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Molecular and Cellular Biology, January 2000, p. 42-53, Vol. 20, No. 1
Centre d'Immunologie de Marseille-Luminy,
Institut National de la Santé et de la Recherche
Médicale-Centre National de la Recherche Scientifique, 13288 Marseille,1 and Laboratoire de
Génétique Médicale et Développement,
Faculté de Médecine, Institut National de la Santé et
de la Recherche Médicale, 13385 Marseille,2 France
Received 9 July 1999/Returned for modification 3 August
1999/Accepted 23 September 1999
V(D)J recombination in differentiating lymphocytes is a highly
regulated process in terms of both cell lineage and the stage of cell
development. Transgenic and knockout mouse studies have demonstrated
that transcriptional enhancers from antigen receptor genes play an
important role in this regulation by activating cis-recombination events. A striking example is the T-cell
receptor Immunoglobulin (Ig) and T-cell
receptor (TCR) genes are assembled from separate variable (V),
diversity (D), or joining (J) gene segments in a process known as V(D)J
recombination (4, 44, 55). Normally, V(D)J recombination is
restricted to, and required for, early B and T lymphocyte development.
It depends on a unique activity, called the V(D)J recombinase, which
targets recombination signal sequences (RSSs; consisting of a heptamer, a spacer of 12 or 23 bp, and a nonamer) flanking the rearranging sides
of all V, D, and J segments. Rearrangement events primarily involve
pairs of segments with RSSs of asymmetrical spacer length (12/23 rule).
The functional core of the V(D)J recombinase consists of the
lymphoid-restricted RAG-1 and RAG-2 gene products which recognize, pair
off, and cleave the RSSs from two rearranging segments, a step also
involving architectural proteins from the high-mobility-group family
(22, 61). These cleavages consist of DNA double-strand breaks (DSBs) introduced precisely at the junction between the heptamer
and adjacent coding sequences, yielding two distinct products: the
hairpin-sealed coding ends (CEs) and the phosphorylated, blunt-ended
signal ends (SEs). Eventually, the cleaved products are assembled
together, a process which requires, in addition to the RAG factors,
components of the general DNA DSB repair machinery (26, 54).
The two CEs are assembled to form a coding joint (CJ) on the rearranged
chromosome following hairpin opening and possible deletion and/or
addition of nucleotides at the free extremities. The SEs are fused
without further processing, yielding a signal joint (SJ) usually
contained within a circular piece of extrachromosomal DNA.
V(D)J recombination is strictly controlled with respect to the lymphoid
cell lineage, stage of cell differentiation, and allele usage. For
example, there is a strong bias for TCR gene rearrangement to occur
only in T cells (and for Ig gene rearrangement to occur only in B
cells). Also, during In studies using transgenic and knockout mouse techniques, it has been
demonstrated that transcriptional cis-regulatory elements affect the recombination potential of adjacent gene segments (29, 63, 67). One striking example has come from studies on the TCR
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Definition of a T-Cell Receptor
Gene Core
Enhancer of V(D)J Recombination by Transgenic Mapping
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ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-chain (TCR
) gene enhancer (E
), which in the mouse
consists of at least seven nuclear factor binding motifs (
E1 to
E7). Here, using a well-characterized transgenic recombination
substrate approach, we define the sequences within E
required for
recombination enhancer activity. The E
core is comprised of a
limited set of motifs (
E3 and
E4) and an additional previously
uncharacterized 20-bp sequence 3' of the
E4 motif. This core element
confers cell lineage- and stage-specific recombination within the
transgenic substrates, although it cannot bypass the suppressive
effects resulting from transgene integration in heterochromatic
centromeres. Strikingly, the core enhancer is heavily occupied by
nuclear factors in immature thymocytes, as shown by in vivo
footprinting analyses. A larger enhancer fragment including the
E1
through
E4 motifs but not the 3' sequences, although active in
inducing germ line transcription within the transgenic array, did not
retain the E
recombinational activity. Our results emphasize the
multifunctionality of the TCR
enhancer and shed some light on the
molecular mechanisms by which transcriptional enhancers and associated
nuclear factors may impact on cis recombination, gene expression, and
lymphoid cell differentiation.
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INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

thymic cell development, TCR
rearrangement initiates in CD4
CD8
double-negative (DN) cells prior to TCR
rearrangement which occurs
during the transition from DN to CD4+ CD8+
double-positive (DP) cells (37). Moreover, during TCR
rearrangement, D
-to-J
recombination usually precedes complete
V
-to-DJ
recombination (in DN CD44+ CD25+
and DN CD44
CD25+ thymocytes, respectively).
Finally, V
-to-DJ
joining is subject to allelic exclusion, a
process whereby productive rearrangement (e.g., those encoding a TCR
chain) on one allele precludes further V
recombination. It has
been hypothesized that these levels of control reflect the ability of
the individual loci and/or segments to serve as substrates for the
recombinase, a concept known as V(D)J recombinational accessibility
(63, 67). Recent evidence has revealed that recombinational
accessibility, as tested by specific DSB formation, is a regulated
property of lymphocyte chromatin (69). However, the
molecular mechanism(s) and structural basis underlying these phenomena
are not yet understood. Proposed activating factors include
transcription across the unrearranged (germ line) region, the
down-modulation of CpG methylation, and/or other epigenetic changes in
chromatin (e.g., level of histone acetylation) affecting nucleosome
positioning (13, 20, 54).
gene locus and the associated enhancer. This large locus (~500 kb in
the mouse) (Fig. 1A) carries two
homologous regions, each containing one D
segment (D
1 or D
2),
six functional J
segments (J
1.1 to 6 or J
2.1 to 6; one
additional pseudo-J is present in each J
cluster), and one constant
(C) region gene (C
1 or C
2). Most of the V
genes are located 5'
of the D
-J
clusters, except for one (V
14) oriented in the
opposite transcriptional direction 3' of C
2. A single
transcriptional enhancer (E
), contained within a 560 bp
HpaI-NcoI DNA fragment (E
560), has been found 5.9 kb downstream of the C
2 exons (38, 50). E
560
activates V(D)J recombination within a transgenic substrate in early
developing thymocytes from both embryonic and adult mice (12,
56). Moreover, deletion of the endogenous E
560 fragment by
using gene targeting in embryonic stem cells and production of mutant
mice results in a drastic inhibition of cis rearrangement of
the mutated TCR
alleles (5, 6), indicating that the
fragment contains regulatory sequences that are absolutely required for
V(D)J recombination to proceed normally at that locus. Finally,
detailed analyses of D
and J
SE intermediates as well as
D
-to-J
CJ and SJ products in thymocytes from mice homozygous for
the targeted deletion (E
/
mice) lead to
the surprising finding that loss of accessibility alone may not fully
account for the defect in recombination, suggesting an unsuspected role
for E
during the recombination reaction (30).

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FIG. 1.
Structural organization of E
and truncated E
variants used in this study. (A) Location within the TCR
locus and
structure of the 560-bp HpaI-NcoI DNA fragment
containing E
(not drawn to scale). The TCR
V, D, and J segments
as well as the C
exons are represented by shaded boxes, and E
is
represented by an oval. 5' and 3' indicate transcriptional orientations
of these various elements within the TCR
locus, with the exception
of the single V
14 gene located downstream of E
, which lies in the
opposite direction. An enlargement of the 560-bp enhancer (E
560)
shows the several nuclear factor binding sites (
E1 to
E7)
previously identified by EMSA within this fragment (71). (B)
Partial restriction endonuclease map of the microinjected inserts and
structure of the E
variants. The TCR
V, D, and J segments are
represented by open boxes, their flanking RSSs are represented by
shaded (23-bp spacer) or open (12- or 13-bp spacer) triangles, the IgH
Cµ exons are represented by shaded boxes, the cosmid sequences are
represented by hatched boxes, and E
is represented by an oval (these
various elements are not drawn to scale). Restriction endonuclease
sites are indicated as B (BamHI), Bg (BglII), Hp
(HpaI), Nc (NcoI), and R (EcoRI). The
structural organization of each E
variant is shown, including the
80- and 30-bp sequences which, in some variants, flank the 5' and/or 3'
sides of the
E1 and
E4 motifs, respectively (delineation of
E
motifs according to Takeda et al. [71]). Locations of
the V
14-hybridizing probe used in Southern analysis of
BglII-restricted genomic DNA from the transgenic mice and of
the 5.5-kb BglII-hybridizing fragment from the unrearranged
substrate are indicated.
Studies of mouse and human E
, using in vitro footprinting analysis
and electrophoretic mobility shift assays (EMSA), have identified
multiple nuclear factor binding sites along this element which are
conserved between the two species, supporting their functional
importance (25, 58, 71). Several such motifs, including
GATA- and helix-loop-helix (HLH)-binding E-box motifs, a cyclic AMP
response element-like sequence, and Ets and core-binding factor (CBF)
sites, are shared by other lymphoid gene enhancers and have been shown
to be required for E
activity (25, 31, 42, 59, 70, 71).
Accordingly, transcription factors known to bind discrete enhancer
motifs were also found to activate reporter genes placed under the
control of related E
sequences, including notably GATA-3
(47) and Ets as well as CBF (36, 59, 70, 78). In
addition, screening of cDNA expression libraries with oligonucleotide
probes spanning E
or related sequences contributed to the
identification of novel factors with homology to POU domain proteins
(51) or to CACCC box binding proteins (73).
Finally, signal transduction pathways involving raf and
ras were proposed to play a role in E
-mediated
transcriptional activation (79). Despite all of these
studies however, E
structural and functional organization remains
less well understood compared to regulatory elements from other
lymphoid genes such as the Ig heavy-chain (IgH) intronic enhancer or
the TCR
enhancer (23, 53). Notably, in contrast to these
other elements, a minimal core E
sequence has not yet been
unequivocally defined (25, 58, 71). Moreover, the functional
importance of individual motifs and their associated factors to act
nonredundantly as positive or negative elements is still unclear; such
motifs include GATA and Ets (31, 47, 59, 70).
In this study, we used transgenic mice to better characterize the
function of the E
element in vivo, focusing especially on its role
in the activation of germ line transcription and V(D)J recombination.
Notably, we have defined a core TCR
enhancer for recombination which
is comprised of two known nuclear factor binding motifs as well as
additional, previously unidentified sequences which we show are
absolutely required to support cis rearrangement of
adjacent TCR
gene segments.
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MATERIALS AND METHODS |
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Construction of recombination substrates.
All recombination
substrates were derived from the construct V
D
J
CµB*R*.
V
D
J
CµB*R* is identical to the V
D
J
Cµ construct (19) except that it lacks the BamHI and
EcoRI restriction sites located within exon 2 of the Cµ
gene and 5.3 kb further downstream, respectively. Both sites were
eliminated following BamHI and EcoRI partial
endonucleolytic cleavages, fill-in of the extremities, and religation.
This allowed direct cloning of the several E
variants, using the
adjacent BamHI and EcoRI sites located 130 bp 3'
of the J
1.2 gene segment in V
D
J
CµB*R*. The E
variants were produced by PCR amplification, using a 4.95-kb E
-containing genomic fragment subcloned into pGEM-4Z (Promega France,
Charbonnières, France) as a template (6), as well as
appropriate forward and reverse primers flanked by BamHI and
EcoRI sites, respectively. With position 1 assigned to the
first nucleotide in the HpaI site from the 560bp
HpaI-NcoI E
-containing fragment
(38), the various truncated variants of E
used in this
study were as follows: E
200 (+124 to +324), E
180 (+124 to +304),
E
169 (+155 to +324), E
149 (+155 to +304), E
98 (+226 to +324),
E
72 (+252 to +324), and E
3' (+300 to +560). Each E
fragment
inserted into the recombination substrate was verified by nucleotide sequencing.
Production of transgenic mice.
Linearization, purification,
microinjection of the individual DNA inserts into fertilized
(C57BL/6 × CBA/J)F2 oocytes, identification of the
transgenic founders, and production and maintenance of transgenic mouse
lines were performed as described previously (12, 18, 19).
The characteristics of the transgenic animals in lines 1003 (carrying
an enhancerless recombination substrate) and E
2 (carrying the
E
560-driven substrate, hereafter referred to as the E
560 line)
have been reported previously (12, 19). Nontransgenic
littermates in the E
560 line were used as the source of wild-type
(WT) genomic DNA and total RNA. All mice were housed in a
specific-pathogen-free animal facility in accordance with institutional
guidelines. Mice were sacrificed for analysis between 4 and 6 weeks of age.
Southern blot analyses.
Preparation of genomic DNA,
restriction enzyme digests, agarose gel electrophoresis, DNA blotting
onto nylon membranes (Hybond-N+; Amersham, Les Ulis,
France), preparation and [
-32P]dCTP labeling of the
V
14 probe, and hybridization procedures were performed as described
previously (12, 19). Images were generated from hybridized
filters by use of a phosphorimager (BAS 1000; Fuji, Raytest France
S.A.R.L., Courbevoie, France) and quantitated with MacBAS software.
DNA and RNA PCR assays.
Genomic DNA and total RNA were
simultaneously extracted from transgenic cell populations (~5 × 105 cells), using TRIzol (GIBCO BRL, Cergy Pontoise,
France) as recommended by the manufacturer. RNA samples were treated
with RNase-free DNase I (Pharmacia, Orsay, France) and were converted
to cDNA by reverse transcription (RT), using the SuperScript II reverse transcriptase (GIBCO BRL). Analysis of transgenic specific V(D)J rearrangements and J
germ line transcripts by, respectively, DNA
long-range PCR (LR-PCR and RNA RT)-PCRs were performed as described
previously (12, 18). LR-PCRs were performed for 25 cycles of
1 min at 94°C, 30 s at 56°C, and 2 min at 72°C; RT-PCRs were
performed for 28 cycles of 30 s at 94°C, 30 s at 58°C,
and 30 s at 72°C. Oligonucleotide primers for amplification of
fragments encompassing substrate D
-to-J
and V
-to-DJ
rearrangements and substrate J
1-IgH Cµ exons, as well as fragments
encompassing
-actin, CD14, and C
2 exons, were as described
previously (12, 30). Sequences of forward and reverse
primers for amplification of fragments encompassing endogenous
D
1-to-J
1.1/J
1.2 rearrangements were
5-CTGGTGGTTTCTTCCAGCCCT-3 and
5-CCTTCCTCTGATTACCAGAAC-3'. PCR amplification of endogenous
V
-to-J
2 rearrangements was performed as described by Schlissel
and Baltimore (62). After amplification, PCR products were
electrophoresed through 1% agarose-0.5% NuSieve gels, transferred to
nylon membranes (Hybond-N+; Amersham), and hybridized with
[
-32P]ATP-labeled locus-specific oligonucleotide
probes internal to the corresponding primers. Images were generated and
quantitated as described above.
Antibodies and fluorescence-activated cell sorting (FACS)
purification of lymphocytes.
Fluorescein isothiocyanate (FITC)-
and phycoerythrin-conjugated monoclonal antibodies (MAbs) against CD25
(7D4), CD44 (Pgp-1), CD3
(145-2C11), and B220 (RA3-6B2) were
purchased from PharMingen (San Diego, Calif.). Lymphocyte preparation
and cell staining with saturating levels of MAbs were performed as
described previously (10, 18). Cell sorting of peripheral T
and B cells was accomplished with anti-CD3
and anti-B220 MAbs. For
cell sorting of DN thymic cell populations, an initial depletion of
CD4+ and/or CD8+ cells was performed with
anti-CD4- and anti-CD8-containing supernatants and rabbit complement,
as described previously (33), before staining with anti-CD44
and anti-CD25 MAbs. Cell sorting was performed with a FACStar Plus
(Becton Dickinson, Mountain View, Calif.).
Fluorescence in situ hybridization (FISH) analysis. Metaphase spreads were prepared from transgenic splenic lymphocytes stimulated with concanavalin A (ConA) at 37°C for 72 h and cultured with 5-bromodeoxyuridine (60 µg/ml) added for the final 6 h of culture to ensure chromosomal R banding. Chromosome spreads were hybridized with a biotinylated 10-kb EcoRI fragment containing the Cµ exons (19), using standard protocols (49, 57). Biotinylation of the Cµ probe was carried out by nick translation using biotin-16-dUTP (Boehringer Mannheim, Meylan, France) as recommended by the manufacturer. Before hybridization, the biotinylated probe was annealed for 45 min at 37°C with a 200-fold excess of murine Cot-1 DNA (GIBCO-BRL) in order to compete with nonspecific repetitive sequences; 200 ng of the probe (at 10 µg/ml in the hybridization solution) was used per slide. The hybridized probe was detected with FITC-conjugated avidin (Vector Laboratories, Burlingame, Calif.). Chromosomes were counterstained with propidium iodide diluted in antifade solution (pH 11.0) as described previously (43). For each chromosomal spread, a total of 30 metaphase cells were analyzed. Over 85% of the cells showed, in addition to the two hybridization signals detectable at the distal end of chromosomes 12 (corresponding to the endogenous IgH loci), a specific signal corresponding to the transgenic site.
In vivo footprinting analyses.
The in vivo genomic footprint
analysis was performed by the dimethyl sulfate (DMS)/ligation-mediated
PCR (LM-PCR) technique (52) essentially as described by
Algarté et al. (1). Briefly, thymocytes from 4- to
6-week-old WT or RAG-1-deficient (RAG-1
/
)
mice (68) were harvested and subjected to DMS treatment, and the DNA was extracted. In parallel, DNA from cells of the same thymuses
was first extracted, deproteinized, and then subjected to DMS treatment
(naked DNA). The resulting DMS-treated DNA samples were cleaved by
pipiridine and subjected to LM-PCR. Specific oligonucleotide primers,
encompassing the E
sequence, were used to carry out a primer
extension reaction for both top and bottom strands. The resulting
double-stranded DNA was ligated to the LM-PCR unidirectional linker
consisting of two strands of dissimilar length: LM-PCR.1 (5'-GGGGTGACCC GGGAGATCTGAATTC-3') and LM-PCR.2
(5'-CTAGACTTAAG-3'). The linker-ligated DNA was
subsequently subjected to PCR using E
-specific oligonucleotide
primers internal to that used for the primer extension step and primer
LM-PCR.1 and a final two cycles of PCR with a third
[
-32P]ATP-labeled E
-specific oligonucleotide
primer. The resulting labeled PCR products were resolved on 5%
Hydrolink Long Ranger sequencing gels (TEBU, Le Perray en Yvelines,
France) and exposed to X-ray film. The primers used for the primer
extension, PCR, and labeling steps were, respectively, as follows:
bottom strand,
E1.1 (5'-GACCGATTCCATCAAAGAG-3'),
E1.2
(5'-GCAACTGAAGAGATGCATTCCTGGG-3') and
E1.3
(5'-GAGATGCATTCCTGGGACTTTTCGGTTCC-3'); top strand,
E2.1 (5'-TTAGAGACCCTCCTCTTGG-3'),
E2.2
(5'-GGTGATAGCTAGAGGCTGAGGTAGA-3'), and
E2.3
(5'-AGAGGCTGAGGTAGAAAGGGCTGCATGAG-3').
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RESULTS |
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Experimental design.
The E
560 mouse DNA fragment (E
560
[Fig. 1]) has been shown by in vitro footprinting and EMSA to include
seven nuclear factor-binding motifs, designated
E1 to
E7
(71). The aim of this study was to define which sequences
within E
560 are involved in targeting TCR
variable segments for
cis recombination and expression in vivo. Thus, we used a
well-characterized transgenic minilocus system in which substrate
rearrangements are strictly dependent on the presence of a
transcriptional enhancer (19, 66, 74). Briefly, the
minilocus (Fig. 1B) is comprised of the unrearranged V
14, D
1,
J
1.1, and J
1.2 gene segments (and flanking RSSs) in the same
transcriptional orientation such that substrate D
-to-J
and
V
-to-(D)J
rearrangements result in deletion of the corresponding intervening sequences; within these transgenes, the former
rearrangements can occur in both T and B cells, whereas the latter are
highly restricted to T cells. The TCR
region is linked to a
downstream DNA fragment containing the IgH Cµ gene, thus conferring a
hybrid structure to the minilocus that facilitates molecular analyses of the integrated transgenes. Truncated versions of E
were inserted between the TCR
and IgH regions in the minilocus, a location in
which the E
560 fragment was previously shown to activate cis recombination and transcription (12, 56). In
preliminary experiments using this transgenic approach, we found that a
200-bp fragment (E
200) comprised of sequences upstream of the
E5
motif (including the
E1 to
E4 motifs and 80 and 30 bp of 5' and
3' flanking sequences, respectively [Fig. 1B]) induces transgene rearrangements, whereas a downstream fragment containing the
E5,
E6, and
E7 motifs (E
3') is essentially inactive (G. Bouvier and P. Ferrier, unpublished data). The present study further dissects the recombinational activity of the 5' region of E
, using several truncated versions of E
200 as schematized at the bottom of Fig. 1B.
Recombination activity of the transgenic substrates.
The
linearized recombination substrates were used to produce transgenic
mice, utilizing standard protocols. The resulting transgenic founders
were backcrossed onto WT (C57BL/6 × CBA/J)F1 mice to
derive transgenic lines. At least three independent transgenic lines
shown to contain from 1 to 18 copies of the intact transgene were
established for each construct (Table 1).
Substrate rearrangements in the individual lines were first analyzed by
Southern blot assay using BglII-restricted genomic DNA from
transgenic thymuses and a V
14-specific hybridization probe to allow
for the identification of site-specific rearrangements within the
integrated transgenes, as previously described (19) (see the
legends to Fig. 1 and 2). DNA from a nonrearranging tissue (kidney) was
used as a negative control. In the case of the E
98 substrate, two
additional founders (E
98 D and E
98 E) were sacrificed and
similarly analyzed. Overall, these analyses demonstrated substantial
differences in the efficiency of V(D)J recombination among the various
transgenes, as judged by the presence or absence of thymus-specific
hybridizing bands, the sizes of which correspond to predicted
rearranged fragments within the transgenic substrates (Fig. 2; results
from all transgenic lines are reported in Table 1). Specifically,
substrate D
-to-J
rearrangement was clearly detected in thymus DNA
carrying the E
200 construct (in three of four lines), the E
169
construct (in all four lines), and the E
98 construct (in three of
five lines) but not in thymus DNA carrying the E
180, E
149, or
E
72 construct (three lines in the first two cases; four lines in the latter). When absent from thymus DNA, substrate D
-to-J
rearrangement was also not detected in DNA from other lymphoid tissues
such as the lymph nodes, spleen, and bone marrow (data not shown). Generally, substrate V
-to-(D)J
rearrangement was barely visible by this assay, including in mice exhibiting substrate D
-to-J
joints (Fig. 2). This may be due to the
fact that V
-to-(D)J
joining in T-lineage cells is frequently less
efficient within this recombination transgene (12, 18, 19,
56), coupled to the related sizes of the unrearranged and
V
-to-(D)J
rearranged hybridizing fragments (Fig. 2, legend; also
see the results from PCR analyses reported below).
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variants in activating rearrangement, we used specific LR-PCR assays as
described previously (12). Depending on the oligonucleotide primers used, the assays allow for amplification of transgene fragments
carrying either the unrearranged D
1, J
1.1, and J
1.2 segments
as well as the rearranged D
1-to-J
1.1 and D
1-to-J
1.2 products or the rearranged V
14-to-(D)J
1.1 and
V
14-to-(D)J
1.2 products, respectively. Representative data are
shown in Fig. 3, top and middle panels;
overall results are reported in Table 1. Significantly, amplified
fragments containing D
-to-J
or V
-to-(D)J
rearrangements
were readily detected in thymus DNA from transgenic mice in the same
E
200, E
169, and E
98 lines that showed substrate recombination
in Southern analyses. Conversely, transgene rearrangements were barely
visible in thymus DNA from transgenic mice in the E
180, E
149, and
E
72 lines (within this group, maximum levels of transgene
rearrangements were observed in the 18-copy line E
149:I [Fig. 3]).
By comparing thymus DNA from transgenic mice of these three types of
lines to serial dilutions of thymus DNA from a rearranging E
200:6t28
transgenic mouse, we estimated that there was an overall 100-fold
reduction in the levels of D
-to-J
and V
-to-(D)J
recombination per copy number in the E
180, E
149, and E
72 lines
[an 18-fold reduction for V
-to-(D)J
rearrangement in the
particular line E
149:I; see the legend to Fig. 3 for calculation].
A similar analysis and calculation indicated that substrate
rearrangements in the E
200 and E
169 lines were roughly equivalent
(when copy number is taken into account, the highest level of
rearrangement is actually found in line E
169:B), whereas they were
reduced two- to threefold in the E
98 lines compared to the other
two. Collectively, these results strongly suggest that when observed,
the recombinational activity is not due to the mere polymerization of
nuclear factor binding motifs in the transgenic array (the synergistic
effect of multimerization of transcription binding sites has been well documented in transfection studies [for example, see reference 15]). Instead, we conclude that a short (98-bp)
region of E
, comprised of the
E3 and
E4 motifs as well as an
additional 30 bp on the 3' side of
E4, is sufficient to promote
V(D)J rearrangement within the integrated minilocus.
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Transcription activity of the transgenic substrates.
A host of
studies, including transgenic analyses of various forms of
recombination substrates, have shown that transcription of germ line Ig
and TCR gene loci and/or segments tightly correlates with their
activation for V(D)J recombination during lymphoid cell development
(29, 67). However, a few examples which support a possible
dissociation between transcriptional and recombinational activities
have been reported (2, 18, 35, 40, 56). Using a previously
described RT-PCR assay (12), we analyzed germ line
transcription through the unrearranged J
segments in the various
integrated transgenes. High levels of germ line J
transcripts were
found in thymus RNA from the rearranging E
200, E
169, and E
98
as well as nonrearranging E
180 and E
149 transgenic mice (Fig.
4A and Table 1). Among these lines,
levels of J
transcripts in thymus RNA varied no more than threefold
according to quantification analysis (Fig. 4 and data not shown).
However, when accounting for the number of nonrearranged copies of the
transgene in the different lines (calculated following densitometric
scanning of Southern blots of thymus DNA similar to those in Fig. 2),
we found that levels of J
transcripts from the nonrearranging lines
were generally the lowest. For example, J
transcription in line
E
180:A was calculated to represent ~20% of that found in the
E
200:6t28 line, whereas levels of transcription in the other two
rearranging mouse lines shown in Fig. 4A were somewhat higher (see
figure legend for details). Thus, the difference between the levels of germ line transcription between line E
180:A and the rearranging lines is relatively modest. A somewhat different picture was found in
assays using thymus RNA from the nonrearranging E
72 transgenic animals. Although J
transcripts could generally be detected, they
were produced at significantly lower levels, exhibiting variations between transgenic animals in the same litter, including some that had
no detectable J
transcription (Fig. 4B, Table 1, and data not
shown). Compared to serial dilutions of thymus cDNA from a
nonrearranging E
180:A transgenic mouse, we estimated the level of
J
transcripts per transgene copy to be decreased 10- to 25-fold, depending on the E
72 line studied (i.e., 0.8 to 2% of J
transcription in the E
200:6t28 line [Table 1]). These data
indicate that all of the truncated E
variants tested in this study
except E
72 are efficient in stimulating J
transcription within
the integrated transgene.
|
180 subfragment activates germ
line transcription but not V(D)J recombination within the transgenic
array. To verify whether transcription is activated in early-developing
thymocytes, we purified DN CD44+ CD25+ and
CD44
CD25+ thymocytes from a transgenic mouse
of line E
180:A by FACS and analyzed their RNA for the presence of
J
transcripts. Purified thymocytes from transgenic mice of lines
E
98:C and E
560 were tested in parallel (DN CD25+
thymocytes in the E
560 line were previously shown to carry transgene recombination [12]). Thus, J
transcripts were
detected in both CD44+ CD25+ and
CD44
CD25+ DN subpopulations from all tested
mice (Fig. 4C). In separate analyses using LR-PCR and genomic DNA from
the same sorted cells, we have confirmed that substrate D
-to-J
rearrangements are not found in the E
180:A transgenic mouse, in
contrast to the E
98:C and E
560 mice (data not shown). We conclude
that substrate J
transcription is activated in the E
180
thymocytes at a developmental stage where TCR
gene recombination
normally takes place.
Effect of transgene integration on enhancer activity.
The
integration of transgenes into the mouse germ line following pronuclear
injection of DNA usually results in an array of tandemly repeated,
head-to-tail copies at a single chromosomal site. Depending on the site
of chromosomal integration, side effects which generally have a
negative effect on transgene expression can result (48). For
example, it has been suggested that silencing of transgene expression
could be a consequence of the spreading of centromeric heterochromatin
within the nearby integrated transgenic array, a mechanism analogous to
position effect variegation (PEV) in drosophila (7, 16, 21).
Among the several E
reporter transgenes which we found to rearrange
and/or be expressed in a majority of independent transgenic lines,
there were a few in which both transgene recombination and
transcription could not be detected (e.g., Table 1, lines E
200:4t26,
E
98:A, and E
98:B). Since the copy number was stable and there was
no evidence of structural alteration of the transgenes within these
lines (R. K. Tripathi, M.-G. Mattei, and P. Ferrier, unpublished
data), it seemed possible that the transgenic arrays had been silenced for expression following integration within or close to
chromosomal centromeres. To test this hypothesis, we performed
FISH analysis on spread chromosomes from ConA-stimulated
splenocytes in transgenic animals of the nonrearranging
E
200:4t26 and E
98:B lines, using as a probe the IgH Cµ
DNA fragment (~10 kb) present on the 3' side of the recombination
substrates. The rearranging lines E
200:6t28 and E
98:C were
similarly analyzed. Under these conditions, the probe is expected to
hybridize to the endogenous IgH loci, thus labeling the telomeric
region of the two homologous chromosomes 12, as well as to the
transgenic array, leading to an additional spot located on an
unpredictable chromosome. In agreement with our hypothesis, we found
that the transgenic arrays in lines E
200:4t26 and E
98:B are
located within the centromeric region of, respectively, a large and a
medium-sized chromosome (Fig. 5, left
panels). Significantly, in both cases, a signal (corresponding
presumably to the transgenic substrates) is associated with
heterochromatin visible as condensed areas in interphase nuclei (Fig.
5, right panels). In marked contrast, the transgenic signal in line
E
200:6t28 is located in the middle part of a medium-sized
chromosome, distant from any possible effect of centromeric
heterochromatin (bottom panels); a similar image of chromosomal midarm
integration was observed in transgenic splenocytes from line E
98:C
(data not shown). As expected, in all cases, fluorescent signals are
visible at the distal end of both chromosomes 12, corresponding to the
endogenous IgH loci.
|
E
98 confers lineage and temporal specificity to transgene
recombination.
The minimal TCR
enhancer fragment that was found
to activate transcription and recombination within the transgenic
substrates integrated at sites distant from centromeric heterochromatin
was E
98. To test whether this element does so in a lineage- and
development stage-restricted manner, we analyzed D
-to-J
rearrangement by LR-PCR using genomic DNA prepared from sorted
populations of T and B peripheral lymphocytes or DN CD44+
CD25+ and DN CD44
CD25+
thymocytes from transgenic mice of line E
98:C. Genomic DNA from the
corresponding cells purified from E
560 transgenic mice was similarly
tested. Previously, we found that transgene rearrangement in this line
is T-cell specific and occurs early during T-cell ontogeny and
differentiation (12). Significantly, fragments containing
substrate D
-to-J
rearrangements were found to predominate in
purified T compared to B cells from the E
98:C mouse, similar to the
recombination profiles observed with a transgenic mouse from line
E
560 (Fig. 6A, top panel). Additional
LR-PCR assays to analyze fragments containing endogenous D
-to-J
and V
-to-J
rearrangements confirmed the T- and B-lineage origin
of the sorted cells (Fig. 6A, middle panels). Moreover, D
-to-J
recombination products were detected in purified DN CD25+
thymocytes from both E
98 and E
560 mice, again with profiles related to those observed for endogenous TCR
gene rearrangement (Fig. 6B). Together with our finding of germ line J
transcripts in
DN thymocytes from the E
98:C mice (see above), these data strongly
suggest that E
98 represents a true TCR
core enhancer for
recombination, as it appears to confer appropriate cell lineage and
temporal specificity.
|
Developing thymocytes show nuclear factor occupancy within
E
98.
The E
98 core enhancer defined in this study contains
the previously identified
E3 and
E4 nuclear factor binding sites
as well as additional sequences 3' of
E4.
E3 contains a typical E-box motif, while
E4 has been shown to be comprised of a composite Ets-CBF binding site (36, 70, 71, 78). Moreover, examination of the
E4 3' flanking sequences by using the TRANSFAC database of
transcription factor binding sites (76, 77) revealed that this region also contains an E-box motif (Fig.
7). To test whether the E
98 element is
bound by nuclear factors in vivo, we analyzed enhancer occupancy within
the endogenous TCR
locus by genomic footprinting using LM-PCR
(52). This technique can reveal nuclear factor binding in
chromosomal DNA following comparison of LM-PCR products between DNA
which is treated in vivo with the membrane-permeable DNA-methylating
agent DMS and that treated in vitro after extraction (thus generating
the complete pattern of G residues across the given region). This is
visualized as either protected G's or hypersensitive G's and A's for
the in vivo versus in vitro DMS-treated DNA. The LM-PCR strategy was
adapted to analyze the 250-nucleotide (nt) region spanning from 5' of
E1 to 3' of
E7. In addition to WT thymocytes, we analyzed
thymocytes from RAG
/
mice. Whereas the
former are comprised of mostly DP cells carrying a productively
rearranged TCR
locus, the latter are made up of DN cells arrested in
development at a stage where TCR
gene rearrangement normally takes
place (e.g., CD25+) (37). The endogenous locus
was chosen for this analysis instead of the transgenic loci because of
technical difficulties encountered in specifically amplifying the E
region from the flanking A-T-rich IgH sequences in the integrated
substrates (R. K. Tripathi, D. Payet, S. Spicuglia, W. M. Hempel, and P. Ferrier, unpublished data).
|
region demonstrated one strong
footprint for both top and bottom strands (Fig. 7). For the top strand,
there was strong occupancy at
E4 visualized as a series of protected
G's and several hypersensitive A's for both WT and
RAG
/
thymocytes (Fig. 7, top panel). As
there are no G's in the
E4 motif on the bottom strand, there was no
apparent footprint in this region (data not shown). On this strand,
however, there was evidence of strong occupancy of
E6, outside the
E
98 region, visualized as a series of protected G's flanking a
hypersensitive A (Fig. 7, lower panel). In addition to the strong
occupancy observed at
E4 and
E6, there was also evidence for
occupancy at
E3, suggested by the presence of a single
hypersensitive A within this element on the top strand. Finally, there
was no evidence of nuclear factor binding throughout the rest of E
,
including the sequence 3' of
E4 (Fig. 7 and data not shown). These
results demonstrate that the
E3 and
E4 sites within the E
98
core are occupied in vivo, in agreement with our functional data. In
addition, the results strongly suggest that (i) nuclear factor
occupancy at E
is established in DN cells (as represented by
RAG
/
thymocytes) and (ii) this occupancy is
not significantly modified in more developed DP cells (as represented
by WT thymocytes).
| |
DISCUSSION |
|---|
|
|
|---|
Previous genetic analyses demonstrate a crucial role of E
in
regulating TCR
gene recombination and, in addition, suggest a
possible dual function for the control of both chromatin structure and
V(D)J recombination at the TCR
gene locus (5, 6, 12, 30,
56). These functions are most likely mediated by the combined action of nuclear factors bound to E
in developing T cells.
Provocatively, several of the identified E
-binding factors also
interact with Ig and other TCR enhancers (39, 42), which
were similarly demonstrated to affect V(D)J recombination at their own
loci (29, 67). Despite these findings, however,
gene-targeting studies have so far failed to reveal an indisputable
role for the relevant factors in regulating V(D)J recombinational
accessibility (14). This may be due to either a redundancy
between members of a given transcription factor family or a pleiotropic
role of the inactivated factor in cell differentiation and/or survival.
The definition of minimal cis-regulatory elements required
to modulate the recombination of linked gene segments may, in addition
to providing molecular clues as to the mechanism(s) involved,
constitute an alternative approach to address the issue of which
trans-acting factors are critical for this process.
We have shown that a 98-bp domain from the 560-bp E
-containing
fragment, comprised of two well-defined nuclear factor binding motifs
(
E3 and
E4) and a 30-bp flanking sequence 3' of
E4, is
sufficient to promote germ line transcription and V(D)J
recombinase-mediated rearrangements within an enhancer-dependent
recombination substrate integrated into the mouse genome in several
transgenic mouse lines. An overlapping 72-bp region that lacks the
upstream
E3 motif was found to be uniformly inactive when tested in
this system, whereas longer E
subfragments containing additional
E1 and
E2 motifs but lacking most of the sequences 3' of
E4
(e.g., E
180 and E
149; both subfragments contain only 10 bp 3' of
E4) were relatively efficient in activating germ line transcription
but not recombination. These data indicate that the E
98 domain
represents a TCR
core enhancer for V(D)J recombination and point
toward a novel E
sequence that plays a critical role in this
process. The implications of these results, in terms of the molecular
mechanisms and nuclear factors involved, are discussed below.
The V(D)J recombination-enhancing E
subfragments identified in this
study, including the E
98 core, promoted rearrangement and germ line
transcription from the integrated transgenes in several independent
transgenic mouse lines, in conformity with the correlation between the
two activities generally observed at antigen receptor gene loci
(20, 67). In a few instances, however, we found that some
E
subfragments (e.g., E
200 and E
98) were not active after
transgene integration within or close to a chromosomal centromere. Most
likely, the subfragments could not antagonize the repressive effect of
centromeric heterochromatin structures. In a study investigating the
function of two enhancer elements, the 5' HS2 enhancer from the human
-globin locus and the metallothionein enhancer, it was found that
instead of regulating the level of expression of a linked reporter
cassette, both enhancers act in cis to protect constructs
from repression by flanking chromatin and to suppress PEV
(72). Strikingly, however, the strength of this effect was
shown to depend on the site of integration, indicating that as in PEV,
chromatin varies in its ability to repress activity in a given gene
unit. These data support a model in which enhancers (and their bound
factors) directly interact with repressive chromatin structures to
permit and stabilize expression rather than to increase the rate of
transcription. While E
activity of the subfragments is suppressed by
centromeric heterochromatin, the possibility remains that it can
overcome the repressive effects of chromatin regions characteristic of
highly regulated loci, such as the TCR
locus, as opposed to those
characteristic of housekeeping genes, somewhat analogous to the
situation just described. We anticipate a similar mode of action for
other recombination enhancers; already, the Ig µ enhancer core has
been shown to establish factor access in chromatin independent of
transcriptional stimulation (34).
Unexpectedly, early-programmed J
transcription (e.g., in DN
CD25+ thymocytes) was detected within transgenes carrying
the rearrangement-defective E
180 subfragment, suggesting that this
element could also confer chromatin accessibility to the transgenic
templates in a significant proportion of cells undergoing TCR
gene
rearrangement. How then could the V(D)J recombinase apparently ignore
the homologous gene segments in the particular transgenes? A trivial
explanation would be that significant differences in accessibility
still exist between rearranging and nonrearranging loci, despite the
modest difference in germ line transcription, and/or that a higher
threshold of chromatin access is required to permit recombination as
opposed to germ line transcription. Alternatively, germ line
transcription in the E
180-containing transgenes may be qualitatively
inadequate, for example, not being initiated at the promoter upstream
of D
1, an element which has been shown by gene targeting to be
required for rearrangement of this gene segment (75).
Finally, in line with our recent report of unresolved DSBs at RSSs from
enhancer-deleted TCR
alleles, it is also possible that specific
nucleoprotein complexes organized at E
regulatory elements may
affect the V(D)J recombination process at a step beyond that of
chromatin access (30). Of note, two potential functions of
E
, increasing chromatin accessibility and affecting directly the
course of the recombination reaction, may not be mutually exclusive. If
this were the case, it would provide an explanation for the apparent
paradox of J
transcription in the absence of recombination in the
E
180 transgenic mouse lines. Current analyses of recombination
intermediates and germ line transcripts in lymphoid cells from
transgenic mouse models, including some established on a
RAG
/
background, may help us to distinguish
among these possibilities. In any case, our data add to the increasing
experimental evidence concerning transgenic and endogenous TCR loci,
suggesting that transcription per se is not sufficient to permit
rearrangement of the given gene segments (5, 18, 45, 56).
It is believed that all lymphoid cells are derived from a common
lymphoid cell progenitor (65). As they differentiate, the developing lymphocytes progressively lose their multilineage potential. In the rearranging E
98:C line, transgene D
-to-J
rearrangements were found to predominate in T as opposed to B lymphocytes.
Accordingly, these products were readily observed in DN
CD44+ CD25+ thymocytes, a subpopulation which
contains 
(and 
) T-cell precursors but can no longer give
rise to B cells (65). Overall, transgene rearrangement
profiles within the E
98:C line (as well as within the E
560 line
[Fig. 6 and reference 12]) mimic those at the
endogenous TCR
locus (11, 24, 46). Coupled to the fact
that the reporter transgenes in the E
98:C and E
560 lines are
located on different chromosomes (Tripathi, Mattei, and Ferrier, unpublished data) thus presumably within independent genomic contexts, these data strongly suggest that the regulatory elements within the
E
core act positively in regulating cis recombination,
independently of the action of adjacent motifs o