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and Dad1 Genes1
Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and 
genes are expressed
specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously
expressed. Between TCR and Dad1 are eight DNase I hypersensitive
sites (HS). HS1 colocalizes with the TCR enhancer
(E) and is T cell-specific; HS2, -3, -4, -5, and -6 map
downstream of HS1 and are tissue-nonspecific. The region spanning
HS2–6 was reported to display chromatin-opening activity and to confer
copy number-dependent and integration site-independent transgene
expression in transgenic mice. Here, we demonstrate that HS2–6 also
displays enhancer-blocking activity, as it can block an enhancer from
activating a promoter when located between the two in a
chromatin-integrated context, and can do so without repressing either
the enhancer or the promoter. Multiple enhancer-blocking elements are
arrayed across HS2–6. We show that HS2–6 by itself does not activate
transcription in chromatin context, but can synergize with an enhancer
when located upstream of an enhancer and promoter. We propose that
HS2–6 primarily functions as an insulator or boundary element that may
be critical for the autonomous regulation of the TCR and Dad1
genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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locus is a complex genetic region that spans more than one
megabase in both mouse and man (1, 2). Contained within this locus are
the variable (V), diversity (D), joining (J), and constant (C) gene
segments that encode two different TCR chains, and . A large
number of V gene segments as well as several
V gene segments are arrayed across the 5'
portion of the locus. A large number of J gene
segments, as well as C, is found at the
extreme 3' end. Nested between the V gene segments and
J gene segments lie the
D, J, and
C gene segments. TCR and TCR gene
segments are assembled by the process of V(D)J recombination during the
differentiation of T lymphocytes in the thymus.
VDJ
rearrangement occurs with retention of the V,
J, and C gene
segments and can lead to the production of a functional TCR
polypeptide, whereas
VJ rearrangement
occurs with deletion of the D,
J, and C gene
segments and can lead to the production of a functional TCR
polypeptide.
Although the TCR and TCR gene segments are tightly linked and
are both rearranged and expressed specifically in T lymphocytes, they
are clearly under distinct regulatory control. TCR gene
rearrangement and expression initiates in the double negative
population of thymocytes and at day 14 of murine embryogenesis, whereas
TCR gene rearrangement and expression initiates in the subsequent
double-positive population of thymocytes and at day 16 of murine
embryogenesis (3, 4, 5, 6). Interestingly, recent studies have identified an
unrelated and distinctly regulated antiapoptosis gene, Dad1, to be
closely linked to the TCR locus, only 12 kb 3' of
C (7). The Dad1 gene is expressed in all
tissues examined and at least as early as day 7 of murine
embryogenesis.
The cis-acting elements that control Dad1 expression remain
to be identified. However, much is known about the
cis-acting elements that control TCR and TCR gene
rearrangement and expression. Of particular importance are the TCR
enhancer
(E)4,
situated in the intron between the J3 and
C gene segments (8, 9, 10), and the TCR
enhancer (E), situated immediately 3' of
C (11, 12). Both enhancers have been shown to
direct the developmental stage-specific activation of V(D)J
recombination in transgenic V(D)J recombination substrates in vivo,
with E activating V(D)J recombination at the
double-negative stage, and E activating V(D)J
recombination at the double-positive stage (13, 14, 15). Further,
E has been shown to be critical for V(D)J
recombination and expression of the endogenous TCR gene (16). Thus
E and E appear to
confer region-specific developmental control to the process of V(D)J
recombination within the endogenous TCR locus. The activation of
V(D)J recombination by these enhancers is thought to occur as a
consequence of their ability to modify local chromatin structure to
allow access to the V(D)J recombinase (17, 18).
The tight linkage of the TCR , TCR , and Dad1 genes raises the
question of how their distinct regulation is maintained. One potential
mechanism is suggested by our recent studies identifying an
enhancer-blocking element, blocking element -1 (BEAD-1), in a
2-kb region between the C and
J gene segments (19). We showed that BEAD-1
could block the TCR enhancer from activating a nearby promoter when
situated between the two in a chromatin-integrated construct in stably
transfected cells. We proposed that BEAD-1 might function in vivo to
prevent E from prematurely activating the
J gene segments for V(D)J recombination during
the double-negative stage of T cell development, thereby helping to
maintain independent regulation of TCR and TCR . BEAD-1 has
properties that are similar to those of a class of regulatory elements,
initially described in Drosophila, that are known as boundary elements
or insulators (20, 21, 22). These elements include scs and scs', which
flank the Drosophila 87A7 hsp70 locus (23, 24), su(Hw) protein binding
sites in the Drosophila gypsy transposon (25, 26), 5' hypersensitive
site (HS) 4 in the chicken ß-globin locus (27, 28), and Fab-7 in the
Drosophila bithorax complex (29, 30, 31, 32). Like BEAD-1, these elements can
block an enhancer from activating a promoter when positioned between
the two. Furthermore, they can insulate a transgene from position
effects. Boundary elements are thought to play a critical role in
dividing chromatin into independently regulated units, or domains,
thereby preventing interlocus regulation. The Fab-7 element in the
Drosophila bithorax complex has been explicitly shown to fulfill these
expectations in vivo, as it is required for normal parasegment-specific
gene expression and parasegment differentiation during Drosophila
development (29, 30).
Recently, a series of eight DNase I HS were mapped between the TCR
and Dad1 genes (7, 33). One of these, HS1, is T cell-specific and
colocalizes with the previously defined E. The
region encompassing HS2, -3, -4, -5, and -6 (hereafter denoted HS2–6)
was originally proposed to represent a locus control region (LCR), or
an essential component of an LCR, that functioned to control V(D)J
recombination and gene expression throughout the entire TCR
locus. In conjunction with E, HS7, and HS8,
HS2–6 was found to confer high-level, integration site-independent,
and copy number-dependent TCR gene expression in the thymus of
transgenic mice, whereas E, HS7, and HS8 alone
could not (33). HS2–6 was reported to have chromatin-opening activity,
as it could activate an erythroid-specific ß-globin reporter in a
tissue-nonspecific manner in transgenic mice (34). However, elimination
of HS2–6 from the endogenous TCR locus had no measurable effect
on either TCR or TCR rearrangement and transcription in
thymocytes and had only a relatively small effect on the relative
proportions of thymocyte subsets that could not be readily attributed
to a specific molecular defect (7). Hence, the true regulatory function
of HS2–6 remains unclear.
LCRs are typically composed of a series of tissue-specific DNase HS
that function in a dominant fashion to open chromatin and activate gene
expression. By comparison, boundary elements are relatively neutral;
although they can interfere with enhancer-promoter communication in a
position-dependent manner, they typically lack an intrinsic and
dominant ability to activate gene expression (20, 21, 35). Either LCR
or boundary activity can be invoked to explain the ability of HS2–6 to
confer copy number-dependent and integration site-independent
expression onto a linked transgene. However, a boundary function for
HS2–6 might best explain the ability of the TCR and Dad1 genes to
maintain dramatically different expression patterns, despite their very
close linkage.
In this report, we show that HS2–6 can block an enhancer from
activating a promoter when located between the two in a
chromatin-integrated substrate. HS2–6, by itself, failed to dominantly
activate transcription from a linked promoter. However, it synergized
with an enhancer to activate the promoter when located upstream of the
two elements. Although position-dependent enhancer blocking and
enhancer synergy may both play a role within the endogenous TCR
locus, we propose that the primary function of HS2–6 is that of an
insulator/boundary element that helps to maintain independent
regulation of the TCR and Dad1 genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The constructs P-Neo, E-P-Neo-scs', and E-2.7-P-Neo-scs' were
described previously (19). In these constructs, E, P, Neo, and scs'
represent the 380-bp E, the 1.6-kb
V1 promoter, the bacterial neomycin resistance
gene, and the Drosophila hsp70 scs' boundary element, respectively
(Fig. 1
A). 2.7 represents two
copies of the 1.35-kb phage 
X HaeIII DNA fragment in
tandem. Plasmid pLCR8.0 carries an 8-kb DNA fragment of the TCR
locus isolated by Dr. P. Lauzurica from a strain 129 mouse genomic DNA
library (kindly provided by Dr. T. Tedder, Duke University, Durham,
NC). The 8.0-kb RSa8.0 and 5.5-kb RSm5.5 DNA fragments were released
from this plasmid by digestion with EcoRI plus
SalI and EcoRI plus SmaI,
respectively. These fragments, as well as the 2.3- and 2.8-kb
HindIII-HindIII (HH2.3 and HH2.8) and 2.4-kb
HindIII-BglII (HB2.4) fragments (Fig. 1B), were inserted into the SalI site between
E and the V1 promoter
of plasmid E-P-Neo-scs' by blunt-end ligation, to generate plasmids
E-RSa8.0-P-Neo-scs', E-RSm5.5-P-Neo-scs', E-HH2.3-P-Neo-scs',
E-HH2.8-P-Neo-scs', and E-HB2.4-P-Neo-scs'. RSm5.5-P-Neo-scs' was
generated by deletion of E from
E-RSm5.5-P-Neo-scs' using NotI and ClaI digestion
followed by religation of blunt ends. HH2.3, HH2.8, and HB2.4 were also
introduced by blunt-end ligation into the XbaI site upstream
of E in E-P-Neo-scs', generating plasmids
HH2.3-E-P-Neo-scs', HH2.8-E-P-Neo-scs', and HB2.4-E-P-Neo-scs',
respectively. Note that all constructs are named in a descriptive
fashion that identifies each of the elements, in order, within the
plasmid. Plasmids were purified by two CsCl density gradient
centrifugation steps and were linearized by NotI or
SacII digestion. Following three phenol and two chloroform
extractions, linearized plasmids were ethanol precipitated and
resuspended in 10 mM Tris (pH 8.0) and 1 mM EDTA.
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Each linearized test DNA construct was transfected into the human T cell leukemia Jurkat by electroporation in triplicate, and transfectants were plated in 30 ml 0.33% agar (Sigma, St. Louis, MO) in RPMI 1640 supplemented with 10% FBS and 1000 µg/ml active G418 (Life Technologies, Gaithersburg, MD), as described previously (19). G418-resistant colonies were counted 3–4 wk after plating and selection.
Cotransfection and cloning by limiting dilution
Jurkat cells were cotransfected with linearized test construct and linearized pTK-hyg (19) at a molar ratio of 6:1 by electroporation. Individual hygromycin B-resistant clones were generated by limiting dilution cloning 24 h after transfection as described previously (19). Test construct integration and copy number were determined by slot blot analysis of genomic DNA isolated from individual clones.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 promoter
(P-Neo) (Fig. 1
A). E is inserted
upstream of the V1 promoter (E-P-Neo) to
measure enhancer activity, and test fragments can be inserted between
E and the V1 promoter
to measure enhancer-blocking activity. The Drosophila boundary element
scs', which was previously demonstrated to be functional as an
enhancer-blocking element in Jurkat cells, is inserted 3' of neo
(E-P-Neo-scs') to prevent activation of the promoter by downstream
copies of the enhancer in tandemly arrayed multicopy integrants.
Using this assay, we asked whether there is enhancer-blocking activity
within HS2–6 of the murine TCR locus. To this end, 8.0-kb
(RSa8.0) and 5.5-kb (RSm5.5) DNA fragments (Fig. 1B), both
of which contain HS2–6, were individually cloned between
E and the V1 promoter
of construct E-P-Neo-scs'. Insertion of either RSa8.0
(E-RSa8.0-P-Neo-scs') or RSm5.5 (E-RSm5.5-P-Neo-scs') between
E and V1 promoter
completely blocked the ability of E to
activate the V1 promoter, suggesting the
existence of enhancer-blocking elements in HS2–6. Enhancer blocking
was independent of the orientation of HS2–6 (Fig. 2A).
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and the
V1 promoter or inhibition of transfection
efficiency due to increased plasmid size. To eliminate these
possibilities and to better localize enhancer-blocking activity, a
2.3-kb HindIII fragment (HH2.3) that includes HS2 and HS3, a
2.8-kb HindIII fragment (HH2.8) that includes HS4, HS5, and
HS6, and a 2.4-kb HindIII-BglII fragment (HB2.4)
that contains HS4 and HS5 (Fig. 1), were individually cloned into
E-P-Neo-scs'. Compared with a 2.7-kb DNA fragment (two tandem copies of
the phage X 1.35 kb HaeIII fragment; E-2.7-P-Neo-scs')
previously shown to have no enhancer-blocking activity (19), HH2.3
(E-HH2.3-P-Neo-scs') reduced the colony number, but not to the level of
P-Neo, indicating partial blockade of E (Fig. 2B). By contrast, HH2.8 (E-HH2.8-P-Neo-scs') was a highly effective
enhancer blocker. Deletion of the HS6 region from HH2.8
(E-HB2.4-P-Neo-scs') resulted in partial loss of enhancer blocking,
indicating that the HS6 region carries a component of the
enhancer-blocking activity. Hence, at least three discrete segments of
HS2–6 (the HS2–3 segment, the HS4–5 segment, and the HS6 segment)
contribute to enhancer-blocking activity in the colony assay. We note
that the colony number for E-HH2.8-P-Neo-scs' was actually slightly
below the level for P-Neo in this experiment; the smaller size of P-Neo
might have resulted in an elevated transfection efficiency relative to
the other constructs. This effect does not compromise our conclusions
about enhancer-blocking relative to E-2.7-P-Neo-scs', as all other
constructs are of similar size.
The enhancer-blocking activity of HH2.8 was further confirmed by a
cotransfection/limiting dilution cloning assay (19). In this assay,
E-HH2.8-P-Neo-scs' and E-2.7-P-Neo-scs' were each cotransfected into
Jurkat cells with a hygromycin resistance gene. Ten hygromycin B
resistant clones containing E-HH2.8-P-Neo-scs' and seven hygromycin B
resistant clones containing E-2.7-P-Neo-scs' were tested for Neo gene
expression by Northern blot analysis. Levels of neo transcripts in the
E-HH2.8-P-Neo-scs' clones were found to be significantly reduced in
comparison to E-2.7-P-Neo-scs' (Fig. 3A). After correction for neo
gene copy number and RNA loading, insertion of HH2.8 between
E and the V1 promoter
resulted in, on average, a 93% decrease of neo gene expression (Fig. 3B).
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did not inhibit
E from activating neo gene expression (Fig. 2C). In contrast, insertion of these elements upstream of
E resulted in 3- to 4-fold increases in colony
number. This result indicates that HS2–6 can either inhibit or augment
neo gene expression in a fashion that is dependent on its position
relative to the enhancer and promoter. The increase in expression in
this experiment could be due to an insulating activity that may protect
the reporter from negative position effects, or could result from an
intrinsic activating property of HS2–6, as has been reported
previously (33, 34). An increase in expression is not intrinsically
associated with the positioning of enhancer-blocking elements in an
upstream position, as no such effect was observed in a previous
study (19).
LCRs activate gene expression in a dominant manner through the
cooperation between multiple cis-elements. Typically, an LCR
contains a classical enhancer that can activate transcription in
transient transfection assays, as well as chromatin-opening elements
that activate gene expression in a chromatin context (20, 35). In the
ß-globin LCR, HS2 is an example of the former, while HS3 and HS4 are
examples of the latter (36, 37). It has been reported that HS2–6
between TCR and Dad1 genes does not activate transcription in a
transient transfection assay (33) but does display chromatin-opening
activity (34). Therefore, we asked whether HS2–6 can activate a
chromatin-integrated V1 promoter in the
absence of an enhancer. HS2–6 fragment RSm5.5 was cloned upstream of
the V1 promoter of P-Neo-scs' in both
orientations. In the soft agar colony forming assay, inclusion of
HS2–6 in either orientation upstream of the
V1 promoter (RSm5.5-P-Neo-scs') failed to
increase colony number over that obtained with P-Neo (Fig. 4A). This result was confirmed
by the cotransfection/limiting dilution assay. In all eight
hygromycin-resistant Jurkat cell clones containing RSm5.5-P-Neo-scs',
neo gene expression was either undetectable or at much lower levels
than in clones containing E-2.7-P-Neo-scs' (Fig. 4B). From
these experiments, we conclude that HS2–6 by itself is unable to
significantly activate transcription from the
V1 promoter. Thus, the increase in neo gene
expression when HS2–6 is introduced upstream of both
E and the V1 promoter
(Fig. 2C) is most likely dependent on the presence of a
linked enhancer (E). From these transfection
experiments, we conclude that HS2–6 possesses an enhancer-blocking
activity that is manifest when it is positioned between an enhancer and
promoter. Further, we conclude that although HS2–6 is unable to
activate a promoter by itself, it can synergize with an enhancer to
up-regulate gene expression when positioned upstream of an enhancer and
promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C). It is
also unclear whether either of these activities are attributable to the
HS per se, or to other associated elements. Further studies will be
required to address whether the two activities can be dissociated from
each other or from the mapped HS.
Our data are compatible with the results of recent gene-targeting
studies in the endogenous TCR locus. A deletion of
E that leaves HS2–6 intact abolishes V(D)J
recombination and transcription of TCR gene (16), indicating that
HS2–6 cannot provide chromatin accessibility for V(D)J recombination
and transcription in the absence of E. In
contrast, a deletion of HS2–6 that leaves E
intact affects neither TCR nor TCR gene expression (7),
indicating that HS2–6 is not required for normal expression of these
genes. Our data are also consistent with the observation by Ortiz et
al. (34) that HS2–6 is a relatively weak activator of heterologous
reporter gene expression in the thymus of transgenic mice. Considering
all of these data, we propose that the primary function of HS2–6 in
the endogenous locus is that of an insulator or boundary element. Such
activity may be at least partially responsible for the LCR-like ability
of HS2–6 to confer copy number-dependent and integration
site-independent expression to an E-containing
TCR transgene, as reported previously (33). Within the endogenous
locus, HS2–6 may prevent the as yet unidentified regulatory elements
responsible for ubiquitous Dad1 expression from activating TCR .
Alternatively, it may prevent E from
superactivating Dad1 expression in T cells. E
is known to be a potent activator of transcription and V(D)J
recombination whose influence extends at least 90 kb in the 5'
direction (16); yet, although Dad1 is positioned only 8 kb 3' of
E, it is striking that Dad1 expression is not
significantly perturbed in double-positive thymocytes of
E-/- mice (B. Sleckman,
personal communication).
The notion that HS2–6 is required for appropriately regulated Dad1 expression in thymocytes must remain a tentative hypothesis, at least in part because the Dad1 promoter has not been defined and was not specifically tested in our assays. A role for HS2–6 in Dad1 expression could best be assessed in mice carrying a targeted deletion of HS2–6 (7). However, several properties of these previously generated mice suggest that they would be inappropriate for this analysis. First, in addition to eliminating HS2–6, the gene-targeting event disrupted the Dad1 transcription unit by eliminating a portion of the Dad1 3' untranslated region. Second, the gene-targeting event resulted in the integration of a PGK-neo cassette, which has been documented to perturb the expression of linked genes in numerous instances (38, 39, 40). Perhaps as a consequence of the above features of the gene-targeting event rather than of HS2–6 deletion per se, no Dad1 transcripts are detectably expressed by the mutant allele, and mice homozygous for the deletion display early embryonic lethality (7). Together, these complexities argue that additional gene-targeting strategies will be required to address the role of HS2–6 in unambiguous fashion.
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Acknowledgments |
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Footnotes |
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2 Current address: Wistar Institute, Philadelphia, PA 19104
3 Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, PO Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address:
4 Abbreviations used in this paper: E, enhancer; BEAD-1, blocking element -1; HS, hypersensitivity site; LCR, locus control region; P, promoter; Neo, neomycin resistance gene.
Received for publication January 29, 1999. Accepted for publication April 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ locus. Genomics 20:419.[Medline]
/C) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19:478.[Medline]
locus and expressed early in T-cell differentiation. Nature 327:677.[Medline]
/ locus impairs T-cell development and reveals the presence of the nearby antiapoptosis gene Dad1. Mol. Cell. Biol. 17:2151.[Abstract]
locus. Science 247:1225. chain gene: a T lineage-specific enhancer element is located in the J3-C intron. J. Exp. Med. 171:75. gene locus. Eur. J. Immunol. 21:807.[Medline]
in the human T-cell receptor locus. Proc. Natl. Acad. Sci. USA 86:6714. locus. EMBO J. 8:729.[Medline]
transgene. J. Exp. Med. 179:43. gene enhancers confer tissue- and stage-specificity on V(D)J recombination events. EMBO J. 12:4335.[Medline]
/ gene rearrangement by T cell receptor and enhancers. J. Exp. Med. 179:1913. enhancer in ß and 
T cells. Immunity 7:505.[Medline]
and gene segments within the human T cell receptor / locus. Proc. Natl. Acad. Sci. USA 94:5219./ locus. Immunity 1:207.[Medline]
chain intron enhancer abolishes chain gene rearrangement in cis but not 
chain gene rearrangement in trans. EMBO J. 12:2329.[Medline]
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