Two forms of loops generate the chromatin conformation of the immunoglobulin heavy-chain gene locus

The immunoglobulin heavy-chain (IgH) gene locus undergoes radial repositioning within the nucleus and locus contraction in preparation for gene recombination. We demonstrate that IgH locus conformation involves two levels of chromosomal compaction. At the first level, the locus folds into several multilooped domains. One such domain at the 3' end of the locus requires an enhancer, Eμ; two other domains at the 5' end are Eμ independent. At the second level, these domains are brought into spatial proximity by Eμ-dependent interactions with specific sites within the V(H) region. Eμ is also required for radial repositioning of IgH alleles, indicating its essential role in large-scale chromosomal movements in developing lymphocytes. Our observations provide a comprehensive view of the conformation of IgH alleles in pro-B cells and the mechanisms by which it is established.     

Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region.

The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid lineage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remains unknown. To gain insights into its function, mouse HS5 was cloned and sequenced. Comparison of the HS5 sequences of mouse, human, and galago revealed two extensively conserved regions, designated HS5A and HS5B. DNase I hypersensitivity mapping revealed that two hypersensitive sites are located within the HS5A region (designated HS5A(major) and HS5A(minor)), and two are located within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, suggesting that these protein binding sites are implicated in the formation of HS5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific. HS5 was detected in murine brain but not in murine kidney or adult liver, suggesting that this site is not ubiquitous. The presence of GATA-1 and NF-E2 motifs (which are common features of the DNase I hypersensitive sites of the LCR) suggests that the HS5 is organized in a manner similar to that of the other HSs. Taken together, our results suggest that HS5 is an inherent component of the beta-globin locus control region.     

A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells

The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5' HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5' upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5' HS region were associated with Kit expression. Therefore, the 5' upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.     

Activation of V(D)J recombination at the IgH chain JH locus occurs within a 6-kilobase chromatin domain and is associated with nucleosomal remodeling

IgH genes are assembled during early B cell development by a series of regulated DNA recombination reactions in which DH and JH segments are first joined followed by V(H) to DJH rearrangement. Recent studies have highlighted the role of chromatin structure in the control of V(D)J recombination. In this study, we show that, in murine pro-B cell precursors, the JH segments are located within a 6-kb DNase I-sensitive chromatin domain containing acetylated histones H3 and H4, which is delimited 5' by the DQ52 promoter element and 3' by the intronic enhancer. Within this domain, the JH segments are covered by phased nucleosomes. High-resolution mapping of nucleosomes reveals that, in pro-B cells, unlike recombination refractory nonlymphoid cells, the recombination signal sequences flanking the four JH segments are located in regions of enhanced micrococcal nuclease and restriction enzyme accessibility, corresponding to either nucleosome-free regions or DNA rendered accessible within a nucleosome. These results support the idea that nucleosome remodeling provides an additional level of control in the regulation of Ig locus accessibility to recombination factors in B cell precursors.     

Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.     

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.     

Tissue-specific DNaseI hypersensitive sites in the 5''-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes.

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells.     

Combination of 3' and 5' IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model

To ensure the B cell differentiation stage specificity of the intronic Emu element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V(H) promoter-GFP reporter gene linked to the 3'LCR region and the Emu element. By flow cytometry, GFP(+) lymphocytes were observed amongst pro-B cells (B220(+)CD43(+)CD117(+)) and at all stages of differentiation up to mature B cells (B220(+)IgM(+)IgD(+)). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP(+)Thy1,2(+) cells (T lymphocytes) and GFP(+)B220(-)CD117(+)CD43(+) cells (uncommitted lymphohematopoietic progenitors). Therefore, the Emu-GFP-3'LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3' and 5' IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.     

Cutting edge: Ig heavy chain 3' HS1-4 directs correct spatial position-independent expression of a linked transgene to B lineage cells.

The Ig H chain locus is regulated by a set of cis-acting elements. Hypersensitive sites (HS) located 3' of the IgH, HS1-4, has been suggested to act as a locus control region (LCR) in cell lines. To assess the proposed role of HS1-4 acting as an LCR, we generated transgenic mice harboring a VH promoter-beta-globin reporter gene linked to the Ig H chain HS1-4 3'regulatory sequences. Transgene expression is strictly confined to B lymphocytes, with no detectable expression outside the B cell lineage in all transgenic founder lines. Furthermore, reporter gene activity is integration independent but not copy number dependent. Thus, additional sequences are required to allow the HS1-4 regulatory region to act as a classical LCR in mice. Our data are discussed in the context of tissue-specific gene expression in B lineage cells.