By
§
§
From * The Howard Hughes Medical Institute and The Children's Hospital, Boston, Massachusetts
02115; and the § Center for Blood Research and
Department of Genetics, Harvard Medical School,
Boston, Massachusetts 02115
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Abstract |
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The 40-kb region downstream of the most 3' immunoglobulin (Ig) heavy chain constant region gene (C) contains a series of transcriptional enhancers speculated to play a role in Ig
heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR
regulatory region, we generated mice with germline mutations in which one or the other of
the two most 5' enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a
loxP sequence (referred to as HS3a
and HS1,2
, respectively). B cells homozygous for the
HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic
similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3a
or HS1,2
mutations. In
addition, induced expression of the specifically targeted pgk-neor genes was regulated similarly
to that of germline CH genes. Our findings implicate a 3' CSR regulatory locus that appears remarkably similar in organization and function to the
-globin gene 5' LCR and which we
propose may regulate differential CSR via a promoter competition mechanism.
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Introduction |
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Immunoglobulin (Ig) variable regions are encoded by
germ-line V, (D), and J gene segments that are assembled
during early B cell differentiation by V(D)J recombination
(for review see reference 1). The Ig heavy (H)1 chain locus
contains eight different constant region (CH) genes with the
organization: 5'V(D)J-Cµ-C-C
3-C
1-C
2b-C
2a-C
-C
-3'. Differentiating B lymphocytes first produce H chains
in the context of an IgM surface receptor. During antigen-driven B cell maturation, B cells can secrete specific antibody
with a different CH region effector function by juxtaposing
the antigen-specific V(D)J gene to a different downstream
CH gene via a recombination/deletion process termed class
switch recombination (CSR; for review see reference 2).
CSR occurs between tandem repetitive switch (S) region
sequences present 5' of individual germline CH genes.
CSR to particular CH genes is directed by different combinations of activators and lymphokines (3). For example, stimulation of B cells with bacterial LPS induces CSR
to the C2b and C
3; whereas LPS plus IL-4 induces CSR
to C
1 and C
. Factors that modulate switching to particular CH genes correspondingly modulate germline transcription of those genes before CSR (7). Thus, LPS treatment of splenic B cells induces germline C
2b and C
3 transcription; whereas LPS plus IL-4 treatment suppresses
germline C
2b and C
3 expression, and induces germline
C
1 and C
gene transcription. All CH gene transcripts initiate at an exon termed I that lies 5' to each S region, proceed through the S region and CH gene, and are processed
to yield noncoding transcripts with the I exon spliced to
the CH exon. Gene-targeted mutational analyses of I region
exons/promoters have confirmed that germline CH transcription and/or transcripts play a direct role in CSR (14-
19). Therefore, control of CSR is directly related to ability to control transcription of the various CH genes.
Understanding of the mechanisms that regulate CSR will require elucidation of both trans- and cis-acting elements that modulate germline CH transcription. In this context, CH gene promoters contain consensus sequences that are responsive to specific lymphokines (reviewed by Coffman et al. [20]). Yet, properly regulated expression of germline CH genes requires sequences beyond those of their proximal promoters (21). In this regard, efficient CSR from Cµ to a downstream CH gene requires the transcriptional enhancer element (iEµ) found in the JH/Cµ intron (22). However, the expression of switched transcripts or translocated oncogenes on alleles in which iEµ is absent suggested the presence of additional downstream positive regulatory sequences (23).
The first candidate for such a downstream transcriptional
enhancer was the so-called 3'CE or 3'EH (Fig. 1, A and
B), which was identified ~15 kb 3' of C
based on ability
to enhance transcription specifically in B lineage cells (26-
28). Homozygous replacement of this element (also referred to as HS1,2, see below) with a pgk-neor gene disrupted CSR and germline transcription of a series of CH
genes, including C
3 which lies 120 kb upstream (29). Assuming that these effects were cis-acting, one hypothesis to
explain this phenotype was that HS1,2 is critical for induction of germline CH gene transcription and that its deletion
was the primary cause of the CSR phenotype (29, 30). A
second possibility is that insertion of the pgk-neor gene cassette in the 3' IgH locus disrupts the normal regulation of
germline transcription/CSR by interfering with the activities of additional required regulatory elements (29).
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The 40-kb region just downstream from C contains
four enhancer elements (that correspond to DNAase1 hypersensitive or HS sites): HS3a (or C
3'E), which lies 4 kb
3' to the C
; HS1,2 (or 3'C
E), which lies 15 kb 3' to
C
; HS3b, which lies 25 kb 3' to C
; and HS4, which lies
~30 kb 3' to C
(26, 31, 32). Like the HS1,2 sequence, HS3a (31) and HS3b (32) enhance reporter gene
expression in activated B cells and plasma cells. On the other hand, HS4 is active throughout B cell development
(32, 33). HS3a, HS1,2, and HS3b appear to represent a unit
with HS3a and HS3b sharing high sequence homology,
but lying in inverted orientation in the chromosome (34,
35). Recent studies have shown these enhancer sequences
are conserved in man, consistent with an important regulatory function (36, 37).
Combinations of HS3b, HS1,2, and HS4 had synergistic
transcriptional enhancing effects when assayed in a transgenic mouse model, and were able to induce copy number
and position-independent reporter gene expression, suggesting that these sequences have locus control region
(LCR) properties (32). In this regard, a similar set of HS
sites that lie upstream of the -globin gene locus constitute
a
-globin LCR, that apparently is responsible for coordinating expression of the various
-globin genes during development (reviewed by Martin et al. [38]). Individual HS
sites within the
-globin LCR also have transcriptional enhancer activity and replacement of 2 of these individually
with an expressed selectable marker gene cassette resulted
in decreased
-globin expression across the locus (39),
an effect reminiscent of what was observed when HS1,2
was replaced with a neor gene (29). Yet "clean" deletion of
the
-globin HS sites had no major effect, indicating that
the effects of the pgk-neor gene insertion resulted from interference with additional regulatory sequences (40, 41).
To elucidate the function of the putative 3' IgH regulatory region and assess the roles of specific enhancers, we have generated mice with germline mutations in which a pgk-neor cassette or a loxP site replaced either the HS3a or the HS1,2 elements and then assayed the effects of these mutations on the CSR process.
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Materials and Methods |
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Vector Construction, Transfection, and Embryonic Stem Cell Screening.
For the generation of HS1,2 mutant mice, a previously described construct (29) was modified by deleting its existing pgk- neor cassette and substituting a loxP flanked pgk-neor cassette in the central Not1 site, by blunt end ligation (Fig. 1 D). The deletion spanned from a Pst-1 site 1.8 kb 5' of the HS1,2 core element (42) to an EcoR1 site 1.7 kb 3' of the element. This construct was linearized using Pvu-1. A 600-bp EcoR1-Xba fragment 5' of HS1,2 was used as a probe in Southern blotting. The HS3a mutant embryonic stem (ES) cells were derived using pLNTK vector P3 (43), cloning the 3-kb H3 fragment containing the alpha membrane exon in the Sal1 site and a 3.4-kb Hind3-Xba fragment into the Xho site, and deleting the 2-kb fragment that contains the previously described 900-bp enhancer (31) HS3a (Fig. 1 C). This construct was linearized using Sal1. A 500-bp EcoR1-Hind3 fragment was used as a probe in Southern blotting. Approximately 20 µg of linearized construct DNA was electroporated into 2 × 107 J1 or E14 ES cells (HS1,2 and HS3a targetings, respectively) and homologous recombinants were selected as previously described (44). One ES clone was injected into C57Bl/6 blastocysts to generate germline chimeric mice, each where a pgk-neor cassette inserted correctly into one allele of either HS3a or HS1,2. Germline transmitting mice were then interbred to produce either mice homozygous for the HS3aN or HS1,2N mutation. Females homozygous for either mutation were bred with males containing an EIIa-Cre transgene (43, 45) to produce mice heterozygous for HS3aSpleen Cell Cultures.
Single-cell suspensions of spleen cells were cultured at 5 × 105 cells/ml in RPMI medium supplemented with 10% FCS and 20 µg/ml LPS with or without 50 ng/ml of mouse recombinant IL-4 as previously described (18). Cultures for IgG2a and IgA production were prepared as previously described (29). Cells were harvested for FACs, and supernatants assayed on days 4-5.Flow Cytometry Analysis.
Single-cell suspensions from spleens were prepared as previously described (18). Cells from day 4 or 5 cultures were washed in PBS, 2% FCS and stained with various antibodies conjugated with fluorescein (IgG1), phycoerythrin (IgM), biotin (IgG2b, IgG3, IgE, IgG2ba/IgG2aa, IgG2aa), or Cychrome (B220; PharMingen, San Diego, CA). Biotin conjugates were revealed by phycoerythrin-streptavidin (PharMingen). The cells were analyzed on a FACScalibur® (Becton Dickinson & Co., Sparks, MD) and analyzed using Cellquest software, and are presented as dot plots after gating for live cells.ELISA Assays.
Supernatants from spleen cell cultures and sera from normal B6/CBA mice or germline mutant mice were analyzed for the presence of different immunoglobulin isotypes by ELISA as described (18). Cultures were established in triplicate for each assay. In total, three independent culture experiments were performed for wild-type and the four different 3' IgH enhancer mutant mice. Mice ranged in age from 6 wk to 3 mo, and culture supernatants were assayed after 5 d of stimulation.Southern and Northern Blot Analyses.
Genomic DNA was prepared as previously described (46). RNA was prepared using the TRIZOL reagent (GIBCO BRL, Gaithersburg, MD) as per the instructions of the manufacturer. Southern and Northern blot analyses were performed as described elsewhere (18). The mb-1 probe was generated from a full-length cDNA. The Pgk probe is a 300-bp R1-H3 fragment, and the neo probe is a 500-bp R1-Pst fragment. The IPCR Amplification of Germline Transcripts.
Total RNA was isolated from 1 × 106 day 3 LPS/IL-4-stimulated splenocytes, using the TRIZOL reagent (GIBCO BRL) as per the manufacturer's instructions. cDNA was generated using Superscript (GIBCO BRL), again according to manufacturer's instructions. For CGeneration of Chimeric Mutant Mice of a Randomly Integrated HS1,2 Targeting Construct.
Four independent ES cell lines that contained randomly inserted HS1,2 targeting constructs (HS1,- 2RAN) and two HS1,2N/+ ES subclones were injected into blastocysts from the RAG-2-deficient mice and transplanted into foster mothers (B6/CBA) as described (49). Chimeric mice with lymphocytes derived from either the randomly or specifically targeted HS1,2N constructs were analyzed in splenic cultures as described above. ![]() |
Results |
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ES cells were transfected separately with
constructs that replace either the entire HS3a or HS1,2
with a loxP-flanked pgk-neor gene cassette (Fig. 1, B-D;
mutations termed HS3aN and HS1,2N, respectively). ES
clones with appropriate targetings of the endogenous loci
were used to introduce each of the mutations into the murine germline. Mice homozygous for either replacement
mutation appeared normal and had B cell numbers in peripheral lymphoid organs that were similar to those of
wild-type controls (data not shown). To generate mice
with a "clean" deletion of each of the respective enhancer
elements, the introduced loxP-flanked pgk-neor gene was
deleted by breeding the mice containing the HS3aN or HS1,2N mutations with a mouse that expresses an EIIa-Cre recombinase transgene at a very early developmental
stage, and which permits generation of progeny that have
deleted the neor gene in the germline (45). From the progeny of this cross, we identified individuals in which the
pgk-neor gene was deleted, resulting in replacement of the
HS3a or HS1,2 sites with only a loxP sequence. Mice homozygous for these deletion mutations (termed HS3a and
HS1,2
, respectively; Fig. 1) again appeared normal, and
contained B cell numbers comparable to those of wild-type
mice (data not shown).
Previously, homozygous replacement of HS1,2 with a pgk-neor cassette in ES cells followed by assay via the RAG-2-deficient blastocyst complementation system showed that this mutation led to chimeric mice with markedly decreased serum levels of IgG3 and IgG2a, but relatively normal levels of other isotypes including IgM (29). To assay the effects of the germline replacement mutations, we quantified serum Ig levels in mice homozygous for the HS1,2N and HS3aN mutations (referred to as HS1,2N/N and HS3aN/N mice, respectively). The serum IgM levels in both mutant lines were similar to those of wild-type control mice (Fig. 2). Levels of IgG1, IgG2b, and IgA also were substantial but there was no detectable IgG3 or IgG2a (Fig. 2). IgE levels were not determined as they were below the detection level of our assay even in normal mice (not shown). Thus, replacement of either HS1,2 or HS3a with a pgk-neor cassette resulted in essentially identical defects in serum Ig expression, even though the sites of the replacement mutations were separated by 12 kb within the downstream IgH region. This finding indicates that either HS3a and HS1,2 are independently essential for promoting CSR to these isotypes or that the neor gene interferes with additional elements when placed at either site.
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Our previous studies showed
that splenic B cells generated from ES cells homozygous for
a HS1,2-pgk-neor gene via replacement mutation by RAG-2-deficient blastocyst complementation had severe defects
in CSR and in transcription of the C3, C
2b, C
2a, and
C
genes (29). To determine whether HS1,2N/N or
HS3aN/N splenic B cells were similarly affected, we cultured both types of mutant B cells for 4 or 5 d in the presence of either LPS, LPS + IL-4, LPS + IFN-
, or LPS + TGF-
, agents that collectively are known to induce class
switching to C
3 and C
2b, C
1 and C
, or C
2a and
C
, respectively. Both mutant cell types proliferated similarly to normal cells after the various treatments (data not shown). Supernatant antibody levels in the cultures after 5 days of treatment were measured by ELISA using isotype
specific antibodies. After appropriate stimulation, secreted
levels of IgM were similar in cultures of HS1,2N/N,
HS3aN/N and wild-type B cells, while the levels of IgG1
and IgA secreted by the mutant cells were significant but
generally lower than those of normal control B cells (Fig. 3). However, IgG3, IgG2b, IgG2a, or IgE were not detectable in the supernatants of the HS1,2N/N and HS3aN/N
B cells, which indicated a reduction of 100-fold or more
from wild-type levels (Fig. 3). Surface stains of day 4 stimulated cells confirmed a substantial inhibition of switching to
IgG3, IgG2b, IgG2a, and IgE, accompanied by an apparently slight inhibition of switching to IgG1 and relatively normal switching to IgA (data not shown).
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To analyze the consequences of replacing HS3a with
pgk-neor cassette on expression of germline CH transcripts,
we assayed total RNA from LPS and LPS plus IL-4-stimulated HS1,2N/N and HS3aN/N B cells for hybridization
to an I2b-region specific probe (Fig. 4 A). These studies
demonstrated major inhibition in the expression of germline I
2b transcripts in LPS cultures of the N/N cells (Fig.
4 A). Additional assays revealed a similar lack of germline transcripts from other CH genes (e.g., C
3 and C
) after
appropriate in vitro stimulation of HS1,2N/N and
HS3aN/N B cells (Fig. 4 C). Together, these findings show
that homozygous replacement of either HS3a or HS1,2
with a pgk-neor gene results in an essentially identical defect
of CSR to IgG3, IgG2b, IgG2a, and IgE after in vitro stimulation, and that this defect corresponds in turn to a block
in the induction of the respective germline CH transcripts.
Again, these findings could be consistent with either a required function for both HS3a and HS1,2 in the CSR process or an effect of the inserted pgk-neor gene on an additional regulatory element.
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The defect in CSR observed in either the HS3aN/N or HS1,2N/N B cells could occur via disruption of a critical cis-acting control element for CSR (29). To directly assay for this, we generated mice harboring one allele with a pgk-neor replaced enhancer element, and one wild-type allele. The ES cells used in our targetings were derived from the 129 mouse strain which carries the IgHa haplotype. The mutant mice were bred with C57Bl/6 mice that carry the IgHb haplotype, allowing the generation of heterozygous mutant progeny that harbor an IgHa allele with the neor mutation and a wild-type IgHb allele. The haplotypic differences between the IgHa and the IgHb alleles can be distinguished at the DNA level (based on nucleotide sequence differences manifested as unique restriction endonuclease sites), as well as at the protein level by antibodies specific for the IgHa and IgHb polymorphisms.
Splenocytes from the wild-type, HS3aN/+, and HS1,2N/+ mutant mice were stimulated in vitro with either LPS or LPS+IL-4, and activated B cells were collected for analysis after 4 d of stimulation. LPS-stimulated cells were doubly stained with a biotin-conjugated antibody that recognizes the IgHa type allele of IgG2b and IgG2a isotypes (IgG2ba and IgG2aa, respectively), and one that recognizes total IgG2b (both IgHa and IgHb allotypes). Wild-type B cells heterozygous for the normal IgHa and IgHb alleles had similar numbers of B cells that stained for either surface IgG2ba or IgG2bb positive cells (Fig. 5 A). However, mice heterozygous for either mutant IgHa allele and the wild-type IgHb allele expressed only surface IgG2bb positive B cells indicating a cis-acting defect of the mutations on class switching to IgG2b (Fig. 5 A, labeled HS3aNa/+b or HS1,2Na/+b).
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To assay for allele-specific transcripts of the C region,
we employed PCR for specific amplification of all transcripts through the C
constant region. The IgEb haplotype
contains an additional Sty-1 restriction site in the 453 base
pairs spanning the first 2 C
constant region exons, as compared with the IgEa haplotype. Therefore, RT-PCR products of transcripts arising from the two alleles can be differentiated based on the size of fragments generated after
digestion with Sty-1. LPS plus IL-4-stimulated B cells from
mice heterozygous for the wild-type IgHa and IgHb alleles
generated an approximately equal ratio of C
-containing transcripts from the two alleles (Fig. 5 B). In contrast,
almost all detectable C
-transcripts in LPS plus IL-4-stimulated B cells from mice heterozygous for either the
HS1,2N or HS3aN IgHa allele and the wild-type IgHb allele were generated from the wild-type (IgHb) allele (Fig. 5 B).
In summary, both pgk-neor replacement of either HS3a or HS1,2 regions resulted in a cis-acting defect in CSR, as measured both by transcripts and surface protein expression, indicating that the effects of the neor gene replacement mutations disrupted a cis-acting control element.
Normal Class Switch Recombination and Germline CH Transcription in HS3aTo further elucidate the mechanism by which the HS3aN and HS1,2N
insertion mutations inhibited CSR, we assayed for class
switching by B cells of HS3a/
and HS1,2
/
mice.
Splenic B cells from both homozygous deletion mutant
mice expressed a wild-type distribution of surface IgM and
IgD (data not shown). Likewise, the levels of serum IgM
and all other assayed downstream isotypes were similar to
those of the wild-type control animals (Fig. 2). Thus, specific deletion of either the HS3a or HS1,2 enhancers had
no readily measurable effect on serum Ig levels. Moreover,
after LPS or LPS plus lymphokine stimulation of HS3a
/
and HS1,2
/
splenic B cells, we observed induction of
surface and secreted Ig isotypes that were generally similar
in level to those of wild-type control mice (Fig. 3 and data
not shown). Although there were potentially small effects
on switching to certain isotypes (e.g., IgG2b and IgE; Fig.
3), these are difficult to assess given the inherent variations
in these population assays. Overall, our results indicate that
neither the HS1,2 nor the HS3a enhancers are required for LPS and lymphokine-stimulated CSR and expression of
IgH isotypes.
To test for the effects of specific deletions of HS3a or
HS1,2 on expression of germline CH transcripts, we assayed
RNA prepared from the LPS or LPS plus IL-4-treated cultures of HS3a/
and HS1,2
/
B cells for hybridization
to an I
2b-specific probe (Fig. 4 B). We readily observed
the induction of I
2b-containing germline transcripts upon
LPS activation of these cells, in striking contrast to the lack
of induction observed in LPS-activated cultures of HS3aN/N
and HS1,2N/N B cells. (compare Fig. 4, A with B). PCR amplification of germline transcripts containing I
3 or I
yielded similar findings (Fig. 4 C). We do note that the extent of induction appeared to be less in the HS1,2
/
B
cells than in wt cells (Fig. 4 B and data not shown). Although the potential for more modest effects on expression
will need to be examined in further detail, we conclude
from these studies that neither the HS3a nor the HS1,2 enhancers are absolutely needed for substantial induction of
germline CH gene transcription or the CSR process that
follows.
Insertion of the pgk-neor cassette into either the HS3a or HS1,2 location in the 3' IgH locus inhibits germline transcription of CH genes up to 120 kb away (Fig. 4). To further analyze the potential mechanisms involved, we assayed for expression of the pgk-neor cassette when it was inserted in place of the HS1,2 sequences. For this purpose, we assayed total splenocyte RNA from HS1,2N/+ mice for hybridization to a neor gene-specific probe. Expression of endogenous pgk sequences (which are ubiquitously expressed) and the Mb-1 sequence (which is B cell specific) was measured as a control. RNA from thymus and from nonlymphoid tissues had very low levels of pgk-neor transcripts, from the specifically inserted pgk-neor gene (data not shown). However, HS1,2N spleen RNA contained significant levels of neor transcripts (Fig. 6, lanes 3 and 5). To determine if these neor transcripts were expressed and inducible in B lymphocytes, we assayed RNA from cultures of HS1,2N/+ spleen cells treated for 4 or 5 d with LPS. These RNA preparations showed a significantly higher level of neor gene transcripts (representing as much as a fivefold induction; Fig. 6; compare lanes 3 and 5 with lanes 9, 11, 14, and 16). Furthermore, we also observed similarly elevated expression levels of the pgk-neor gene in LPS-treated HS3aN/N splenic B cells (data not shown), indicating that such induction occurs with respect to neor genes targeted at two independent sites in the 3' IgH locus.
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These findings suggested that the pgk-neor sequences inserted into the 3' IgH region are specifically expressed and LPS-inducible in B cells. As a control for this experiment, we used ES cell lines that contained randomly inserted HS1,2 targeting constructs to generate splenic B cell populations by the RAG-2-deficient blastocyst complementation method (49). Spleen cells from these chimeric mice were then assayed for neor and control gene transcription before and after 5 d of culture in LPS. These cells proliferated and underwent class switching to IgG3 and IgG2b in a manner similar to that of normal splenic B cells. However, none of four independent populations of spleen cells with random integrations of the HS1,2 targeting construct expressed levels of the pgk-neor gene above the very low levels found in thymus or non-lymphoid tissues, either before or after LPS treatment (Fig. 6, compare lanes 1, 2, and 4 with lanes 7, 8, 10 and 12, 13, 15).
Together, these data indicate that specific targeting of the pgk-neor gene into the 3' IgH locus leads to its upregulated expression in total splenic B cells and that this expression level is further augmented in LPS-stimulated B cell populations.
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Discussion |
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The 40-kb region directly 3' of the IgH locus contains four known transcriptional enhancer sequences, including HS3a, HS1,2, HS3b, and HS4 (reviewed by Birshtein[42]) and is referred to as the 3' IgH regulatory region. Cell transfection and transgenic studies indicated that the individual enhancers are active in stimulated or terminally differentiated B cells, suggesting they are involved in controlling late B cell differentiation events such as CSR and high level IgH gene expression (26, 30, 32, 50). However, until now, there has been no direct examination of the potential role of any of these elements during normal B cell development. Although we previously demonstrated that replacement of the HS1,2 enhancer with a pgk-neor cassette disrupted CSR to multiple CH genes, the mechanism was not clear. Thus, the block in CSR could have resulted from the deletion of the HS1,2 enhancer, an inhibitory effect of the inserted pgk-neor cassette on other elements, or both (29). Furthermore, the effects of the previous mutation could have resulted from disrupting expression of a gene encoding a trans-acting factor necessary for CSR (29). Our current work clearly resolves these possibilities by showing that neither HS3a nor HS1,2 are necessary for CSR or IgH gene expression; whereas replacement of either with a pgk- neor cassette interferes with CSR and germline transcription of distant, cis-linked CH genes.
HS3a and HS1,2 Are Not Required for CSR or IgH Expression.HS1,2 was identified as a strong transcriptional enhancer that is active specifically in stimulated B cells and
plasma cells (26, 28). Previous studies demonstrated that
HS1,2 increased expression of linked transgenes and rendered them LPS-inducible in B lineage cells (50, 51).
HS1,2 also contains numerous transcription factor binding
sites (52), further implicating it in transcriptional regulation or related processes. HS3a, which lies 12 kb upstream of HS1,2 and immediately downstream of the C
gene, was similarly defined based on ability to enhance reporter gene expression specifically in late-stage B lineage
cell lines (31). This activity, combined with the finding that
HS3a also contains numerous factor-binding motifs, again
led to speculation about its potential roles in Ig HC gene
expression or CSR (31). Our current studies are consistent
with the possibility that these enhancers, in particular
HS1,2, may have some role in influencing expression (Fig.
4). However, our finding that neither HS1,2 nor HS3a is
essential for V(D)J recombination, substantial expression of
germline CH transcripts, or IgH CSR and IgH expression is
surprising and necessitates consideration of other potential
functions for these elements. One possibility is in the somatic hypermutation process as 3' IgH region sequences
have been implicated by the finding of an increased occurrence of mutations in VHDJH transgenes recombined into
the IgH locus (56). The availability of mice containing germline deletions of the individual 3' IgH enhancers will facilitate searches for more specific functions.
The lack of major effect of the HS3a or HS1,2 deletions
on measured processes might result from redundancy.
HS3a and/or HS1,2 share potential transcription factor
binding sites with HS3b and/or HS4 (36). Therefore, these
enhancers may have overlapping or redundant functions,
which could obviate major effects of the deletion of a single element. In this context, disruption of the NF-B p50
gene led to a phenotype reminiscent of the homozygous HS1,2N/N or HS3aN/N phenotype, with impaired CSR
to an overlapping set of CH genes (57). As there are NF-
B-binding sites in both the HS1,2 and HS4 enhancers
(58), it is possible that the NF-
B KO phenotype could result, at least in part, by interfering with the function of both
enhancers. Enhancer redundancy is found in the Ig
locus,
where the intronic and 3' enhancers appear to have overlapping functions with respect to Ig
gene rearrangement
and expression (43, 59). For some processes, the intronic
Eµ also may work in conjunction with 3' IgH sequences,
as expression of rearranged V(D)J sequences in iEµ deleted
cell lines was suppressed upon replacement of HS1,2 with a
pgk-neor gene (30).
Our studies also demonstrate that expression of the C1
and C
genes are less affected in vitro or in vivo even with
respect to the HS1,2N or HS3aN mutations, suggesting
that expression of these genes may be positively influenced
by control elements not influenced by the 3' IgH regulatory locus (29). Likewise, the finding that IgG2b levels are
suppressed with respect to in vitro LPS stimulation of
HS3aN/N and HS1,2N/N B cells but are relatively normal
in vivo in the corresponding mice, supports the notion that
C
2b can be activated in vivo by a novel pathway which is independent of the 3' IgH regulatory region. The availability of the germline mutant mice will facilitate the search for
such novel activating pathways and elements.
The 3' IgH regulatory region is quite
reminiscent in organization to the LCR region 5' of the globin locus which similarly contains four erythroid-specific HS sites that individually harbor distinct transcriptional enhancer activity (60). The
-globin LCR confers
tissue-specific, high level, position-independent expression
to cis-linked genes (61, 62), enhances transcription as far as
70 kb away, and influences chromatin structure and timing of replication over >200 kb (63, 64). Similarly, the combined HS1,2, HS3b, and HS4 sequences induced copy
number and position independence to transfected constructs, suggesting the 3' IgH region also may function as
an LCR (32). In another striking parallel to our current
findings, replacement of either the HS3 or HS2 enhancers
of the
-globin LCR with an expressed selectable marker
gene resulted in a severe block in the expression of the linked
-globin locus; but, in both cases,
-globin locus
gene expression was substantially restored upon removal of
the selectable marker gene (40, 41). Heterozygous deletion
of the complete
-globin LCR in human cells of a thallassemia carrier eliminated
-globin gene expression in cis,
implicating its essential role in
-globin expression and
suggesting that the individual enhancer elements are redundant (65). By analogy, more significant effects on CSR or
expression may occur upon simultaneous deletion of multiple 3' enhancer elements (66). As insertion of the pgk-neor
cassette into both the 3' IgH HS3a and HS1,2 locations
blocks CSR to the same upstream genes and, in both cases
renders the inserted pgk-neor cassette LPS inducible, it
seems likely that major distal elements necessary for LPS induction of germline CH transcription still lie downstream of
HS1,2, with HS3b and HS4 being prime candidates.
In addition to the 3' IgH locus and the -globin locus,
inhibition of expression due to an inserted pgk-neor cassette
has been observed in several other loci (41, 43, 59, 67).
Although the effects of the inserted pgk-neor gene in different loci may not necessarily occur via a single mechanism, a
common theme is the potential of complex, long-range mechanisms that have evolved to control expression of developmentally regulated multi-gene loci. The effect of the
pgk-neor gene insertion in the
-globin LCR or the 3' IgH
region could be via the inhibition of the neighboring enhancer elements (38, 40, 41); for example, the HS1,2 replacement might inhibit transcription factors binding to the
HS3a, HS3b, or HS4 elements, and thereby inhibit all
three enhancer elements. However, a more likely scenario
is that the pgk-neor gene insertion results in interference
with long-range transcriptional control elements involved
directly with promoter-LCR interactions (71, 72) or long-range effects on chromatin structure that modulate distal
promoter function (73). Recent studies of a transgenic
-globin locus showed that a second
-globin gene competed more efficiently with other genes to which it was
LCR-proximal (74), supporting the looping model of
LCR function in which one gene interacts with the LCR
at a time (75).
Additional insight into the potential mechanisms by
which the 3' IgH regulatory locus may modulate germline
CH transcription and/or CSR comes from recent studies of
lymphocytes in which the C or the I
2b exons were replaced with a pgk-neor gene; class switching to CH genes
upstream, but not downstream of the pgk-neor insertion
was inhibited, again with the exception of switching to
IgG1 (Seidl, K., H. Oettgen, and F. Alt, manuscript in
preparation). On the other hand, other recent studies of B
cells in which the intronic Eµ element was replaced with a
pgk-neor cassette showed that the inserted pgk-neor cassette
maintained CSR to downstream CH genes at relatively normal levels even in the absence of Eµ (76). In this case, the expressed pgk-neor cassette may have provided the necessary transcriptional functions to promote CSR at the Sµ
region whereas downstream germline transcription units
were unaffected by this 5' insertion. Together, all of these studies are consistent with the notion that the pgk-neor insertions inhibit CSR in a polarized fashion, primarily affecting germline transcription units 5' to the insertion site.
In this regard, the
-globin LCR has been speculated to
regulate differential
-globin locus gene expression via a
promoter competition mechanism (71, 77). The 3' IgH
regulatory region might employ a similar mechanism to
regulate differential expression of CH genes dependent on
this region (29). Whatever the absolute mechanism, the
many similarities between the overall organization of the
-globin LCR and the 3' IgH region suggest that these
two loci have evolved similar strategies to regulate differential gene expression.
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Footnotes |
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Address correspondence to Frederick W. Alt, The Howard Hughes Medical Institute, The Children's Hospital, 861, 320 Longwood Ave., Boston, MA 02115. Phone: 617-355-7290; Fax: 617-730-0432; E-mail: alt{at}rascal.med.harvard.edu
Received for publication 24 April 1998 and in revised form 28 July 1998.
The first three authors contributed equally to this work.We thank W. Forrester for critical reading of the manuscript.
This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grants AI-240047 and AI-31541 (to F.W. Alt) and AI-01285 to (J.P. Manis). N. van der Stoep was supported in part by European Molecular Biology Organization fellowship AltF-300-1994, and M. Tian was supported in part by an Irvington Institute fellowship.
Abbreviations used in this paper CSR, class switch recombination; ES, embryonic stem; H, Ig heavy; LCR, locus control region.
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References |
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---|
1. | Lansford, R., A. Okada, J. Chen, E. Oltz, T. Blackwell, F. Alt, and G. Rathburn. 1996. Mechanism and control of immunoglobulin gene rearrangement. In Molecular Immunology. Second edition. B. Hames and D. Glover, editors. Oxford University Press, Oxford. 248-282. |
2. | Zhang, J., F.W. Alt, and T. Honjo. 1995. Regulation of class switch recombination of the immunoglobulin heavy chain genes. In Immunoglobulin Genes. Second edition. T. Honjo and F.W. Alt, editors. Academic Press Limited, London. 235-265. |
3. | Snapper, C.M., and W.E. Paul. 1987. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science. 236: 944-947 [Medline]. |
4. |
Coffman, R.L.,
J. Ohara,
M.W. Bond,
J. Carty,
A. Zlotnik, and
W.E. Paul.
1986.
B cell stimulatory factor-1 enhances
the IgE response of lipopolysaccharide-activated B cells.
J.
Immunol.
136:
4538-4541
|
5. | Kearney, J.F., M.D. Cooper, and A.R. Lawton. 1976. B lymphocyte differentiation induced by lipopolysaccharide. III. Suppression of B cell maturation by anti-mouse immunoglobulin antibodies. J. Immunol. 116: 1664-1668 [Abstract]. |
6. | Layton, J.E., E.S. Vitetta, J.W. Uhr, and P.H. Krammer. 1984. Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s). J. Exp. Med. 160: 1850-1863 [Abstract]. |
7. | Yancopoulos, G.D., R.A. DePinho, K.A. Zimmerman, S.G. Lutzker, N. Rosenberg, and F.W. Alt. 1986. Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching. EMBO (Eur. Mol. Biol. Organ.) J. 5: 3259-3266 [Abstract]. |
8. |
Lutzker, S.,
P. Rothman,
R. Pollock,
R. Coffman, and
F.W. Alt.
1988.
Mitogen- and IL-4-regulated expression of germ-line Ig ![]() |
9. | Stavnezer, J., G. Radcliffe, Y.C. Lin, J. Nietupski, L. Berggren, R. Sitia, and E. Severinson. 1988. Immunoglobulin heavy-chain switching may be directed by prior induction of transcripts from constant-region genes. Proc. Natl. Acad. Sci. USA. 85: 7704-7708 [Abstract]. |
10. | Esser, C., and A. Radbruch. 1989. Rapid induction of transcription of unrearranged S gamma 1 switch regions in activated murine B cells by interleukin 4. EMBO (Eur. Mol. Biol. Organ.) J. 8: 483-488 [Abstract]. |
11. | Berton, M.T., J.W. Uhr, and E.S. Vitetta. 1989. Synthesis of germ-line gamma 1 immunoglobulin heavy-chain transcripts in resting B cells: induction by interleukin 4 and inhibition by interferon gamma. Proc. Natl. Acad. Sci. USA. 86: 2829-2833 [Abstract]. |
12. | Rothman, P., S. Lutzker, B. Gorham, V. Stewart, R. Coffman, and F.W. Alt. 1990. Structure and expression of germline immunoglobulin gamma 3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching. Int. Immunol. 2: 621-627 [Medline]. |
13. | Rothman, P., Y.Y. Chen, S. Lutzker, S.C. Li, V. Stewart, R. Coffman, and F.W. Alt. 1990. Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon. Mol. Cell. Biol. 10: 1672-1679 [Medline]. |
14. |
Harriman, G.R.,
A. Bradley,
S. Das,
P. Rogers-Fani, and
A.C. Davis.
1996.
IgA class switch in I ![]() |
15. | Xu, L., B. Gorham, S.C. Li, A. Bottaro, F.W. Alt, and P. Rothman. 1993. Replacement of germ-line epsilon promoter by gene targeting alters control of immunoglobulin heavy chain class switching. Proc. Natl. Acad. Sci. USA. 90: 3705-3709 [Abstract]. |
16. | Jung, S., K. Rajewsky, and A. Radbruch. 1993. Shutdown of class switch recombination by deletion of a switch region control element. Science. 259: 984-987 [Medline]. |
17. | Zhang, J., A. Bottaro, S. Li, V. Stewart, and F.W. Alt. 1993. A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon. EMBO (Eur. Mol. Biol. Organ.) J. 12: 3529-3537 [Abstract]. |
18. | Bottaro, A., R. Lansford, L. Xu, J. Zhang, P. Rothman, and F.W. Alt. 1994. S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process. EMBO (Eur. Mol. Biol. Organ.) J. 13: 665-674 [Abstract]. |
19. | Lorenz, M., S. Jung, and A. Radbruch. 1995. Switch transcripts in immunoglobulin class switching. Science. 267: 1825-1828 [Medline]. |
20. | Coffman, R.L., D.A. Lebman, and P. Rothman. 1993. Mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol. 54: 229-270 [Medline]. |
21. | Bottaro, A., and F.W. Alt. 1997. Local and General Control Elements of Immunoglobulin Class Switch Recombination. IgE Regulation: Molecular Mechanisms. D. Vercelli, editor. John Wiley & Sons LTD, London. 155-177. |
22. | Gu, H., Y.R. Zou, and K. Rajewsky. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 73: 1155-1164 [Medline]. |
23. | Eckhardt, L.A., and B.K. Birshtein. 1985. Independent immunoglobulin class-switch events occurring in a single myeloma cell line. Mol. Cell. Biol. 5: 856-868 [Medline]. |
24. | Klein, S., T. Gerster, D. Picard, A. Radbruch, and W. Schaffner. 1985. Evidence for transient requirement of the IgH enhancer. Nucleic Acids Res. 13: 8901-8912 [Abstract]. |
25. | Wabl, M.R., and P.D. Burrows. 1984. Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis. Proc. Natl. Acad. Sci. USA. 81: 2452-2455 [Abstract]. |
26. | Pettersson, S., G.P. Cook, M. Bruggemann, G.T. Williams, and M.S. Neuberger. 1990. A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus. Nature. 344: 165-168 [Medline]. |
27. | Lieberson, R., S.L. Giannini, B.K. Birshtein, and L.A. Eckhardt. 1991. An enhancer at the 3' end of the mouse immunoglobulin heavy chain locus. Nucleic Acids Res. 19: 933-937 [Abstract]. |
28. | Dariavach, P., G.T. Williams, K. Campbell, S. Pettersson, and M.S. Neuberger. 1991. The mouse IgH 3'-enhancer. Eur. J. Immunol. 21: 1499-1504 [Medline]. |
29. | Cogne, M., R. Lansford, A. Bottaro, J. Zhang, J. Gorman, F. Young, H.L. Cheng, and F.W. Alt. 1994. A class switch control region at the 3' end of the immunoglobulin heavy chain locus. Cell. 77: 737-747 [Medline]. |
30. | Lieberson, R., J. Ong, X. Shi, and L.A. Eckhardt. 1995. Immunoglobulin gene transcription ceases upon deletion of a distant enhancer. EMBO (Eur. Mol. Biol. Organ.) J. 14: 6229-6238 [Abstract]. |
31. | Matthias, P., and D. Baltimore. 1993. The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the alpha constant region. Mol. Cell. Biol. 13: 1547-1553 [Abstract]. |
32. | Madisen, L., and M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells. Genes Dev. 8: 2212-2226 [Abstract]. |
33. | Michaelson, J.S., S.L. Giannini, and B.K. Birshtein. 1995. Identification of 3' alpha-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23: 975-981 [Abstract]. |
34. | Chauveau, C., and M. Cogne. 1996. Palindromic structure of the IgH 3' locus control region [letter]. Nat. Genet. 14: 15-16 [Medline]. |
35. | Saleque, S., M. Singh, R.D. Little, S.L. Giannini, J.S. Michaelson, and B.K. Birshtein. 1997. Dyad symmetry within the mouse 3' IgH regulatory region includes two virtually identical enhancers (C alpha3'E and hs3). J. Immunol. 158: 4780-4787 [Abstract]. |
36. | Chen, C., and B.K. Birshtein. 1997. Virtually identical enhancers containing a segment of homology to murine 3' IgH-E(hs1,2) lie downstream of human Ig C alpha 1 and C alpha 2 genes. J. Immunol. 159: 1310-1318 [Abstract]. |
37. |
Mills, F.C.,
N. Harindranath,
M. Mitchell, and
E.E. Max.
1997.
Enhancer complexes located downstream of both human immunoglobulin C ![]() |
38. | Martin, D.I., S. Fiering, and M. Groudine. 1996. Regulation of beta-globin gene expression: straightening out the locus. Curr. Opin. Genet. Dev. 6: 488-495 [Medline]. |
39. | Kim, C.G., E.M. Epner, W.C. Forrester, and M. Groudine. 1992. Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region. Genes Dev. 6: 928-938 [Abstract]. |
40. | Fiering, S., E. Epner, K. Robinson, Y. Zhuang, A. Telling, M. Hu, D.I. Martin, T. Enver, T.J. Ley, and M. Groudine. 1995. Targeted deletion of 5'HS2 of the murine beta-globin LCR reveals that it is not essential for proper regulation of the beta-globin locus. Genes Dev. 9: 2203-2213 [Abstract]. |
41. | Hug, B.A., R.L. Wesselschmidt, S. Fiering, M.A. Bender, E. Epner, M. Groudine, and T.J. Ley. 1996. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3. Mol. Cell. Biol. 16: 2906-2912 [Abstract]. |
42. | Birshtein, B.K., C. Chen, S. Saleque, J.S. Michaelson, M. Singh, and R.D. Little. 1997. Murine and human 3' IgH regulatory sequences. Curr. Top. Microbiol. Immunol. 224: 73-80 [Medline]. |
43. | Gorman, J.R., N. van der Stoep, R. Monroe, M. Cogne, L. Davidson, and F.W. Alt. 1996. The Ig(kappa) enhancer influences the ratio of Ig(kappa) versus Ig(lambda) B lymphocytes. Immunity. 5: 241-252 [Medline]. |
44. | Shinkai, Y., G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A.M. Stall, et al . 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 68: 855-867 [Medline]. |
45. |
Lakso, M.,
J.G. Pichel,
J.R. Gorman,
B. Sauer,
Y. Okamoto,
E. Lee,
F.W. Alt, and
H. Westphal.
1996.
Efficient in vivo
manipulation of mouse genomic sequences at the zygote
stage.
Proc. Natl. Acad. Sci. USA.
93:
5860-5865
|
46. | Laird, P.W., A. Zijderveld, K. Linders, M.A. Rudnicki, R. Jaenisch, and A. Berns. 1991. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19: 4293 [Medline]. |
47. | Zelazowski, P., D. Carrasco, F.R. Rosas, M.A. Moorman, R. Bravo, and C.M. Snapper. 1997. B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching. J. Immunol. 159: 3133-3139 [Abstract]. |
48. | Li, Y.S., K. Hayakawa, and R.R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178: 951-960 [Abstract]. |
49. | Chen, J., R. Lansford, V. Stewart, F. Young, and F.W. Alt. 1993. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc. Natl. Acad. Sci. USA. 90: 4528-4532 [Abstract]. |
50. | Arulampalam, V., C. Furebring, A. Samuelsson, U. Lendahl, C. Borrebaeck, I. Lundkvist, and S. Pettersson. 1996. Elevated expression levels of an Ig transgene in mice links the IgH 3' enhancer to the regulation of IgH expression. Int. Immunol. 8: 1149-1157 [Abstract]. |
51. | Arulampalam, V., P.A. Grant, A. Samuelsson, U. Lendahl, and S. Pettersson. 1994. Lipopolysaccharide-dependent transactivation of the temporally regulated immunoglobulin heavy chain 3' enhancer. Eur. J. Immunol. 24: 1671-1677 [Medline]. |
52. | Grant, P.A., T. Andersson, M.F. Neurath, V. Arulampalam, A. Bauch, R. Muller, M. Reth, and S. Pettersson. 1996. A T cell controlled molecular pathway regulating the IgH locus: CD40-mediated activation of the IgH 3' enhancer. EMBO (Eur. Mol. Biol. Organ.) J. 15: 6691-6700 [Abstract]. |
53. | Neurath, M.F., E.E. Max, and W. Strober. 1995. Pax5 (BSAP) regulates the murine immunoglobulin 3' alpha enhancer by suppressing binding of NF-alpha P, a protein that controls heavy chain transcription. Proc. Natl. Acad. Sci. USA. 92: 5336-5340 [Abstract]. |
54. | Meyer, K.B., M. Skogberg, C. Margenfeld, J. Ireland, and S. Pettersson. 1995. Repression of the immunoglobulin heavy chain 3' enhancer by helix-loop-helix protein Id3 via a functionally important E47/E12 binding site: implications for developmental control of enhancer function. Eur. J. Immunol. 25: 1770-1777 [Medline]. |
55. |
Singh, M., and
B.K. Birshtein.
1996.
Concerted repression of
an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).
Proc. Natl. Acad. Sci. USA.
93:
4392-4397
|
56. | Tumas-Brundage, K.M., K.A. Vora, and T. Manser. 1997. Evaluation of the role of the 3' alpha heavy chain enhancer [3' alpha E(hs1,2)] in Vh gene somatic hypermutation. Mol. Immunol. 34: 367-378 [Medline]. |
57. | Sha, W.C., H.C. Liou, E.I. Tuomanen, and D. Baltimore. 1995. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell. 80: 321-330 [Medline]. |
58. | Michaelson, J.S., M. Singh, C.M. Snapper, W.C. Sha, D. Baltimore, and B.K. Birshtein. 1996. Regulation of 3' IgH enhancers by a common set of factors, including kappa B-binding proteins. J. Immunol. 156: 2828-2839 [Abstract]. |
59. | Xu, Y., L. Davidson, F.W. Alt, and D. Baltimore. 1996. Deletion of the Ig kappa light chain intronic enhancer/matrix attachment region impairs but does not abolish V kappa J kappa rearrangement. Immunity. 4: 377-385 [Medline]. |
60. | Baron, M.H.. 1997. Transcriptional control of globin gene switching during vertebrate development. Biochim. Biophys. Acta. 1351: 51-72 [Medline]. |
61. | Blom van Assendelft, G., O. Hanscombe, F. Grosveld, and D.R. Greaves. 1989. The beta-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell. 56: 969-977 [Medline]. |
62. | Grosveld, F., G.B. van Assendelft, D.R. Greaves, and G. Kollias. 1987. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell. 51: 975-985 [Medline]. |
63. | Behringer, R.R., T.M. Ryan, R.D. Palmiter, R.L. Brinster, and T.M. Townes. 1990. Human gamma- to beta-globin gene switching in transgenic mice. Genes Dev. 4: 380-389 [Abstract]. |
64. | Forrester, W.C., E. Epner, M.C. Driscoll, T. Enver, M. Brice, T. Papayannopoulou, and M. Groudine. 1990. A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus. Genes Dev. 4: 1637-1649 [Abstract]. |
65. | Driscoll, M.C., C.S. Dobkin, and B.P. Alter. 1989. Gamma delta beta-thalassemia due to a de novo mutation deleting the 5' beta-globin gene activation-region hypersensitive sites. Proc. Natl. Acad. Sci. USA. 86: 7470-7474 [Abstract]. |
66. | Gregor, P.D., and S.L. Morrison. 1986. Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products. Mol. Cell. Biol. 6: 1903-1916 [Medline]. |
67. | Chen, J., F. Young, A. Bottaro, V. Stewart, R.K. Smith, and F.W. Alt. 1993. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus. EMBO (Eur. Mol. Biol. Organ.) J. 12: 4635-4645 [Abstract]. |
68. | Serwe, M., and F. Sablitzky. 1993. V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer. EMBO (Eur. Mol. Biol. Organ.) J. 12: 2321-2327 [Abstract]. |
69. | Olson, E.N., H.H. Arnold, P.W. Rigby, and B.J. Wold. 1996. Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell. 85: 1-4 [Medline]. |
70. |
Pham, C.T.,
D.M. MacIvor,
B.A. Hug,
J.W. Heusel, and
T.J. Ley.
1996.
Long-range disruption of gene expression by
a selectable marker cassette.
Proc. Natl. Acad. Sci. USA.
93:
13090-13095
|
71. | Hanscombe, O., D. Whyatt, P. Fraser, N. Yannoutsos, D. Greaves, N. Dillon, and F. Grosveld. 1991. Importance of globin gene order for correct developmental expression. Genes Dev. 5: 1387-1394 [Abstract]. |
72. | Wijgerde, M., F. Grosveld, and P. Fraser. 1995. Transcription complex stability and chromatin dynamics in vivo. Nature. 377: 209-213 [Medline]. |
73. | Walters, M.C., S. Fiering, J. Eidemiller, W. Magis, M. Groudine, and D.I. Martin. 1995. Enhancers increase the probability but not the level of gene expression. Proc. Natl. Acad. Sci. USA. 92: 7125-7129 [Abstract]. |
74. | Dillon, N., T. Trimborn, J. Strouboulis, P. Fraser, and F. Grosveld. 1998. The effect of distance on long-range chromatin interactions. Mol. Cell. 1: 131-139 . |
75. | Walters, M.C., W. Magis, S. Fiering, J. Eidemiller, D. Scalzo, M. Groudine, and D.I. Martin. 1996. Transcriptional enhancers act in cis to suppress position-effect variegation. Genes Dev. 10: 185-195 [Abstract]. |
76. | Bottaro, A., F. Young, J. Chen, M. Serwe, F. Sablitzky, and F. Alt. 1998. Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish class switching at the µ locus. Int. Immunol. 10: 799-806 [Abstract]. |
77. | Enver, T., N. Raich, A.J. Ebens, T. Papayannopoulou, F. Costantini, and G. Stamatoyannopoulos. 1990. Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice. Nature. 344: 309-313 [Medline]. |