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hs1,2 Enhancer in the Rabbit IgH Locus1
Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153
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Abstract |
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enhancer (3'E) regulate expression of the Ig
heavy chain genes during B cell development. A 3'E is composed of
DNase I-hypersensitive sites, hs1,2, hs3a,b, and hs4, found 3' of the
murine C gene as well as 3' of both human C genes, C1 and
C2. Rabbits have 13 C genes, and we tested whether a 3'E is
associated with each of these genes. To identify 3'E regions we
developed a rabbit hs1,2 probe and used this to search for enhancer
homologues of human hs1,2 in a genomic fosmid library. We identified a
single hs1,2 fragment 8-kb downstream of C13, the presumed 3'-most
C gene. We also identified and partially sequenced a new C gene,
C14, located 6 kb upstream of C13. Genomic Southern blot analysis
confirmed that the rabbit genome contains only one hs1,2 enhancer
region. We tested the enhancer activity of the hs1,2 with the SV40,
VH, and I promoters using the luciferase reporter gene
in transient transfection assays and found that it significantly
enhanced the activity of SV40 and VH promoters and slightly
enhanced an I promoter. We conclude that the rabbit has a single
hs1,2 enhancer that resides at the 3' end of the IgH gene cluster and
may constitute one of the cis-elements regulating the
expression of IgH genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhancers (3'E), are important
for this regulation (4, 5, 6, 7, 8). The 3'E regions are located
downstream of the single C gene in mice and rats (4, 5, 6)
and downstream of each C gene, C1 and C2, in humans (9, 10). The 3'E contains four DNase I hypersensitivity sites
(hs), denoted hs1,2, hs3a, hs3b, and hs4 (8, 11). Three of
these hypersensitive sites, hs1,2, hs3a, and hs3b, are detected within
DNA regions that have significant enhancer activity. The hs3a and 3b
have a high degree of nucleotide sequence identity to each other, but
not to hs1,2. All three enhancers appear to function late in B cell
differentiation. The hs4, the most distal enhancer, does not share
homology with hs1,2 and hs3a,b and appears to be active throughout B
cell development (12, 13).
Each enhancer region includes a core segment containing a single
octamer binding motif (ATTTGCAT) (9, 10, 14). The core
region of hs1,2 is highly conserved among mouse, human, and rat
(8). The nucleotide sequences flanking the core region,
however, are less conserved and contain inverted repeats and GC-rich
segments (4, 9, 11). Multiple binding sites for B
cell-specific transcription factors were identified in the 3'E,
including Oct-1 and Oct-2 binding sites (6, 15, 16), as
well as B cell lineage-specific activator protein and NF-
B binding
sites (17, 18). The 3'E has been shown to be important
for both transcription and isotype switching. Murine, rat, and human
3'E were shown in transfection assays to increase the transcription
from various promoters (4, 5, 6). In vivo studies also showed
that 3'E increased the transcription of
VH-dependent reporter genes in transgenic mice
(19, 20, 21). Further, Alt and colleagues showed that
disruption of the 3'E by neo led to deficiencies in the
secretion of some IgG isotypes, suggesting that 3'E affects
recombination during isotype switching (7). The 3'E
region was accessible to DNase I and was differentially methylated
during B cell development (22, 23). Thus, 3'E has been
shown to influence the control of IgH genes and B cell development. The
specific role of 3'E, however, remains a mystery.
In the rabbit the IgH locus contains 13 nonallelic germline C genes,
all of which are expressible (24, 25, 26). Within the rabbit
IgH gene cluster, Eµ has been identified as an enhancer region
(27, 28), but 3'E has not been described. Because
3'E is important for IgH gene expression and class switching, and
because a 3'E resides downstream of each of two C genes in the
human genome, we investigated whether homologous regions are associated
with each of the 13 rabbit C genes.
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Materials and Methods |
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genes
A 70-bp 3'E hs1,2 probe was PCR amplified from rabbit genomic
DNA with primers taken from sequences of the core region of human and
murine hs1,2:
5'-TATTTT/CTGGAAACAA/GCCT-3'
and
3'-T/ACA/GGGCACATGCAAAT-5'
(10). The hs1,2 sequence was determined using an ABI Prism
310 Genetic Analyzer and was >70% similar to mouse and human hs1,2.
This region contained the core region with the octamer binding motif.
Human and mouse hs4 probes (
100-bp) were PCR amplified from genomic
DNA with the following primers: human,
5'-TTTGGATCCAGGTATTTCTAAAAATGCT-3' and
5'-TTGGATCCACCTCCCCCAATGCAAAT-3'; mouse, 5'-AAACATTTCTAAAAATGAT-3'
and 5'-CCCCAGCACCATGCAAAT-'. The identity of the probes was
established by nucleotide sequence analysis. The mouse hs3b probe was
obtained from a QM293Luc plasmid (gift from B. B. Birshtein,
Albert Einstein College of Medicine, Bronx, NY). For PCR amplification
of hs3 from rabbit genomic DNA we used as 5' primer,
5'-ATCACCTGCAGAAA-3', and as 3' primers
5'-TGA/TTTCTCAGAACAG-3' or
5'-AGGCA/CCACCTGGCC-3'.
A partial MboI genomic fosmid library from DNA of rabbit IgH
E haplotype was prepared and screened with the rabbit 70-bp hs1,2 and
550-bp C probes as previously described (24, 29, 30).
Fosmid clones that hybridized with these probes were purified, and the
DNA was restriction mapped and analyzed by Southern blot with the C,
3' hs1,2, hs3b (mouse), and hs4 (mouse and human) probes.
Hybridization with the hs3b and hs4 probes was performed at low
stringency in 6x hybridization solution (31) and washed
in 2x wash buffer at 56oC. The nucleotide
sequences of the identified C genes and 3'E regions were
determined.
pGL3-based luciferase reporter-gene constructs
Constructs containing both a promoter and the 900-bp
XbaI/PstI putative 3'E fragment were developed
using the pGL3 basic vector with the luciferase reporter gene (Promega,
Madison, WI). The plasmid constructs with either the putative rabbit
3'E or one of several promoters were used as controls. The 900-bp
XbaI/PstI fragment containing the putative hs1,2
was amplified by PCR from Fos15B with primers
5'-CGGGGATCCTCTAGAAGGA-3' and 3'- TTTGGATCCAGGACCAGTGCTGAGTGC-5'
and inserted into the BamHI site of the pGL3 basic vector
with and without a promoter in the 5' to 3' and 3' to 5' orientations
relative to the reporter gene. The 700-bp
EcoRI/BamHI fragment containing rabbit Eµ
(27) was also inserted into the
BamHI/SalI sites of pGL3 with and without a
VH promoter.
The rabbit VH1a2 promoter fragment (240
bp) was amplified by PCR with primers
5'-TAACAAGCTTAAAAATTCATATGATCTGAATC-3' and
3'-TCCTAAGCTTGGTGAGCGTCTGTGTTGA-5' from the 2.3-kb HindIII
fragment of cosmid clone cos 8.2 (32) containing
VH1a2. This 240-bp fragment (-240 to -1
relative to the ATG start codon) containing the TATA box, heptamer,
pyrimidine tract, and octamer was inserted into the pGL3 basic vector
with and without 3'E using HindIII sites in the 5' to 3'
orientation relative to the reporter gene. For the I promoter we
used the 180- and 450-bp fragments containing the rabbit I4 promoter
region: -180 to -2 bp and -450 to -2 bp upstream of the putative
ATG start site, respectively (33). Both I fragments
contained TATA-less promoter regions and a putative TGF-
-responsive
element. The 180- and 450-bp I promoter fragments were subcloned
into SacI/XhoI sites of the pGL3 vector with and
without the 900-bp fragment containing a putative enhancer.
After confirming the nucleotide sequences of all constructs, plasmid DNA was purified on CsCl gradients and used in transient transfection assays.
Cell lines
The rabbit B cell line 55D1, the plasmacytoma cell line 240E (27, 34), the murine A20 B cell line (American Type Culture Collection, Manassas, VA), and the rabbit CD4+ T cell line 484.3 (a gift from P. Medveczky, University of South Florida, Tampa, FL) were grown in RPMI 1640 supplemented with 10% FCS. The kidney cell line RK13 was maintained in DMEM with 10% FCS.
Transient transfection assays
The reporter constructs (20 µg) were transfected by electroporation into the various cell lines (20 x 106 logarithmically growing cells). Rabbit RK13 was transfected with Lipofectamine (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol (Life Technologies). Transfections were performed three times with each construct, except for transfections with 484.3 T cells, which were transfected twice. Luciferase activity was measured in duplicate in each experiment, and values were normalized to Renilla luciferase activity according to the manufacturer’s protocol (Promega). To estimate enhancer activity, the luciferase activities of constructs containing both enhancer and promoter were compared with reporter gene activity of the promoter-only control plasmid and are reported as the fold increase in luciferase activity. The data from transient transfection assays were statistically analyzed by Student’s t test.
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Results |
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E region(s)
To identify rabbit 3'E regions, a genomic DNA fosmid library
was screened with a C probe and with a rabbit 70-bp hs1,2 probe. We
identified 12 fosmid clones that hybridized with the C probe; two of
those fosmids also hybridized with hs1,2 probes. None of the fosmid
clones hybridized with mouse and human hs4 and hs3b probes.
The DNA of the two clones, Fos15B and Fos 3D, that hybridized with the
hs1,2 probe were overlapping and encompassed a total of 60 kb of DNA
(Fig. 1
). Hybridization with a C probe
showed the DNA from Fos 3D contained two C genes, and nucleotide
sequence analysis revealed that one of the C genes was identical
with C13 (30). The other C
gene has not been identified previously and is designated
C14 (GenBank accession no. AF314407).
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-containing fosmid clones, we attempted to PCR-amplify hs3 and
hs4 homologues from rabbit genomic DNA and C-containing fosmids
using primers from the most conserved region of both mouse and human
hs3a,b and hs4. We did not obtain a PCR product, and we conclude that
the rabbit genome either does not contain hs3 and hs4 homologues, or if
it does, they are highly divergent from mouse and human hs3 and hs4.
Because we did not identify hs3- and hs4-like elements, we focused the
remainder of our study on the hs1,2 putative enhancer.
We cloned and determined the nucleotide sequence from the 1.8-kb
XbaI fragment located 8 kb 3' of C13 that hybridized with
the hs1,2 probe. From the nucleotide sequence of the 900-bp
XbaI/PstI fragment (GenBank accession no.
AF314408), we found a 123-bp segment that was 84% identical with the
core region of human hs1,2 and 69% identical with that of mouse hs1,2
(Figs. 1
and 2). Significant homology was
not found between rabbit putative hs1,2 and other enhancers (human and
mouse hs3 and hs4). The region flanking hs1,2 was GC rich and contained
single and dinucleotide repeats (GA) similar to those seen in human and
mouse hs1,2 (8, 9). Based on sequence similarity to human
and rodent hs1,2 we conclude that the 900-bp
XbaI/PstI fragment contains a putative hs1,2
located 8 kb downstream of C13.
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To determine whether the rabbit genome contains more than one
hs1,2 homologue to the core region of human hs1,2, we performed
Southern blot analysis of XbaI-digested rabbit appendix
genomic DNA with the 900-bp XbaI/PstI fragment as
a probe (35). We found a single 1.8-kb band that
hybridized to this probe, a size corresponding to that in the fosmid
clones (Fig. 3). Similarly, the probe
hybridized to a single 2-kb BamHI fragment identical with
the hs1,2-hybridizing fragment in the fosmid clones (data not shown).
We conclude that the rabbit genome contains a single hs1,2 enhancer
homologue of human hs1,2.
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13 and hs1,2 is correct and that no other
enhancers similar to hs1,2 are present within 40 kb downstream of
C13.
Chromosomal organization of the C genes
The physical map of the previously identified 13 C genes showed
several clusters of C genes that could not be linked within the IgH
locus (24). To determine whether the 3'E is located at
the 3' end of the IgH locus, as in mouse and human, we attempted to
complete the physical map of the IgH region by identifying those
regions of DNA missing from the original map.
A fosmid clone, Fos 6, from a previous study (36)
contained a C gene downstream of C5. We
analyzed the nucleotide sequence of this gene and identified it as
C1, thus linking the
C1-containing cluster to the upstream genes
(Fig. 4). Ten additional C-hybridizing
fosmid clones spanning about 165 kb of the C region were obtained
from the fosmid library in this study and by restriction mapping and
nucleotide sequence analysis were found to overlap each other as well
as the clones identified in the previous map of the region. Fos 3B was
found to have additional DNA downstream of C1
that contained the I exon associated with C12,
thereby linking C12 within the locus (Fig. 4).
We were not able to clone from this library the DNA that spans the
region between C12 or Ca9 and the
next downstream genes; however, we propose the genetic map shown in
Fig. 4. According to this map, the hs1,2 resides at the 3' end of the
C gene cluster.
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The 900-bp XbaI/PstI fragment containing the
putative hs1,2 was tested for enhancer activity after cloning it into
the luciferase reporter vector pGL3 with various promoters: SV40,
rabbit VH1, and rabbit I4. We first
established that these promoters were active in the reporter gene
constructs following transient transfections of several B-lineage cell
lines and a rabbit T cell line and measured luciferase activity. We
determined the increase in luciferase activity over that for the pGL3
basic construct without any promoter (Fig. 5). The I promoters increased
luciferase expression up to 27-fold in B cells and 20-fold in a T cell
line, whereas the VH promoter increased
expression by 4-fold. We conclude that both VH
and I4 promoters are active in B and T cell lines.
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The hs1,2 was tested first with the SV40 promoter (Fig. 6A), because this promoter is
active with heterologous enhancers. The 900-bp fragment in the 5' to 3'
orientation increased expression of the luciferase gene 2.5- to
4.5-fold in rabbit B cells (55D1), plasmacytoma cells (240E), kidney
epithelial cells (RK13), and T cells (484.3); a 14-fold increase was
found in the murine A20 B cell line. In the 3' to 5' orientation, the
900-bp hs1,2 fragment demonstrated somewhat lower enhancer activity in
240E and 55D1 cells; 484.3 and A20 cells were not tested in this assay.
Analysis of these data by Student’s t test indicated that
the 900-bp XbaI/PstI fragment had significant
enhancer activity in the cell lines tested (p
< 0.02).
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hs1,2 in the germline. In
constructs with the VH promoter (Fig. 6B), the hs1,2 increased luciferase expression 6-fold in
55D1 B cells, 3-fold in 240E plasmacytoma cells, and >20-fold in
murine A20 B cells; the enhancement was independent of the orientation.
By Student’s t test these differences are significant
(p < 0.01), and we conclude that the 900-bp
fragment has significant enhancer activity with the
VH promoter in B cells.
We compared the enhancer activity of hs1,2 on the
VH promoter with that of Eµ, which is
considered a strong transcriptional enhancer, previously shown to
enhance the activity of a VH promoter (3, 37). We found that Eµ enhanced VH
promoter activity 12-fold in A20, 4-fold in 240E, and <2-fold in
55D1 (Fig. 6B). Overall, the activity of hs1,2 was similar
to that of Eµ in all cells tested, and we conclude that with the
VH promoter, the rabbit hs1,2 has enhancer
activity comparable to that of Eµ.
We also tested the hs1,2 with the I promoter, because I promoters
are in close proximity to the 3'E region and are potential targets
for hs1,2 activity. We found that the hs1,2 had a slight effect (up to
4-fold) on a 180-bp region of the I promoter in B cells (55D1 and
A20; Fig. 6C). These data suggest that the 900-bp fragment
has weak enhancer activity with the I4 promoter in the tested B
cells.
To assess the tissue specificity of hs1,2, we transfected a rabbit
kidney epithelial cell line, RK13, with the reporter constructs
containing VH, I, and SV40 promoters (Fig. 6).
We found a <2-fold increase in luciferase activity with the
VH promoter regardless of the orientation (Fig. 6B) and almost no activity with I promoters (Fig. 6C).
However, the hs1,2 in both orientations enhanced activity of the SV40
promoter (2.5-fold) in both B cells and epithelial cells (Fig. 6A). Similarly, a rabbit CD4+ T cell
line transfected with the same set of constructs (Fig. 6) showed a
2-fold increase in luciferase expression with the SV40 promoter and a
low level of activity with VH and I promoters.
The data indicate that, depending on the promoter, hs1,2 can function
in both B and non-B cells.
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Discussion |
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E. Several studies have shown that the 3'E is important for IgH
gene expression and class switching (7, 38). Because each
of the germline C genes in the human genome is associated with its
own 3'E, we thought that the rabbit genome with 13 C genes may
have multiple C regions and hence multiple enhancers. Instead, the
rabbit has a single hs1,2 homologue of the murine and human hs1,2
elements. We did not find a region homologous to the murine and human
hs3a and 3b as well as hs4 regions. It is possible, however, that other
enhancer elements that are not similar to hs3a,b and hs4
(12) are present in the germline.
While hs1,2 mapped 8 kb 3' of C13 in our
study, no hs1,2 was found downstream of the previously cloned
C13 (24). Because the size of the
HindIII restriction fragment (21 kb) on which both
C13 and hs1,2 are found in each of two
overlapping fosmid clones is the same as that found by a genomic
Southern blot analysis, we are confident that hs1,2 resides 8 kb 3'of
C13. In addition, the 8-kb XbaI
fragment immediately 5' of hs1,2 in Fos 15B and Fos 3D is identical
with that found by genomic Southern blot analysis (data not shown). We
suspect that in the previous study hs1,2 was not present in the
C13-containing recombinant phage due to either
a restriction polymorphism or deletion of DNA in the recombinant phage
library.
Although we have not completed the physical map of the C chromosomal
region, we think C13 is the 3'-most C gene.
Burnet et al. (24) previously identified several clusters
of C genes and by analyzing the DNA of 12 overlapping
C-containing fosmid clones, we linked some of these clusters
together (Fig. 4). We found that the order of the C genes 3'
of C
is
5'-C4-C5-C1-C2-C3-C7-C10-C11-C12//C8-C9-//C14-C13-3'E-3'.Although
we could not directly link either the C8 and C9 gene cluster or
the C14 and C13 gene
cluster to the other C genes, we suggest that
C13 is the 3'-most C gene because most of
the C genes are generally separated by 10 kb, and no C genes are
found within 30 kb 3' of C13.
The organization and nucleotide sequence of rabbit hs1,2 are similar to
those of hs1,2 in other species (8, 10). We found a 123-bp
region containing an octamer motif that had significant homology to the
human and mouse hs1,2 core regions and was flanked by segments with
single nucleotide repeats and GA-rich repeats (8, 9). In
fact, the rabbit hs1,2 contains nucleotide sequences similar to those
in the human that serve as binding sites for Oct-1 and NF-B, a set
of transcription factors that regulates human hs1,2 (6, 14, 16, 17, 18). The NF-B site in rabbit hs1,2 is located outside the
core region, similar to that of mouse hs1,2. We think that this site is
functional because a rabbit probe containing the NF-B site of hs1,2
was shifted by nuclear extracts from anti-µ-activated primary
rabbit B cells as tested by EMSA (unpublished observations). We did
not, however, identify binding sites for B-cell lineage-specific
activator protein within the 900-bp fragment, suggesting that in the
rabbit, control of the IgH locus involves sets of transcription factors
binding to hs1,2 different from those found in the mouse.
Functional assays showed that the rabbit hs1,2 enhances transcription
from a viral promoter (SV40) and the promoter of rabbit
VH1, and modestly from the I promoter
region of rabbit C4. It is not clear which
promoters are targets for hs1,2 in vivo, although in vitro 3'E has
been shown to act on several Ig promoters, including
VH, V, and V
(9, 10, 20). We
also tested rabbit hs1,2 with an I promoter because these are
located in close proximity to 3'E. Rabbit hs1,2 weakly enhanced the
I promoter. In contrast, Yanzhong et al. (39) recently
showed that human hs1,2 strongly enhanced I1 and I2 promoters.
While hs1,2 enhanced the activity of the rabbit 180-bp I promoter
more than that of the 450-bp I promoter, we suggest that the overall
low level of enhancement in our experiments may be because the hs1,2
enhancer and I promoter require a combination of transcription
factors that is not expressed in the cell lines used in this study.
The rabbit hs1,2 enhanced the activity of the VH
promoter in vitro at a level similar to or greater than that of Eµ,
which is known to regulate Ig gene expression. The enhancement of
VH and I
promoter activity by 3'E has been
shown in transgenic mice, suggesting that 3'E can regulate promoters
as far as 100 kb upstream (19, 20, 21). Further
characterization of hs1,2 and its targets is required for determining
how hs1,2 contributes to immune responses in vivo.
In mice and humans hs1,2 enhancer activity is B cell specific (5, 6, 8, 9, 10). In our experiments we found that while hs1,2 had a high level of activity in murine A20 B cells, the activity in rabbit B cells was considerably lower. Further, the activity in rabbit T and epithelial cells was nearly equal to that in rabbit B cells, raising the possibility that hs1,2 is not B cell specific. We think, however, that the hs1,2 enhancer probably is B cell specific, but that because the rabbit B cell lines were obtained from c-myc and c-myc/v-abl transgenic rabbits, in which expression of the oncogenes was controlled by Eµ (27, 34), the low level of activity may result from inhibition of hs1,2 by a high level expression of c-myc/v-abl. However, we cannot exclude that the difference in hs1,2 activity in mouse and rabbit cells is due to inherent differences between rabbit and mouse cell lines.
We undertook this study with the idea that a 3'E would be located
adjacent to each of the 13 C genes and that differences in the
3'E associated with each gene might explain the differential
expression of the C genes in various tissue. Our results
demonstrate that only one hs1,2 resides in the germline. While
additional functional analyses of hs1,2 will be required to elucidate
the specific role of IgH 3'E, we suggest that hs1,2 may interact
with I promoter and regulate in part C gene expression in vivo.
Studies to directly test this idea need to be performed.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Katherine L. Knight, Loyola University Chicago, Department of Microbiology and Immunology, 2160 South First Avenue, Maywood, IL 60153.
3 Abbreviations used in this paper: Eµ, intronic enhancer; 3'E, 3' enhancer; hs, hypersensitivity site.
Received for publication January 18, 2000. Accepted for publication September 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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constant region. Mol. Cell Biol. 13:1547. genes. J. Exp. Med. 186:845.1 and C2 genes. J. Immunol. 159:1310.[Abstract]
3'E and hs3). J. Immunol. 158:4780.[Abstract]
-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23:975.E(hs1, 2). Proc. Natl. Acad. Sci. USA 93:4392.B-binding proteins. J. Immunol. 156:2828.[Abstract]
enhancer by suppressing binding of NF-P, a protein that controls heavy chain transcription. Proc. Natl. Acad. Sci. USA 92:5336. regulated germline transcripts are expressed from 2a transgenes independently of the heavy chain 3' enhancers. J. Immunol. 163:5758. enhancer are differentially methylated and DNase I hypersensitive during B cell differentiation. J. Immunol. 150:1772.[Abstract]
genes. EMBO J. 8:4041.[Medline]
genes as chimeric rabbit-mouse IgA antibodies. Proc. Natl. Acad. Sci. USA 86:7561. and IL-2. J. Immunol. 162:5380. enhancers. J. Immunol. 164:6380.This article has been cited by other articles:
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  V. Volgina, P.-C. Yam, and K. L. Knight A negative regulatory element in the rabbit 3'IgH chromosomal region Int. Immunol., August 1, 2005; 17(8): 973 - 982. [Abstract] [Full Text] [PDF]
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 H. Spieker-Polet, P.-C. Yam, and K. L. Knight Functional Analysis of I{alpha} Promoter Regions of Multiple IgA Heavy Chain Genes J. Immunol., April 1, 2002; 168(7): 3360 - 3368. [Abstract] [Full Text] [PDF]
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), VH
(
), I
), I
). Bars represent luciferase
activity in cells transfected with promoter-containing plasmid compared
with cells transfected with basic pGL3 vector (fold increase was
calculated by: activity of pGL3 with promoter/activity of pGL3 basic).
The cells used for transfection were 55D1 (rabbit B cell line), 240E
(rabbit plasmacytoma), 484.3 (rabbit T cell line), and A20 (mouse B
cell line). The SD was determined from duplicates of three independent
transfections.
3';
, 5'
, 3'