By
From the * Department of Pathology, and the Biostatistics Unit, Comprehensive Cancer Center,
University of Alabama at Birmingham, Birmingham, Alabama 35233-7331
Control of the rearrangement and expression of the T cell receptor and
chains is critical for
determining T cell type. The process of
deletion is a candidate mechanism for maintaining separation of the
and
loci. Mice harboring a transgenic reporter
deletion construct show
/
T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed
HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement
control of transgenic elements, with abundant rearrangements of transgenic
-deleting elements now in
/
T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active
rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with
deletion playing an important role in maintaining separate TCR
and
loci.
Two types of mature T cells possessing different tissue
distributions and apparent functions have been described (1). These T cells have TCR During the cloning and sequencing of the human TCR
A model was proposed in which Because In this report, we extend the observations with these
transgenic molecular tags, and identify a DNA binding complex and DNA recognition motif that are candidates for
controlling the lineage-specific use of the PCR.
Nonquantitative PCR was carried out essentially as
described (22) using 0.5 µg of genomic DNA per reaction tube.
The primers for all PCR reactions are listed in reference 21. Parameters for amplification of human Transgenic Mouse Construction.
The human transgenic reporter
construct (TG)2 was identical to TG1 as described (21), except for
a 50-bp deletion within the 1.9-kb SalI to XhoI fragment, which
was subcloned into pBluescript (Stratagene Corp., La Jolla, CA)
and an oligonucleotide spanning this region, but that did not
contain the 48-bp potential signal sequence region HPS1A,
which was used to make the deletion by the oligonucleotide-directed site-specific mutagenesis method of Kunkel (23). A sequence confirmed deletion mutant was isolated, and the 1.85-kb
SalI-Xho I HPS1A minus fragment was subcloned into the base
transgene construct used to make TG1. PCR was used to confirm that isolated transgenic constructs contained the 48-bp deletion (data not shown). Transgenic founder lines were created by
injecting construct DNA into day 1 fertilized embryos by the University of Alabama at Birmingham (Birmingham, AL) transgenic mouse facility.
Isolation and Expansion of Extracting DNA for PCR.
Single cell suspensions from thymus or spleen were spun in eppendorf tubes at 1-2,000 g. Nuclei
were prepared by 1% triton lysis, centrifuged, and solubilized in
1% SDS. DNA was cleaned by overnight digestion with proteinase K and phenol extraction. Liver DNA was prepared as described (22).
Nuclear Extract Preparation.
Nuclear extracts were prepared
essentially as described (25). For human thymus, a single cell suspension of thymocytes was made with a Dounce homogenizer in
nuclear extract buffer I (15 mM Hepes, pH 7.6, 10 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, 1 mM DTT, 10 mM Na2S2O5, and 2 mM
PMSF). After removing large debris and centrifugation, cells were
resuspended in 20 ml buffer I. Nuclei were prepared by passing
cells through a 27-guage needle five times with high velocity.
The nuclei were centrifuged and resuspended in 10-20 ml buffer
II (15 mM Hepes, pH 7.6, 115 mM KCl, 5 mM MgCl2, 0.1 mM
EDTA, 1 mM dithiothreitol (DTT), 10 mM Na2S205, and 2 mM PMSF). A 10% volume of 4M (NH4)2SO4 was added to solubilize
the nuclei, and the mixture rocked gently at 4°C for 30 min.
DNA was spun out at 30,000 g for 1 h at 4°C. For each milliliter
of the supernatant, 0.3 g solid (NH4)2SO4 was added and the solution rocked gently for 15 min at 4°C. The precipitate was centrifuged at 10,000 g for 20 min and resuspended in 3-5 ml of
buffer III (25 mM Hepes, pH 7.6, 50 mM KC1, 12.5 mM
MgCl2, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol). The
extract was dialyzed against buffer III and stored at Cell Lines.
Cell lines used for extract preparation were pre-T
cells RPMI 8402 (26; gift of Stan Korsmeyer, Washington University, St. Louis, MO), CCRF-CEM (No. CCL 119; American
Type Culture Collection, Rockville, MD), and 2052 (27; gift of
Michael Lieber, Washington University, St. Louis, MO); pre-B cell
line Nalm-6 (28; gift of Stan Korsmeyer); mature B cell line SU-DHL-6 (29; gift of Stan Korsmeyer); HELA cells (No. CCL2;
American Type Culture Collection); and the erythroid leukemia line
MEL (gift of Tim Ley, Washington University, St. Louis, MO).
Mature B cells and T cells (unseparated tissues) were isolated as
single cell suspensions from BALB/c mice of 3-6 mo of age. Line
38B9 (27) for in vitro recombination assays was a gift of John
Kearney (University of Alabama at Birmingham, Birmingham, AL).
Mobility Shift Assay.
The mobility shift assay used was a modification of Heberlein and Tjian (25). In brief, the desired probe
was end labeled with 32P, ethanol precipitated, washed extensively
to remove unincorporated nucleotides, and counted. Reactions are
performed in 1.5-ml eppendorf tubes by adding 10,000-50,000
cpm of probe (1-2 ng DNA) in 1-2 µl, 1.5 µl of 10 × gel shift
buffer (40% glycerol, 10 mM EDTA, pH 8.0, 100 mM 2-mercaptoethanol, 100 mM Tris, pH 8.0, 500 mM NaCl), 2.5 µl 200 ng/µl poly dI.dC, x µl extract, and 10-x µl H2O in a total volume of 15 µl. The reaction mixture was allowed to sit at room
temperature for 30 min. A 4% nondenaturing acrylamide gel was
used for fractionation of products in a running buffer of 1× TBE.
Gels were run for 1-2 h at 100-150 volts. After electrophoresis, gels were transferred to Whatman 3-MM paper and dried under
vacuum. Autoradiography was performed for 6-12 h.
In Vitro Recombination Assay.
Plasmids pLH103 and pLH104
were created by replacement subcloning the heptamer-spacer-nonamer (h-s-n)1 of Table 4.
Quantitation of In Vitro Rearrangements
and
chains or
TCR
and
chains, associated with T3. Molecular cloning
and sequencing of all four TCR chains led to the discovery
of the unique localization of the TCR
chain on chromosome 14 in both mice and humans (10). The surprising feature of this locus is that its internal coding segments, D, J, and C
, are contained within the much larger TCR
chain locus. This unusual feature among rearranging loci in
the immunoglobulin supergene family leads to the conundrum of how to regulate the recombination of the
and
chain gene segments. In spite of the proximity of the two
receptors, cross-utilization of segments between the receptors infrequently occurs (17, 18). These two receptor chains
are expressed in a mutually exclusive manner in different
subsets of T cells, suggesting that separate alleles do not behave independently. Furthermore, in
/
T cells, TCR
chain recombinations would delete the functional
chain
and could not be allowed. These results imply a mechanism
to ensure that in
/
T cells,
gene rearrangement is inhibited and in
/
T cells, internal
gene segments are not
used.
chain, a novel rearrangement in early thymocytes was described that is a candidate mechanism for discrimination
between using the TCR
versus TCR
chains. This
deletion rearrangement step occurs in immature thymocytes destined to become
/
-bearing T cells. An upstream
-deleting element,
REC, preferentially recombines with
a downstream
-deleting element,
J
, at very high frequency in polyclonal human thymus (13, 19), and to a
lesser degree in murine thymus (20). This recombination
deletes all internal
coding segments, and occurs with high
frequency on both alleles in mature
/
T cells, suggesting
that the recombination would lead to subsequent rearrangements in the remaining TCR
chain locus.
deletion is a preliminary step in the formation of TCR
chains (19, 21). Use
of the
deleting elements would be an intermediary step in
the implementation of a signal to rearrange and express
TCR
chains, thus becoming an
/
-bearing T cell. This
model does not dictate which receptor rearranges first,
or
, nor in which order they rearrange. It only requires that
deletion, when it occurs, occurs before V
-J
joining.
The model would also necessitate that
-deleting element recombinations would be restricted to the
/
T cell lineage, for obvious reasons.
REC was frequently deleted on both alleles in
mature
/
T cells, (13, 19, 20) assessing mature T cells for the occurrence of
deletion was unproductive. Therefore,
a
deletion transgenic reporter construct was designed to
molecularly "tag" mature
/
and
/
T cells for the occurrence of
deletion (21). The construct contained the 5
and 3
-deleting elements, as well as some intervening
TCR
chain J and C segments. Since the transgene was
independent of the endogenous
/
locus, the status of
transgenic
-deleting elements could be assessed in
/
and
/
T cells. Analysis of mature transgenic
/
T cells demonstrated a high degree of rearrangement within this
transgenic reporter construct, whereas mature transgenic
/
T cells were essentially devoid of
-deleting element
rearrangements (21). This lineage-specific use of
-deleting
elements suggested that the machinery responsible for
deletion is present in developing
/
T cells and is absent
from the
/
T cell population, supporting the model for
deletion in the formation of
/
T cells. Furthermore, the
transgenic reporter approach allows the manipulation of
discrete DNA segments in the hope of identifying molecular mechanisms controlling the use of transgenic elements.
-deleting elements.
REC-J
x were 94°C for 30 s,
55°C for 1 min, and 72°C for 30 s for 30 cycles. Internal human
REC probe was obtained by PCR amplification of cloned plasmid DNA with primers internal to those used for detecting rearrangements of transgenic human
REC. PCR product blots,
representing 20% of reaction volume, were electrophoresed in
1% agarose gels, transferred, and hybridized as described above for
high molecular weight DNA. Quantitative PCR for
REC-J
x rearrangements using competitor H
REC1 was as described (21).
/
T Cells.
/
T cells were isolated from the thymus and/or spleen of four animals at a time.
Single cell suspensions of thymocytes or splenocytes in DMEM
were isolated on 100 × 15-mm petri dishes (Fisher Scientific,
Pittsburgh, PA) that had been coated with the anti-
TCR antibody (403A10; supplied by Osami Kanagawa, Washington University, St. Louis, MO). Plates were washed to remove remaining
/
T cells and immature T cells. DMEM with 10% fetal calf serum and 50 IU/ml rIL-2 was added, and the plates were incubated for 5-7 d. At this time, the cells were no longer adherent to
the plates. Cells were collected, nuclei prepared (see extracting DNA for PCR below), 2 mg proteinase K was added, and the solution incubated at 52°C overnight. Extraction, precipitation, and
analysis was as described (24).
80°C.
REC into the BamHI site of pJH299 (30).
Oligonucleotides flanking the h-s-n of
REC with BamHI-clonable ends were used to generate BamHI-clonable DNA segments by
PCR from cloned
REC DNA. For pLH103, the 5
oligonucleotide was positioned to also include HPS1A in the PCR fragment;
for pLH104, the 5
primer was between HPS1A and the h-s-n.
Subcloning each PCR product into the BamHI site of pJH299
resulted in a swap of recombination signals with 23-bp spacers, and
like pJH299, pLH vectors rearrange by inversion. Each newly generated plasmid was confirmed by sequencing. The in vitro recombination system of Hesse et al. (27, 31) was performed essentially as described. In brief, 150 ng of plasmid was introduced into
2052 or 38B9 by osmotic transfection using dextran sulfate for 5 min at 37°C. Transfected cells were washed and then allowed to
grow for 48 h in DMEM with 10% FCS for 2052 or RPMI with
20% FCS for 38B9, and then the plasmid DNA was harvested by
alkaline lysis as described (32) except that the precipitation step
after addition of acetate solution III was extended to 60 min. Before
transforming into bacteria for selection, the plasmids were digested with DpnI to ensure that only the plasmids that actually replicated within the mammalian cells were tabulated (27). The DpnI
digested plasmids were electroporated into DH5
cells using a manipulator (BTX Electrocell Manipulator 600; BTX, San Diego,
CA) at the following settings: T = 2.5 kV/resistance high voltage,
R5 (129 ohm), S = 1.3
1.5 kV, C not used in this mode, cuvette gap was 1 mm. 1-10% of the transformation mixture was
plated onto 100 µg/ml ampicillin (AMP) LB plates, and the remainder of the transfection mixture was plated onto 100 µg/ml ampicillin + 11 µg/ml chloramphenicol (AMP-CAM) LB plates.
Colonies were counted after 24 h from the AMP plates and 48 h
from the AMP-CAM plates. Rearrangement frequencies were
tabulated by comparing the number of replicated, unrearranged
plasmids (AMP plates) to replicated, rearranged plasmids (AMP-
CAM plates). To confirm that the colonies appearing on the
AMP-CAM plates were true recombinants, 51 of pLH104 and
69 of pLH103 AMP-CAM-resistant colonies were sequenced.
88% of pLH104 AMP-CAM colonies and 74% of pLH103
AMP-CAM colonies were found to be true rearrangements. The
AMP-CAM data in Table 4 has been adjusted by, and the statistical analysis reflects, the estimated number of recombinants based
upon the sequencing results. Based upon the estimated frequency
of rearrangements of 88 and 74%, respectively, and the number of
rearrangements sequenced, we have 90% confidence of <10% error in the estimated rearrangement frequency.
Cell line
Colony status
Plasmid
Fold increase
pLH103/pLH104
pLH103 (+ HPS1A)
pLH104 (
HPS1A)
38B9
No. recombined* (AMP-CAM)
185
75
(pre-B)
Total Dpnl-resistant colonies (AMP)
34,407
55,353
Combined
2.90
2052
No. recombined* (AMP-CAM)
119
98
(pre-T)
Total Dpnl-resistant colonies (AMP)
19,151
32,455
*
69 pLH103 colonies and 51 pLH104 colonies were sequenced to obtain an estimate of the number of true recombinants (see Materials and Methods).
Significant by logistic regression model to P <<0.00001.
Statistical Analysis.
To assess the effect of cell type and plasmid type on rearrangement rate, logistic regression was used. The
appropriateness of the model is established by likelihood ratio test
(P = 1032) and that both plasmid type and cell type are significant factors in the model (P = 8 × 10
29 and P = 3 × 10
5, respectively). From this model it was found that plasmid pLH103 is
2.90 times more likely to rearrange than plasmid pLH104 (95% C-I for odds ratio 2.40-3.49) and B cells are 1.47 times more likely to rearrange than T cells (95% C-I for odds ratio 1.23- 1.76). Statistical analysis of Table 3 for TGl and TG2 T cells populations with sample sizes of two and three, respectively, is not
appropriate.
One possible mechanism for regulating lineage-specific use
of the -deleting elements would be control of the process
by sequence-specific DNA binding proteins. Any such
protein(s) would be responsible for recognizing DNA elements near
REC, and, by DNA-protein or protein-protein interactions, regulate the use of
REC. Evidence for
the role of any such protein(s) in
deletion would be confirmed by constructing a second transgene identical to TG1
(21), but lacking the DNA recognition sequence.
A candidate region for controlling lineage specificity in
TG1 was discovered by first comparing sequences between
human and murine REC. The rationale was to search for
areas of high DNA homology between
RECs, and then
look for DNA binding proteins in the developing T cells of
the thymus. Two areas of very high sequence homology,
outside of the h-s-n canonical recombination motif, were
noted 5
and 3
to the recombination signal (Fig. 1 a; 20).
Nuclear extracts from murine thymus were used as probes to determine reactivity by mobility shift assay to both regions. DNA binding proteins were found in murine thymus to a 97-bp upstream region, designated HPS1, that is
not detected in mature T cells of the spleen, nor in a HeLa
cell nuclear extract (Fig. 2 a, arrow).
The 97-bp segment, HPS1, is underlined in Fig. 1 a.
Two areas of near identity are seen within this region of
DNA, HPS1A and HPS1B (Fig. 1 b). Each segment, 48 bp
for HPS1A and 36 bp for HPS1B, was used as a probe for
mobility shift assay and as cold competitor during a mobility shift assay with the whole 97-bp segment as probe. In
Fig. 2 c, the 48-bp segment recognizes a similar complex in
thymic extracts, as did the 97-bp segment. The 36-bp segment failed to detect any specific DNA binding proteins (data not shown). In addition, Fig. 2 d demonstrates that
the reactivity in this 97-bp stretch of DNA has been localized to the 48 most 5 bases of HPS1, termed HPS1A, by
mobility shift competition assay. When using the 48 mer as
cold competitor, the reactivity localized to the 97-bp segment was abolished. In contrast, when the 36-bp HPS1B
segment was used as a cold competitor, essentially all reactivity to HPS1 remained.
DNaase footprinting analysis using a 230-bp segment of DNA containing HPS1A indicated that the area protected within this 48 mer was roughly 24 bp (Fig. 1 b and data not shown). This is a rather large footprint for a single DNA binding protein, suggesting this might be a multimember complex.
HPS1A Recognizing Proteins Are Found only in Lymphoid Cells and Tissues.To determine if the protein binding complex was restricted to the T cell lineage, mobility shift analysis was performed on several different cell types. Table 1 summarizes this data. It appears that the DNA binding complex recognizing HPS1A is present in pre-T cells (8402, CEM, and 2052) and pre-B cells (Nalm-6). Proteins recognizing the 48-bp segment were not found in mature B or T cells (spleen and SU-DHL-6), HeLa cells, or the erythroid leukemia line, MEL.
|
Results from TG1
strongly suggested that regulatory signal sequences around
REC, if they exist, must be contained within the relatively small 1.9-kb DNA segment containing human
REC (Fig. 3 a; 21). Because of its proximity to the h-s-n
and its temporal restriction to early T cells, HPS1A became
the prime candidate for a region controlling lineage-specific use of
-deleting elements. In an attempt to determine
if HPS1A is important in the
deletion process, TG2 was
assembled using oligonucleotide-directed site-specific mutagenesis. The 1.9-kb SalI-XhoI
REC-containing fragment in Fig. 3 b was mutated to delete the 48-bp segment, HPS1A (black box). The newly generated 1.85-kb mutant
fragment was ligated into the base construct (Fig. 3 a) to
make TG2. TG2 was injected by the University of Alabama at Birmingham (Birmingham, AL) Transgenic Facility and seven founder lines containing the construct were
created. All seven lines had expected transgenic bands by
Southern analysis, and ranged in copy number from 2 to 20 copies (data not shown). The estimated copy number of
each transgene is indicated in Table 2.
|
DNA was isolated from the thymus,
spleen, and liver of all seven transgenic lines. None of the
transgenic lines of TG2 contain detectable rearrangements
by Southern blot analysis, even when the lanes are overloaded (data not shown). However, PCR performed on
the same DNA sources easily detected REC-
J
and
REC-j
56-60 recombinants in 7/7 TG2 lines (Fig. 4 and
Table 2). As was noted in TG1,
REC 3
-deleting element recombinations are detected within liver DNA in
those lines that have consistent
REC recombinations in
thymus and spleen DNA. The likely source for these recombinants is contamination from circulating blood T
cells.
In contrast to TG1, REC-
J
and
REC-J
56-60 rearrangements are detected in the
/
populations of 7 out of
7 TG2 lines by PCR analysis (Fig. 4 and Table 2). It thus
appears that the use of
-deleting elements has been significantly altered in the construct that has deleted the 48-bp
segment just 5
to
REC.
Although the presence of REC recombinants in
/
T cells of TG2 by noncompetitive PCR and the undetectable
REC recombinants in TG2
/
T cell populations by Southern blot (data not shown) suggested altered
transgenic
REC use in TG2 lines, these assays did not indicate the extent of the alteration. To better elucidate the
differences between transgenic constructs, quantitative PCR was used to investigate
REC rearrangements. A competitor
containing
REC and several J
segments was constructed
(21) and used with the enzyme immunoassay procedure
described (33). IL-10 primers of construct pQPCR.MCC7
(33) are situated within a single exon of that gene and serve
as a control for the number of alleles within each DNA preparation. Since the seven transgenic lines showing consistent
REC recombinations appeared to be identical, only three
lines were used for quantitative PCR on tissues and purified
/
T cell populations (lines 4-4, 5-5, and 6-1). The thymus was used for a relatively pure population of T cells,
predominantly
/
T cells. Table 3 summarizes the TG2
lines quantitative rearrangement data, and indicates that
REC recombinations are more frequent in the TG2
/
T cell population when compared to TGl lines 14 and 22. The ratio of recombinations found in T cells of TG2 lines 4-4, 5-5, and 6-1 were 0.03-4-fold more frequent in the
thymic T cell population than in the purified
/
T cell
population. Conversely, in the TG1 lines, the ratio of
REC recombinations in
/
versus
/
T cells ranges
from 109 to 186. This recombination ratio represents an
~100-fold lower
/
:
/
recombination frequency ratio
in TG2 T cells than that seen in TG1 lines 14 and 22 T cells. In comparing the numbers from both TG1 and TG2 populations, the major change was the increased frequency of
transgenic
REC recombinations in TG2
/
T cells from
<3
REC rearrangements/100,000 alleles in TG1 lines to
>25
REC rearrangements/100,000 alleles in TG2 lines.
The data contained in Table 3 also suggests an altered use
of transgenic REC in polyclonal
/
T cell populations
in TG2 lines compared to TG1 lines. On average, there are
more rearrangements in TG1 thymus (190/100,000 alleles)
versus TG2 thymus (33/100,000 alleles). This apparent reduced incidence of quantifiable rearrangements in TG2
/
T cells (~fivefold decrease compared to TG1 lines) and the
absence of detectable rearrangements in TG2 DNA by Southern blot analysis (data not shown) suggested that the DNA
segment HPS1A also functions as an enhancer of recombination in
/
T cells. However, due to probable insertional effects within both transgenic lines, leading to a wide
range of
REC recombinations within these lines (note the
difference in thymic
REC recombinations in TG2 lines
4-4, 5-5, and 6-1), ~20-30 different founders for each
construct would need to be analyzed to reach statistical significance. To circumvent the need for multiple transgenic
founder lines, we used the in vitro recombination system of
Hesse et al. (31) to follow the effects of HPS1A on recombination.
Plasmids pLH103 and pLH104 were generated
from the base construct pJH299 (30) by the addition of the
REC h-s-n with and without the DNA segment HPS1A
(Fig. 5). These rearranging substrates were independently
transfected into the putative
/
T cell line 2052 (27) and
the B cell line 38B9 (27) and analyzed for rearrangements (see Materials and Methods). Table 4 is a compilation of
four separate experiments of each plasmid into both the T
and B cell lines. Construct pLH103 (containing HPS1A) consistently produces more rearrangements in both cell lines, when
compared to construct pLH104 (significant to P <<0.00001).
The threefold increase in recombinations seen with pLH103
is very close to the ~fivefold increase in recombinations
seen when comparing
/
T cells in TG1 lines to
/
T
cells from TG2 lines (Table 3). Curiously, the B cell line
38B9 rearranges the substrates more frequently than the T
cell line 2052. Nonetheless, the presence of HPS1A on
nonchromosomal rearranging constructs in both T and B
cells increases recombination by ~threefold.
The unique positioning of the locus within the
TCR locus has clear implications for T cell lineage development. Mechanisms must exist to ensure the separation
and control of recombinations within this combined locus.
Two models have been proposed for
versus
receptor
development: (a) precommitment of T cell precursors, and
(b) ordered rearrangement of receptors. Precommitment is
defined as choosing which T cell type,
/
or
/
, to become by some unknown mechanism before rearranging
the receptor genes (34). This choice could take place at
any time before commencing TCR rearrangement, including the earliest stages of development in the bone marrow or fetal liver. This model implies that a signal for particular T cell lineage is generated, by other unknown mechanisms,
and then implemented by controlling the use of TCR
or
chain rearrangement and expression.
The second hypothesis, the ordered rearrangement hypothesis, states that control of the /
locus occurs through
an ordered rearrangement of the
chain first, and, if failed
or inappropriate, then commencing
chain recombination
(38, 39). This model implies that functional
chain rearrangement and expression essentially precludes
chain recombination. Recent evidence from several groups favors
this ordered rearrangement hypothesis (40, 41). Data presented seems to demonstrate a high degree of
chain rearrangements have occurred in
/
T cells, a finding that is
incompatible with a precommitment model.
At present, it is not clear how the data supporting each
of these models will be reconciled. It is undoubtedly a
complex process for choosing receptor type, and more data
is needed before precise mechanisms can be defined. How
then, does deletion fit into these paradigms? Several
pieces of data suggest
deletion plays a central role in the
process of maintaining separate
and
receptors. (a) The
frequency of
deletion, most notable in human thymus, is
consistent with
deletion often occurring on both alleles
in cells destined to rearrange TCR
chains (19). (b) The
high degree of evolutionary conservation in location, sequence identity, and apparent function indicates a significant role for
deletion in T cell development (20). (c) The
maintenance of lineage specificity for
deletion in the first
transgenic construct implies tight regulatory control over the
use of
-deleting elements (21). (d) A discrete population of
developing CD3, CD4, CD8 negative thymocytes containing
REC-
J
recombinations but not V
-J
joining implies that
deletion occurs before TCR
chain recombination (21).
Corroborating data for deletion as an antecedent to
V
-J
joining has been reported (42). In this in vitro system, immature thymocytes progress to undergo
REC rearrangements before forming functional TCR
chains.
However, none of this data proves that
deletion occurs
before V
-J
joining on the same allele, in individual cells.
To try and understand the potential role of deletion in
the formation of T cell receptors, we developed a transgenic
reporter construct for the detection of
-deleting element
recombinations. This transgene was designed to molecularly
"tag" cells having undergone
deletion, for subsequent
analysis within mature T cell populations. The model of
deletion implies that use of deleting elements would be restricted to the
/
T cell population. If
deletion was a
random phenomenon, then rearrangements would be expected in all T cell lineages. Human TG1 displayed a remarkable degree of lineage specificity for the use of the
-deleting elements (Table 3), as predicted by the model (21). This
data strongly suggests that
deletion is involved, in some
fashion, with developing
/
T cells. It further implies that
tight regulatory control of
deletion in
/
T cells exists,
even on random integration of transgenic
-deleting elements. The data also suggested that DNA segments mediating the tight control of
deletion would be contained
within the transgene reporter construct, TG1.
The next obvious step was to define the molecular basis
for lineage-specific control of this process. The identification of a DNA binding protein, specific to a region near
REC and restricted to rearranging cells, allowed the precise targeting of a DNA region that might be responsible
for this lineage-specific control of transgenic elements. When
TG2 was designed, one of three outcomes was anticipated:
(a) no differences noted between TG1 and TG2, (b) loss of
all or most recombination in
/
T cells of TG2, or (c) loss
of lineage specificity with recombination seen in both
/
and
/
T cell populations of TG2. Either the loss of recombination or the loss of lineage specificity would substantiate the presence of signal sequences for control of
deletion within the 48-bp segment, HPS1A, whose deletion was the only change between the two transgenic constructs. We report here the loss of tight lineage specificity
for
deletion, with frequent rearrangements present in
TG2
/
T cells. Moreover, the TG2 transgenic construct
in
/
T cells also demonstrates alterations when HPS1A is
removed, with a decreased frequency of transgenic
REC
rearrangements (an ~fivefold decrease in peripheral T cells
[Table 3] confirmed by in vitro recombinatorial analysis
[Table 4]). Therefore, we propose that the 48-bp DNA element, HPS1A, is a recognition motif for a DNA binding
protein complex that controls recombination at
REC.
Two lines of evidence supports HPS1A's role in deletion. As mentioned above, the removal of this recognition
sequence in TG2 resulted in transgenic
REC rearrangements in both
/
and
/
T cells, and appears to alter the
frequency of
REC recombinations in the
/
T cell subsets, and more importantly in in vitro recombination systems. This data suggests that this DNA motif has two functions: in
/
T cells, HPS1A seems to promote and
facilitate recombination; in
/
T cells it seems to inhibit
REC recombination (see below).
Secondly, the presence of specific DNA binding proteins
recognizing HPS1A is limited to early lymphoid lineages
(pre-B and pre-T cells, Table 1) where active recombination of TCR genes is occurring (27). It is interesting that
proteins recognizing HPS1A are also found in pre-B cells.
Precedent for mechanisms separating two independent rearranging loci has been suggested between and
immunoglobulin light chains. In this system,
recombination
precedes
. When failed or inappropriate
rearrangement has occurred, an element termed the
-deleting element in
humans (43) or RS in the mouse (41, 46, 47), deletes
the C
segment and
rearrangement commences. The deleting element resides ~10 kb 3
to C
and is composed of
the canonical recognition sequence of the putative recombinase complex, a h-s-n motif. In
producing B cells,
deletion has occurred on both
alleles and appears to occur
before the rearrangement and expression of
light chains in
most
-producing B cells. A preliminary search around the
-deleting element and RS sequences has not revealed any
homology with
REC or HPS1A (data not shown). A more
extensive search is currently underway.
The described effects of HPS1A on recombination of
REC are compatible with either positive or negative regulation. For example, in
/
T cells, the presence of proteins recognizing HPS1A may actively promote recombination of
REC, and the absence of such proteins in
/
T cells would be the basis for lineage specificity. This promotion of recombination, presumably through protein-
protein interaction with the recombinase complex, would
focus recombination at
REC and facilitate
REC rearrangements (we prefer "facilitation" to "enhancement" because of the relatively modest [~60-80%] decrease in
transgenic
REC rearrangements when HPS1A is deleted).
The positive regulatory model would have an absolute requirement for the presence of HPS1A-recognizing proteins
before recombination could occur because of the scarcity
of
REC rearrangements detected in the
/
population of
TG1 (Table 3). If such a model were true, it would imply
another DNA segment, other than the h-s-n of immunoglobulins and T cell receptors, is directing a recombinase mediated event. Two other such sites, termed KI and KII,
were recently described in the murine
light chain locus
(48). Mutation of both elements resulted in an ~80% decrease in rearrangements into the cis
chain locus. Interestingly, the decrease seen by removal of HPS1A closely matches
the decrease seen when both
locus elements are mutated.
However, a comparison of DNA binding regions for KI,
KII, and HPS1A shows no significant homology (data not
shown) suggesting the two processes are not related.
Conversely, recombination of REC may be a default
pathway in T cell development, and proteins inhibiting this
recombination in
/
T cells could result in the maintenance of lineage specificity. The default pathway in this
negative regulatory model would need to be highly efficient to explain the high degree of
REC recombination found in
/
T cells (21). In addition, removal of a DNA
recognition sequence (HPS1A) in the default pathway would
also be required to effect the efficiency of
REC recombination, to explain the frequency change of transgenic
REC
rearrangements seen in in vitro recombination assays and
thymic
/
T cells of TG2 (Tables 3 and 4).
Although the data is not conclusive, we favor a model in
which both positive and negative regulation occurs in the
different lineages. The signal to become an /
T cell
would generate proteins that would interact with HPS1A,
thereby promoting
REC recombinations leading to
deletion. The signal to become a
/
T cell would result in
the generation of proteins that would interact with HPS1A
and prevent rearrangements involving
REC. It is also possible that positive or negative regulation of
REC recombination is one or more steps removed from interaction
with HPS1A, protein-protein interactions with HPS1A
binding proteins mediate the desired effect.
Nonetheless, the data from TG2 strongly suggests that
HPS1A is the focal point for a signal that directs lineage-specific use of the -deleting elements. Understanding this
signal will require the isolation, partial sequencing, and
cloning of the DNA binding proteins recognizing HPS1A.
We have successfully isolated the 70-kd member of this
complex (data not shown). Partial amino acid sequencing
has revealed no homology to known proteins, and we are
actively pursuing the cloning of its gene.
deletion seems to play a central role in the process of
maintaining separate TCR
and
loci. The evolutionary
presence of such seemingly redundant systems for deleting
coding segments (V
to J
joining would also delete
-coding segments) implies the choice between
or
receptors needs to be tightly regulated, and that a complex interaction between inhibition and induction of recombination
is involved in the process of choosing receptor type. For
instance, the induction of
chain rearrangement in
/
T cell precursors also requires the inhibition of V
to J
joining or the functional
chain would be deleted. Therefore, the
/
locus seems to be the focal point for the separation of
/
and
/
T cell lineages. Control of the rearrangement process within this combined locus appears to
be the implementation of signals for determination of T cell
type. Understanding
deletion may lead to a description of
the molecular events underlying T cell lineage commitment.
Address correspondence to R.D. Hockett, Jr., UAB Dept. of Pathology, WP230, 618 S. 18th St., Birmingham, AL 35233. Phone: 205-934-6246; FAX: 205-975-7074; E-mail: Hockett{at}wp.path.uab.edu
Received for publication 14 January 1997 and in revised form 24 April 1997.
1Abbreviations used in this paper: AMP, ampicillin; CAM, chloramphenicol; DTT, dithiothreitol; h-s-n, heptamer-spacer-nonamer; TG, transgenic reporter construct.The authors wish to thank Drs. Casey Weaver and Pat Bucy for critical reading of the manuscript and helpful discussions. We also thank Dr. Michael Lieber for supplying reagents and technical assistance.
This work was supported by grant AI35107 from the National Institutes of Health.
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