Changes in replication, nuclear location, and expression of the Igh locus after fusion of a pre-B cell line with a T cell line

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3' end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3' to 5' unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) x BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3' Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.     

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.     

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.     

Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.     

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.     

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.     

β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications

Increasing evidence points to nuclear compartmentalization as a contributing mechanism for gene regulation, yet mechanisms for compartmentalization remain unclear. In this paper, we use autonomous targeting of bacterial artificial chromosome (BAC) transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs) within an HBB BAC bias a competition between pericentric versus peripheral heterochromatin targeting toward the nuclear periphery, which correlates with increased H3K9me3 across the β-globin gene cluster and locus control region. Targeting to both heterochromatin compartments is dependent on Suv39H-mediated H3K9me3 methylation. In different chromosomal contexts, PTRs confer no targeting, targeting to pericentric heterochromatin, or targeting to the periphery. A combination of fluorescent in situ hybridization, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over hundreds of kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent lamin-associated domain. Our results demonstrate that multiple cis-elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.     

Unusual germline DSP2 gene accounts for all apparent V-D-D-J rearrangements in newborn, but not adult, MRL mice.

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline D(H) gene in mice of the Igh(j) haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Igh(j) mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Igh(j) mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had approximately 2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.     

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus

Regulatory elements located within an ～28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.     

Global reorganization of budding yeast chromosome conformation in different physiological conditions

The organization of the genome is nonrandom and important for correct function. Specifically, the nuclear envelope plays a critical role in gene regulation. It generally constitutes a repressive environment, but several genes, including the GAL locus in budding yeast, are recruited to the nuclear periphery on activation. Here, we combine imaging and computational modeling to ask how the association of a single gene locus with the nuclear envelope influences the surrounding chromosome architecture. Systematic analysis of an entire yeast chromosome establishes that peripheral recruitment of the GAL locus is part of a large-scale rearrangement that shifts many chromosomal regions closer to the nuclear envelope. This process is likely caused by the presence of several independent anchoring points. To identify novel factors required for peripheral anchoring, we performed a genome-wide screen and demonstrated that the histone acetyltransferase SAGA and the activity of histone deacetylases are needed for this extensive gene recruitment to the nuclear periphery.     

A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.     

Isolation and restriction map of the V-J interval of the human T cell receptor gamma chain locus

The human T cell receptor gamma chain locus encodes the immunoglobulin-like gamma chain polypeptide and spans a distance of approximately 150 kb. Previous studies have not precisely characterized the interval separating variable regions from joining--constant regions which is excised during gamma gene rearrangement. We report a series of overlapping cosmids which includes the portion of the gamma chain locus beginning with V2 and extends to the second exon of C2. Sixteen kilobases separate the most 3' variable region gene, V4, from the most 5' joining segment, J1.1.     

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.