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3 Interval Exhibits B Cell-Specific Enhancer Function in Early Development1
Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and C
3, which is unstable and may be a recombination hot spot.
We isolated a pBAC clone (pHuIgH3'-3) that established a 52-kb
distance between C and C3. Sequence analysis identified a high
number of repeat elements, explaining the instability of the region,
and an unusually large accumulation of transcription factor-binding
motifs, for both lymphocyte-specific and ubiquitous transcription
activators (IKAROS, E47, Oct-1, USF, Myc/Max), and for factors that may
repress transcription (
EF1, Gfi-1, E4BP4, C/EBP
). Functional
analysis in reporter gene assays revealed the importance of the
C-C3 interval in lymphocyte differentiation and identified
independent regions capable of either enhancement or silencing of
reporter gene expression and interaction with the IgH intron enhancer
Eµ. In transgenic mice, carrying a construct that links the
-globin reporter to the novel -3 intron enhancer (E-3),
transgene transcription is exclusively found in bone marrow B cells
from the early stage when IgH rearrangement is initiated up to the
successful completion of H and L locus recombination, resulting in Ab
expression. These findings suggest that the C-C3 interval exerts
regulatory control on Ig gene activation and expression during early
lymphoid development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-C3-C1-
C
-C
1-C-C2-C4-C-C2-3',
over a 350-kb region of chromosome 14 (1). Overlapping
phage and cosmid clones established the C gene organization but
attempts to obtain the entire region on overlapping clones or a single
yeast artificial chromosome
(YAC)5 have been
unsuccessful (Ref. 2 and our own results). PCR-based
approaches identified highly repetitive regions downstream of C1 and
C2 which include virtually identical 3'-enhancers made up from
different numbers of short motifs (3, 4). Similar
repetitiveness, leading to instability, was also assumed for the
estimated 40- to 70-kb gap between C and C3, which could not be
cloned to establish a C gene contig. Indirect results from transgenic
mice lacking different regions 3' of Cµ and C further suggested
that this particular downstream region might be important for high
expression and switching of IgH genes (5, 6). Analysis of
recombination in the C-C3 interval showed a lack of association
between these genes, which may indicate a potential hot spot for
recombination (7, 8). The potential significance of this
region is further supported by the finding that a large area between
C and C3 is deleted in certain leukemias, which may be linked to
a pathogenic mechanism active at an early stage of B cell development
(9).
In the mouse, the IgH locus has been completely cloned
(10) and in DNA-binding assays a cluster of matrix
association regions (MARs) was found in the C-C3 intron
(11). Although the region was not extensively
characterized by sequencing, the presence of long interspersed
repetitive elements in the vicinity of MARs may lead to the high
recombination observed in this region of the IgH locus. Probes derived
from bacteriophage clones covering the mouse C-C3 region failed
to identify corresponding sequences in the human locus
(9). During B lymphocyte development, it is generally
thought that transcriptional activation of the IgH locus is regulated
using two enhancer arrays that flank the constant region cluster (Refs.
12, 13, 14); reviewed in Ref. 15). These arrays,
the Eµ intron enhancer and the 3' enhancer downstream of C,
contain multiple sites for the binding of both tissue specific and
ubiquitous trans-acting factors (13, 16).
Enhancer-mediated activation appears to be controlled by the
interaction of both negative and positive regulatory elements
(17, 18). The Eµ intron enhancer provides potential
protein binding sites for several regulatory elements that are
essential for lymphocyte differentiation, including E47, PU.1, Ets-1,
TFE3, USF, and Oct (19, 20). The 3' enhancer shares
some DNA sequence elements with the Eµ enhancer but also has
additional motifs for factors involved in transcriptional regulation
(13, 16). Activation and sequential DNA rearrangement of
the Ig loci are crucial steps in Ab expression, and
cis-acting locus elements like enhancers, which accommodate
various combinations of factor-binding sites, have been implicated in
IgH locus recombination and transcription (17). Although
important information about enhancer core functions has been obtained
from mutant mouse strains, these are poorly understood processes
because there appears to be no activity of either enhancer in early B
cell development when IgH heavy (H) chain rearrangement is initiated.
For example, deletion of the H chain intron enhancer Eµ showed severe
impairment of VH to DJH rearrangement while the
earlier D to JH rearrangement was much less affected (21).
In chimeric mice, in which the C 3'-enhancer was replaced by a
marker gene, isotype deficiency and impairment of H chain class
switching were observed (18).
Here we show that 21 kb of the unstable region in the human IgH locus
between C and C3 contain a highly clustered array of a large
number of transcription factor-binding motifs interspersed with repeat
sequences. Transfection assays revealed transcription enhancement and
silencing activity at the pre-B cell stage, and in transgenic mice
strong enhancer function was identified in the bone marrow, the primary
site of B cell differentiation. Flow cytometry analysis of early B cell
populations showed that this enhancer is already active at the
pro/pre-B cell stage where DNA rearrangement is initiated. We discuss
the possibility that the region accommodating E-3 exerts locus
control function at an early developmental stage that may be critical
in normal and aberrant B cell development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A size-selected library was constructed from human DNA using the
pBeloBACII vector (22). An MluI linker was
added to the pBeloBACII vector after linearization with SphI
in the multiple cloning site. Human genomic DNA was prepared from the
fibroblast cell line KB (23) and digested to completion
with MluI. The fragments were size fractionated by pulsed
field gel electrophoresis (PFGE) in 1% low melting temperature agarose
(SeaPlaque FCM; Flowgen, Shenstone, U.K.) at 12 /cm in 0.5x
Tris-buffered EDTA with a 40-s pulse for 24 h at 3.5°C. Gel
slices containing DNA fragments from 
30–80 kb were excised, melted
at 67°C, and digested for 1 h at 40°C with 1 unit Gelase
(Epicentre Technologies, Madison, WI) per 0.1 g gel. Size-selected
DNA (100–200 ng) was ligated with dephosphorylated
MluI-restricted pBeloBACII vector (25–50 ng) using 4 U T4
ligase (NEB, Beverly, MA). Ligation mixtures were dialyzed against 30
ml TE and 1 mM polyamines for 4 h at room temperature using
Millipore 30000 NMWL filters (Millipore, Bedford, MA). Aliquots of the
dialyzed ligation mixture (1 µl) were used to transform
Escherichia coli DH10B by electroporation at 120 V, 25 µF,
and 100 ohms (24). Transformed cells were incubated for 90
min at 37°C in SOC medium (2% Bactotryptone, 0.5% Bacto yeast
extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM
MgSO4, 20 mM glucose), shaken at 250 rpm, and plated on
Luria broth-agar containing 12.5 µg/ml chloramphenicol, 50 µg/ml
5-bromo-4-chloro-3-indolyl--D-galactoside, and 25
µg/ml isopropyl--D-thiogalactoside. Distinguishable
white and blue colonies appeared after 24 h, and 5000 white
clones were isolated and analyzed by colony-filter hybridization with
the 0.4-kb EcoRI fragment of pM5-1-23 (25) and
human C3 (Ref. 26 ; see also below). The insert size was
determined by MluI digestion and PFGE.
Hybridization and sequencing
For hybridization analysis, genomic DNA (10 µg) or pBAC DNA
(0.5 ng) was digested with the desired restriction enzyme and separated
on 0.8% agarose gels in TAE. DNA was transferred to nylon membranes
(Hybond-N; Amersham, Arlington Heights, IL) by alkaline transfer and
the blots were hybridized with oligolabeled probes at 65°C in Church
buffer. Hybridization probes were a 7.4-kb HindIII fragment
containing human 3 (26) and a 0.4-kb EcoRI
fragment obtained from a 3.4-kb BamHI fragment located 20
kb downstream of human C from the 3'-end of the human IgH (HuIgH)
YAC, derived by plasmid rescue after digestion (clone pM5-1-23
(25)). The 5.9-kb BamHI fragment of
pHuIgH3'-3 (see Fig. 1
) was subcloned, and a 0.5-kb
SspI-BamHI fragment was derived therefrom for
further mapping.
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Cell lines and transfection assay
The cell lines used for the reporter gene analysis were from the Institute’s collection or a kind gift from collaborators, and their origin is described in the American Tissue Culture Collection catalog. The human cell lines used were NALM-6 pre-B cells, DG-75 plasma cells, Jurkat T lymphoblast cells, and KB fibroblasts (Ref. 27 and references therein; Refs. 23, 28 , and 29), and the mouse cell lines were 3-1 and 18-81 pre-B cells and the plasma cell lines MPC11 (producing IgG2b) and AH (producing IgM) (30). The cell lines were maintained in RPMI (Life Technologies, Paisley, U.K.) supplemented with 10% FCS and 50 µM 2-ME.
Transcriptional activity of fragments from the pHuIg3'-3
5'-region was analyzed with a luciferase reporter assay system
according to the manufacturer’s protocol using a BioOrbit 1253
luminometer (Promega, U.K.). The pGL3 vector ensures low background
luciferase expression and this allowed the unambiguous measurement of
enhancer function. The overlapping fragments from the pHuIg3'-3
5'-region (see Figs. 1
and 5) tested were: 1) 10.7-kb
MluI-HindIII; 2) 7.2-kb
MluI-BamHI; 3) 8.5-kb HindIII; and 4)
5.9-kb BamHI. These were inserted 5' of the SV40 promoter of
the pGL3 reporter gene construct. The size of fragment 2 in the
reporter gene construct was reduced by creating the necessary 5' and 3'
overhang by MluI and KpnI restriction and
exonuclease III treatment. As a positive control, pGL3 control vector
containing the SV40 enhancer was used. To test the interaction with
other IgH enhancers, the human Eµ intron enhancer (25)
was added 5' of an inserted fragment. Pre-B cells, pro-B cells, Jurkat
cells, and KB fibroblasts (2.5 x 106) were
transfected by electroporation using 7 µg pGL3 containing fragment 1,
2, 3, or 4 with or without Eµ and with 1 µg pSV--galactosidase
control plasmid as an internal standard. Electroporation conditions
were 960 µF, 200 ohms, and 270–300 V. Cells were harvested after
20–22 h incubation. Plasma cells (1–3 x
105) were transfected with the above constructs
by lipofection with DOTAP (Boehringer Mannheim, Mannheim, Germany)
according to the manufacturer’s protocol. The -galactosidase assay
was performed in 100-µl reaction buffer (0.1 M
Na2HPO4/NaH2PO4,
pH 7.3; 1 mM MgCl; 50 mM 2-ME; 1.33 mg/ml
o-nitrophenyl--D-galactopyranoside)
for 30–120 min. The reaction was stopped with 150 µl 1 M
Na2CO3, and the conversion
of substrate was measured in a Titertek Multiscan MCC/340 at 410
nm.
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The 1.3-kb E-3 fragment (positions 5885 to 7185 in pGL3)
obtained by PCR was added to a 3-kb human -globin gene
subcloned in pUC12 (31) by blunt end ligation into the
XbaI site in the linker. A 4.3-kb
SacI-HindIII DNA fragment containing E-3
linked to the -globin reporter gene was gel purified and injected
into the pronuclei of fertilized (C57BL/6 x
CBA)F1 eggs at 1–2 pg/ml (32).
Transgenic mice were obtained with high and low copy number verified by
tail blot analysis with a transgene probe. RNA from different tissues
was prepared as described by the manufacturer using the RNAqueous Kit
(Ambion, Abingdon, U.K.) or the RNAzol B method (AMS
Biotechnology, Oxford, U.K.) for bone marrow. Cell
preparations were essentially free of erythrocytes with the exception
of liver cells where a low percentage remained. Hybridization probes
were the -globin transgene and a 540-bp actin gene fragment obtained
by PCR (33). For the isolation of B cell subpopulations by
flow cytometry, bone marrow cells were stained as described
(34). Multicolor staining was conducted with the following
reagents in combinations shown in Fig. 7B: PE-conjugated
anti-mouse CD25 (P3317; Sigma, St. Louis, MO); PE-conjugated anti-mouse
CD45R (B220) (P3567; Sigma); biotinylated anti-mouse IgM (No. 02082D;
PharMingen, San Diego, CA); PE-conjugated anti-mouse c-kit
(CD117) (No. 09995B; PharMingen); FITC-conjugated anti-mouse CD19 (No.
09654D; PharMingen); FITC-conjugated anti-mouse IgD (No. 02214D,
PharMingen) and biotinylated anti-mouse CD43 (No. 01602D; PharMingen).
Binding of biotinylated Ab was developed with streptavidin quantum red
(S2899; Sigma). The oligonucleotides for RT-PCR of -globin and HPRT
as a control have been described (31). RT-PCR was
performed with the One-Step System (Life Technologies) under the
following conditions: 50°C for 30 min followed by 94°C for 2 min
for cDNA synthesis, followed by 30 PCR cycles (15 s at 92°C, 30
s at 55°C, 30 s at 72°C) and 5 min at 72°C to complete the
reaction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and C3 is 52 kb
Southern blot analysis suggested that the region between C and
C3 is 40–70 kb in size (1, 8). We analyzed human sperm
DNA and the human fibroblast cell line KB (23) by
digestion with rare cutting enzymes and hybridization with probes from
the 3'-end of the human IgH YAC and from human C3
(see Fig. 1). Southern blots of
MluI digests showed a possible region of 50 kb capable of
spanning the gap between C and C3 (data not shown). A
size-selected bacterial artificial chromosome (BAC) library was
constructed and hybridized with the 0.4-kb EcoRI fragment of
pM5-1-23, from the 3'-end of the IgH YAC (25), and human
C3 (26). Of eight clones, pHuIgH3'-3, hybridized
with both of these probes and MluI restriction digestion
followed by PFGE identified a 48-kb insert. Comparison of the
restriction patterns of pHuIgH3'-3 with the HuIgH YAC
(5) and cosIg6 (35) revealed substantial
homologies at the 5'- and 3'-ends, respectively, which suggested that
pHuIgH3'-3 spanned the gap between the C and C3 constant
region genes (Fig. 1A). The 5' MluI cloning site
of pHuIgH3'-3 lies 4 kb 3' of the membrane exon 2, a
region not yet characterized by sequence analysis, whereas the 3'
MluI cloning site is located in the hinge region of C3
(36). Alignment of restriction maps of pHuIgH3'-3
with the HuIgH YAC and cosIg6 identified a novel 11-kb region.
To verify that this novel region is in the correct genomic
configuration, different digests of human DNA and pHuIgH3'-3 DNA
were analyzed in Southern blots using different hybridization probes.
An example (Fig. 1B) shows that hybridization with the
0.5-kb SspI-BamHI fragment from pHuIgH3'-3
identified a 5.9-kb BamHI fragment and a 8.5-kb
HindIII fragment, both of which are present in human genomic
DNA and pHuIgH3'-3. The C-proximal BamHI site and
the C3-proximal HindIII site of the hybridization
fragments are also present on the IgH YAC and cos Ig6, respectively.
These results confirm that the cloned region on pHuIgH3'-3
completes the C-C3 sequence gap and establishes the distance
between Cm2 and C3 as 52 kb.
The C-C3 interval region is highly repetitive
The nucleotide sequence of the C-proximal 5'-region of
pHuIgH3'-3 (EMBL accession number AJ303052), indicated in Fig. 1A by the hatched line, was determined by subcloning of
overlapping fragments, exonuclease III digestion and primer walking.
Self-alignment of this 21-kb region (Fig. 2A) identified many regions of
similarity as Alu motifs (37), with a pronounced cluster
at the 3'-end (Fig. 2B) that incorporated several residual
fragments of Alu motifs as well as complete motifs, in various
orientations. In addition, we found transposon-like elements (long
interspersed nuclear elements, long terminal repeats, etc.) in
abundance and of different complexity (38, 39, 40); three
40-bp MstII-like repeats were arranged in a tandem
configuration, which may highlight transposon activity, and a 76-bp
region comprising 23 (CT) repeats was followed immediately by 15 (AT)
repeats. The CT and AT repeats encode the amino acid pairs Leu/Ser and
Ile/Tyr independently of the reading frame. The presence of this large
number of repetitive elements may be the reason for the instability of
the locus observed during cloning efforts.
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Potential binding factor recognition sites of regulatory proteins
were identified by comparison of the pHuIgH3'-3 sequence with the
TRANSFAC transcription factor database using Matinspector (41, 42). As shown in Fig. 3, many
binding motifs were represented very frequently despite the high score
used for the database search, which was 1.0 for core similarity and
0.90 or greater for matrix similarity. The transcription factors
identified can operate either to enhance or suppress Ig transcription,
and many are also present in the Eµ and 3' enhancer regions
(reviewed in Refs. 43 and 44). Binding sites
were found for the activating proteins Ikaros, E47, Oct1, USF, and
Myc/Max (45, 46, 47, 48, 49, 50, 51). The Ikaros gene products belong to the
group of zinc finger DNA binding proteins (52) and have a
complex role in the early stages of lymphocyte development with
establishing the maintenance and differentiation of multipotent
progenitors (53). In the group of repressor proteins,
binding sites for EF1, Gfi-1, E4BP4, and C/EBP were identified
(54, 55, 56, 57). The potential binding site of the nuclear zinc
finger protein Gfi-1 (growth factor independence 1) has been identified
in a large number of eukaryotic promoter-enhancers (58, 59).
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and C3 exerts
previously unrecognized Ig locus control function.
Pre-B cell-specific enhancer activity downstream of C
To determine any functional significance of the C-C3
interval in development and whether the region contained new
cis-acting regulatory sequences, we analyzed transcriptional
promoter activity of subcloned fragments from pHuIgH3'-3 using
the luciferase reporter gene assay. The analysis identified a 7.2-kb
MluI-BamHI fragment from the 5'-C-proximal
region which showed good enhancer activity in human and mouse pre-B
cells (NALM-6 and 3-1) when placed 5' of the luciferase reporter but
did not promote luciferase activity in human or mouse plasma cells
(Figs. 4 and 5). Because other fragments did not
exhibit B cell-specific enhancer activity, we wondered whether the
failure to demonstrate enhancer activity in Ig2b producing MPC11, in
which the C-C3 intron regions are deleted, was a consequence of
the IgH locus having undergone isotype switching in this cell line.
This was not the case in that IgM-producing AH myeloma cells produced
very similar results for all fragments (data not shown). In addition,
the orientation of the subcloned fragments did not alter their
functional activity and enhancer function of the
MluI-BamHI fragment in pre-B cells was maintained
in both transcriptional orientations.
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-3 intron
enhancer, we dissected the MluI-BamHI fragment by
MluI and exonuclease digestion which removed the 5'-region
and resulted in various smaller size fragments (Fig. 4A). In
murine pre-B cells, enhancer activity of the whole region (positions
1–7185) exceeds that of the Eµ enhancer, but the most dramatic
increase, twice the activity of Eµ, was obtained with a 1.3-kb
fragment from positions 5885 to 7185 (Fig. 4B). In
comparison with mouse cells, human NALM-6 pre-B cells showed marked
reductions in transcriptional activation. This may simply be a
reflection of reduced transfection efficiencies of the human cells but
could also indicate concerted interaction of several functional motifs
which are recognized differently in mouse and human pre-B cells.
However, the strongest transcriptional activation in both human and
mouse pre-B cells was obtained by the fragment from positions
5885–7185. Comparable enhancer activities, dependent on the
developmental stage of the cell rather than its species of origin,
agree with what has been found for other human Ig enhancers analyzed in
vitro and in transgenic mice (31, 60, 61). No enhancer
activity was obtained at the later developmental stage of the plasma
cell (Fig. 4C). In addition, neither fibroblasts (as control
cells) nor T lymphocytes showed any enhancer activity (data not shown).
These results from reporter gene transfection assays define a novel
region which we named E-3 with strong cis-acting
enhancer function operative at the pre-B cell stage. A transcription silencer is located adjacent to the enhancer
The proximity of Eµ and E-3 led us to speculate about
possible enhancer cooperation during B cell development. For this we
added the human Eµ intron enhancer to four separate but overlapping
fragments of the 21-kb sequenced 5'-region of pHuIgH3'-3 and
measured transcriptional activity in luciferase reporter assays (Fig. 5). Except in one combination (Eµ + fragment 1), enhancer activity
remained by and large as identified in single enhancer/fragment
constructs analyzed in B cell subsets. This suggests separate functions
of Eµ and E-3 and that their enhancer activities are not simply
additive. Enhancer activity was maintained when Eµ was combined with
fragments 2, 3, and 4, whereas the combination of Eµ with fragment 1
completely abolished enhancer activity in pre-B cells and significantly
reduced enhancer activity in plasma cells (Fig. 5B). This
suggests that fragment 1 contains a B cell-specific transcription
silencer which is likely to be located at the 3'-end of fragment 1.
Fragment 4 shares a corresponding region, and this may explain why
transcription levels are somewhat reduced in pre-B cells when coupled
to Eµ. Furthermore, fragment 1 contains the silencer in close
proximity to the E-3 enhancer identified on fragment 2. Thus, the
lack of enhancer activity of fragment 1 can be explained by the
presence of a strong B cell-specific silencer in this region. It
emerged that further dissection of the fragments was ineffective, as
E-3 enhancer and silencer activity was reduced or abolished, and
suggests that recognition motif combinations are essential. The results
show that the 5'C-C3 region accommodates a previously
unidentified B cell-specific enhancer-silencer array which may interact
with Eµ to control Ig expression during developmental processes.
However, the many varied sequence recognition motifs in the C-C3
interval, together with the coordinated enhancer interaction
identified, indicate a more complex activity with perhaps other
transcription modifiers.
In transgenic mice, E-3 is active in the developing B cell
To determine in vivo specificity of the novel enhancer, we
constructed a transgene composed of E-3 linked to human
-globin as a reporter gene (Fig. 6A). A high and a low copy
number transgenic mouse line with head to tail integration of the
transgene was identified by Southern blot (Fig. 6B), and RNA
was prepared from different tissues for Northern analysis and RT-PCR.
For the assays, actin or HPRT transcripts were used as internal
controls. In Northern hybridization, we were surprised to find
-globin expression in the bone marrow and not in other B lineage
tissue (Fig. 6C), which suggests an essential role of
E-3 in B cell development. We then used RT-PCR as a more
sensitive method to analyze various B lineage, T lineage, and
nonlymphoid tissues. The result was the same; the E-3-driven
transgene is silent in all nonlymphoid tissue as well as in the T cell
compartment, and extensive -globin specific transcription is found
only in bone marrow RNA. These results and the in vitro reporter gene
assays suggest differentiation stage-specific activity of E-3
from pro-B cell development (initiation of DNA rearrangement) to a
mature B cell (surface IgM) before migration from the bone marrow.
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-3 is active, we used flow cytometry and RT-PCR (Fig. 7). The progressive stages in B cell
development are well characterized by cell surface markers and DNA
configuration (62). We isolated several distinct cell
populations from the bone marrow identified by staining with labeled
Abs specific for the B cell markers B220 or CD19 in combination with
specific Abs for c-kit and CD43 (pro-B and pre-B-I stage),
CD25 (pre-B-II stage) and IgM/IgD (immature to mature B cell) (Fig. 7B). RT-PCR identified -globin activity in
B220+ or CD19+ B cells from
bone marrow up to the immature
IgM+IgD- population that
had completed the DNA rearrangement process. Transgene activity may be
initiated at the early pro-B cell stage where a faint
-globin-specific PCR band (relative to the HPRT signal obtained in
the same reaction) was found. Because the number of
c-kit+CD19- cells
was very low, this result may have been obtained from a small
cross-contamination of the more abundant CD19+
cell population. However, at the stage when DNA rearrangement, D to
JH joining, is initiated in
c-kit+CD19+ and
CD43+B220+ B cells
(63) the E-3 enhancer is active. H chain and largely
L chain rearrangement is finalized at the immature B cell stage, and
cells expressing surface IgM show that E-3-driven -globin
expression is extinguished at this maturation stage. This concludes
that the E-3 enhancer is active at the earliest DNA rearrangement
event and silent when the rearrangement process (V-D-J for the H chain
and V-J for a L chain) has been successfully completed. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-C3 interval
Use of the BAC cloning system, capable of maintaining highly
repetitive large insert DNA (22, 64), enabled us to
establish the distance between Cm2 and C3 as 52 kb. Southern
analysis and alignment of pHuIgH3'-3 with the 3'-end of the HuIgH
YAC (5) and the 5'-end of cosIg6 (35)
confirmed the overlap and identified a novel region of 11 kb. The
failure of previous attempts to clone the C-C3 contig in cosmids
or YACs can now be fully explained by the extensive repetitiveness of
the intronic sequence. Such repetitive regions are prone to deletion
through homologous recombination when cloned in yeast
(65). It is estimated that Alu-like repeat sequences are
represented on average every 4 kb throughout the genome (66, 67). In the interval from Cm2 to C3, 11 Alu-like repeats
were identified in a 15-kb region. In contrast, the number of Alu
motifs identified in the 950-kb region containing the variable genes of
the human IgH locus was much less than expected by random distribution
(68). The presence of Alu repeat elements immediately
adjacent to protooncogene translocations has led to the suggestion that
frequent Alu motifs may predispose a region as a hot spot for
recombination (69, 70). Indeed, the MstII-like
sequences, identified in tandem in the C-C3 interval, align with
the consensus sequence of transposon-like elements in the human genome
(39, 40). The instability of the region is further
supported by linkage data, which indicate a lack of association between
C and C3 (7) and that certain leukemias have
deletion boundaries in the C-C3 region (9). Thus,
translocations near the enhancer-silencer array in the C-C3
interval may result in transcriptional alterations of the rearranged
genes or loci leading to a malignant phenotype of the cell subset where
enhancer activity can be identified. Interestingly, chromosomal
translocations in Burkitt’s lymphoma where c-myc expression
is deregulated by linkage to a known enhancer may represent tumors of a
developmental stage in which the enhancer is active.
A factor-binding site cluster
The frequency of potential transcription factor-binding sites in
the 21 kb of the C-C3 interval region sequenced was unexpected.
Using the default scores for the analysis, 0.75 for core similarity and
0.85 for matrix similarity (41), identified too many
motifs to be useful. Our analysis used scores for the core similarity
of 1 and matrix similarity of 0.90. This higher score allowed the
unambiguous identification of multiple motifs for nine transcription
factor-binding sites, five of which are recognized by proteins that
have been shown to increase transcription in lymphocytes (Ikaros, E47,
Oct-1, USF, and Myc/Max) and four motifs shown to be recognized by
transcription silencer or repressor proteins (EF1, Gfi-1, E4BP4, and
C/EBP). It has been shown that the repressor proteins -EF1 and
Gfi-1 interact with elements of the Eµ enhancer (59, 58). However, it is not obvious how the lymphocyte-specific
enhancer and repressor activity identified by functional analysis of
C-3 interval fragments relates to this accumulation of potential
transcription factor-binding motifs. Core sequence motifs for
factor-binding sites found in Eµ also occur frequently in this novel
enhancer region and, indeed, in the whole 21-kb region analyzed (see
Fig. 3). That all nine binding sites appear frequently throughout the
E-3 interval makes it impossible to predict the activity of a
particular region solely based on the nucleotide sequence. Furthermore,
besides short sequence motifs, no homology to draw conclusions about
functional similarity was found between the
MluI-HindIII fragment accommodating E-3 and
the Eµ or 3' enhancers (71, 72, 73, 74). Similarly, the
region responsible for the transcription silencer activity could not be
deduced from sequence comparison. However, because of the significance
of the functional activity identified in the C-3 interval, it is
unlikely that the motifs in this cluster are randomly distributed. In
addition, recognition sequences essential for suppressing the function
of the mouse 3'-enhancer have been reported (30) but were
not evident in the sequenced region. Sequence comparison of human
E-3 with the available mouse -3 interval sequence did not
allow identification of an equivalent mouse enhancer; however, a reason
for this could be the apparent sequence gaps.
An essential role of the C-C3 interval in early lymphocyte
development
The location of the C-C3 interval means that it will be
deleted after switching from Cµ to other isotypes. This implies that
the regulatory activity of this region must be important during early
developmental steps, which is supported by our finding that E-3
enhancer control is operative at the pro/pre-B cell stage (see Fig. 7).
Reporter gene assays suggest that the C-C3 interval region
provides strong B cell-specific enhancer and repressor function. The
strength of E-3, positions 5885–7185, can be 2-fold greater than
Eµ enhancer activity in pre-B cells, but unlike Eµ and E3' the
-3 interval enhancer does not exhibit any activity in mature B
cells. Interestingly, E-3 activity is solely B cell specific,
unlike that of Eµ which shows some activity in T cells at the
developmental timing of rearrangement (75). At
developmental stages where neither the Eµ nor the E3' enhancer was
particularly active by itself, enhancer combinations identified
synergistic transcriptional activity (76). A different
picture emerged when C-3 interval fragments in combination with
Eµ were transfected (Fig. 5). Here individual enhancer function
remained cell type specific rather than synergistically increased; in
E-3 + Eµ constructs the activity equaled that of E-3 in
pre-B cells whereas Eµ levels were obtained in plasma cells. The
identified silencer extinguished both enhancers at the pre-B cell stage
which suggests a complex, yet to be explored, regulatory function of
elements in the E-3 interval which appear to coordinate
stage-specific H chain activation. When linked to a -globin gene
(that does not contain any cell type-specific intragenic regulatory
elements active in hemopoietic cells) and assayed in transgenic mice,
E-3 is a B cell-specific transcription enhancer element active
during B cell development in the bone marrow. This pattern of activity
complements the transcriptional activities of Eµ and E3',
contributory to V to DJ joining, switching, and H chain expression
(18, 21, 77, 78), and may suggest a role of E-3 in H
chain activation and/or initiation of DNA rearrangement. A view that
the presence of several enhancers in the Ig loci simply reflects
redundancy cannot be supported with these results which put the
functional activity of the IgH enhancers in a possible order. Starting
with the earliest; E-3 may be important for H chain activation
with initiation of DNA recombination; Eµ may then complete the
joining process to allow H chain expression; Eµ may also be involved
in facilitating isotype switching; E3' may complete the switching
process, which also deletes Eµ and E-3, and may have a role in
influencing expression.
The high frequency of transcription factor-binding sites and the
functional activity of the C-C3 intron, which includes an
enhancer-silencer array, is reminiscent of the locus control regions
described for the globin locus (79, 80). It is
characteristic of locus control regions that they are essential for the
correct activation of a locus to ensure that physiological expression
levels are obtained. In transgenic mice carrying part of the human IgH
locus, high level expression of the transgenes independent of copy
number and integration site was not achieved (5). This
suggests that neither the Eµ nor the 3' enhancer region are
sufficient in themselves to ensure the full activity of a single copy
translocus. In transgenic mice, it will be interesting to see whether a
translocus that includes a complete C-C3 interval allows correct
chromatin opening and gene activation. Identification of an equivalent
region in the mouse and its removal by a knockout approach may shed
further light on IgH locus activation and the DNA recombination
processes, which are still poorly understood.
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Acknowledgments |
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Footnotes |
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2 Current address: Child Health Research Institute, Womens and Childrens Hospital, North Adelaide, Australia.
3 Current address: Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Germany.
4 Address correspondence and reprint requests to Dr. Marianne Brüggemann, Laboratory of Developmental Immunology, The Babraham Institute, Babraham, Cambridge, CB2 4AT U.K.
5 Abbreviations used in this paper: YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; MAR, matrix association region; PFGE, pulsed field gel electrophoresis; HuIgH, human IgH.
Received for publication July 17, 2000. Accepted for publication December 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene and association with other constant-region genes. Am. J. Hum. Genet. 45:606.[Medline]
B-binding proteins. J. Immunol. 156:2828.[Abstract]
L) in the absence of µ on human pro-B cell lines and normal pro-B cells. Eur. J. Immunol. 26:2172.[Medline]
enhancer at early stages of B-cell differentiation. Mol. Cell. Biol. 13:3611. 3' enhancer triggers gene expression in early B lymphocytes but its activity is enhanced on B cell activation. Int. Immunol. 8:1561.
locus is similarly well expressed in mice and humans. J. Exp. Med. 189:1611., and genes. Nature 300:23.
genes. Nucleic Acids Res. 14:1779.-cell-specific repression of insulin gene transcription by CCAAT/enhancer-binding protein : inhibitory interactions with basic helix-loop-helix transcription factor E47. J. Biol. Chem. 272:28349.-crystallin enhancer-binding protein EF1 is a repressor of E2-box-mediated gene activation. Mol. Cell. Biol. 14:5692. immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements. Mol. Immunol. 33:427.[Medline]
and globin gene. Mol. Cell. Biol. 12:4919.This article has been cited by other articles:
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  R. Afshar, S. Pierce, D. J. Bolland, A. Corcoran, and E. M. Oltz Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5'DQ52 Region in Targeting DHJH Recombination J. Immunol., February 15, 2006; 176(4): 2439 - 2447. [Abstract] [Full Text] [PDF]
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 S. S. Park, J. S. Kim, L. Tessarollo, J. D. Owens, L. Peng, S. S. Han, S. Tae Chung, T. A. Torrey, W. C. Cheung, R. D. Polakiewicz, et al. Insertion of c-Myc into Igh Induces B-Cell and Plasma-Cell Neoplasms in Mice Cancer Res., February 15, 2005; 65(4): 1306 - 1315. [Abstract] [Full Text] [PDF]
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, 400-bp
EcoRI fragment of plasmid pM5-1-23; •, 
, 500-bp SspI-BamHI fragment
from pHuIgH3'
), and without Eµ (
) was
determined in pre-B cells and plasma cells. Assay details are described
in Fig. 4
0.9 for matrix similarity (
), and human pre-B cells (NALM-6, 
) (B) and
murine plasma cells (MPC11, 
)
(C). Experiments using murine 18-81 pre-B cells gave
similarly positive results. No enhancer-dependent activity was found
for AH murine plasma cells, the human T lymphoblast cell line Jurkat,
or human KB fibroblasts (data not shown). Bars indicate luciferase
activity for the different fragments, 4510-B, 5555-B, 5885-B, 6461-B,
and 6884-B, which are different length 3'-clones obtained by
exonuclease digest of the 1-B fragment subcloned in pGL3 (see Fig. 5