoglobulin heavy chain 3![]() |
40. | Janknecht, R., and A. Nordheim. 1993. Gene regulation by Ets proteins. Biochim. Biophys. Acta. 155: 346-356 . |
41. | Amin, J., J. Ananthan, and R. Voellmy. 1988. Key features of heat shock regulatory elements. Mol. Cell. Biol. 8: 3761-3769 [Medline]. |
42. | Xiao, H., and J.T. Lis. 1988. Germline transformation used to define key features of heat-shock response elements. Science (Wash. DC). 239: 1139-1142 [Medline]. |
43. | Zimarino, V., C. Tsai, and C. Wu. 1990. Complex modes of heat shock factor activation. Mol. Cell. Biol. 10: 752-759 [Medline]. |
44. |
Schuetz, T.J.,
G. Gallo,
J.L. Sheldon,
P. Tempst, and
R.E. Kingston.
1991.
Isolation of ![]() |
45. | Rabindran, S.K., G. Giorgi, J. Clos, and C. Wu. 1991. Molecular cloning and expression of a human heat shock factor, HSF1. Proc. Natl. Acad. Sci. USA. 88: 6906-6910 [Abstract]. |
46. | Bondeson, M.L., N. Dahl, H. Malmgren, W.J. Kleijer, T. Tonnesen, B.M. Carlberg, and U. Pettersson. 1995. Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome. Hum. Mol. Genet. 4: 615-621 [Abstract]. |
47. | Naylor, J.A., D. Buck, P. Green, H. Williamson, D. Bentley, and F. Giannelli. 1995. Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions. Hum. Mol. Genet. 4: 1217-1224 [Abstract]. |
48. |
Michaelson, J.S.,
S.L. Giannini, and
B.K. Birshtein.
1995.
Identification of 3![]() ![]() |
49. | Erkine, A.M., C.C. Adams, T. Dikaen, and D.S. Gross. 1996. Heat shock factor gains access to the yeast HSC82 promoter independently of other sequence-specific factors and antagonizes nucleosomal repression of basal and induced transcription. Mol. Cell. Biol. 16: 7004-7017 [Abstract]. |
50. | Tsukiyama, T., P.B. Becker, and C. Wu. 1994. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature (Lond.). 367: 525-532 [Medline]. |
51. | Metz, K., J. Ezernieks, W. Sebald, and A. Duschl. 1996. Interleukin-4 upregulates the heat shock protein Hsp90a and enhances transcription of a reporter gene coupled to a single heat shock element. FEBS (Fed. Eur. Biochem. Soc.) Lett. 385: 25-28 . |
52. | Jenuwein, T., W.C. Forrester, R.G. Qiu, and R. Grosschedl. 1993. The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation. Genes Dev. 7: 2016-2032 [Abstract]. |
53. | Forrester, W.C., C. van Genderen, T. Jenuwein, and R. Grosschedl. 1994. Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions. Science (Wash. DC). 265: 1221-1225 [Medline]. |
54. |
Chung, J.H.,
M. Whitely, and
G. Felsenfeld.
1993.
A 5![]() ![]() |