Intergenic Control Region Research Articles

Altered 3D chromatin structure permits inversional recombination at the IgH locus.

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombination activating gene products (RAG) 1 and 2. Diversity (DH) gene segments rearrange first, followed by variable (VH) gene rearrangements. Here, we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, which recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on wild-type IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by distinct mechanisms from those that apply to DH gene segments. These distinctions may underlie differential allelic choice associated with each step of IgH gene assembly.

Altered 3D chromatin structure permits inversional recombination at the IgH locus

Abstract Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2. Diversity gene segments (DH) rearrange first, followed by variable (VH) gene rearrangements. Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by diffusion-controlled mechanisms. These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.

Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus

Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eμ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eμ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eμ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eμ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.

A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory.

In nature, plants often encounter chronic or recurring stressful conditions. Recent results indicate that plants can remember a past exposure to stress to be better prepared for a future stress incident. However, the molecular basis of this is poorly understood. Here, we report the involvement of chromatin modifications in the maintenance of acquired thermotolerance (heat stress [HS] memory). HS memory is associated with the accumulation of histone H3 lysine 4 di- and trimethylation at memory-related loci. This accumulation outlasts their transcriptional activity and marks them as recently transcriptionally active. High accumulation of H3K4 methylation is associated with hyper-induction of gene expression upon a recurring HS. This transcriptional memory and the sustained accumulation of H3K4 methylation depend on HSFA2, a transcription factor that is required for HS memory, but not initial heat responses. Interestingly, HSFA2 associates with memory-related loci transiently during the early stages following HS. In summary, we show that transcriptional memory after HS is associated with sustained H3K4 hyper-methylation and depends on a hit-and-run transcription factor, thus providing a molecular framework for HS memory.

CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination

Ig heavy chain (IgH) variable region exons are assembled from V, D, and J gene segments during early B-lymphocyte differentiation. A several megabase region at the "distal" end of the mouse IgH locus (Igh) contains hundreds of V(H)s, separated by an intergenic region from Igh Ds, J(H)s, and constant region exons. Diverse primary Igh repertoires are generated by joining Vs, Ds, and Js in different combinations, with a given B cell productively assembling only one combination. The intergenic control region 1 (IGCR1) in the V(H)-to-D intergenic region regulates Igh V(D)J recombination in the contexts of developmental order, lineage specificity, and feedback from productive rearrangements. IGCR1 also diversifies IgH repertoires by balancing proximal and distal V(H) use. IGCR1 functions in all these regulatory contexts by suppressing predominant rearrangement of D-proximal V(H)s. Such IGCR1 functions were neutralized by simultaneous mutation of two CCCTC-binding factor (CTCF)-binding elements (CBE1 and CBE2) within it. However, it was unknown whether only one CBE mediates IGCR1 functions or whether both function in this context. To address these questions, we generated mice in which either IGCR1 CBE1 or CBE2 was replaced with scrambled sequences that do not bind CTCF. We found that inactivation of CBE1 or CBE2 individually led to only partial impairment of various IGCR1 functions relative to the far greater effects of inactivating both binding elements simultaneously, demonstrating that they function cooperatively to achieve full IGCR1 regulatory activity. Based on these and other findings, we propose an orientation-specific looping model for synergistic CBE1 and CBE2 functions.

Enhancers Located in Heavy Chain Regulatory Region (hs3a, hs1,2, hs3b, and hs4) Are Dispensable for Diversity of VDJ Recombination

V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

CTCF-binding elements mediate control of V(D)J recombination

Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7 megabase (Mb) mouse IgH locus (IgH), V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Herein, we report a key IgH V(D)J recombination regulatory region, termed InterGenic Control Region-1 (IGCR1), that lies between the VH and D clusters. Functionally, IGCR1 employs CTCF looping/insulator factor binding elements and, correspondingly, mediates IgH loops containing distant enhancers. IGCR1 promotes normal B cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VHs and promoting rearrangement of distal VHs. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination, respectively, by suppressing VH joining to Ds not joined to JHs and VH to DJH joins in thymocytes. IGCR1 also is required to allow feedback regulation and allelic exclusion of proximal VH to DJH recombination. Our studies elucidate a long-sought IgH V(D)J recombination control region and implicate a new role for the generally expressed CTCF protein.

Nuclear DNA-binding proteins which recognize the intergenic control region of penicillin biosynthetic genes

The biosynthesis of penicillin, a secondary metabolite produced by Penicillium chrysogenum, is subject to sophisticated genetic and metabolic regulation. The structural genes, pcbC and pcbAB, which encode two of the penicillin biosynthetic enzymes are separated by a 1.16-kb intergenic region and transcribed divergently from one another. To identify and characterize nuclear proteins which interact with the pcbAB-pcbC intergenic promoter region, crude and partially purified nuclear extracts were used in mobility shift and DNA footprinting assays. Multiple DNA-binding proteins appear to bind to different regions of this DNA segment. An abundant nuclear protein, nuclear factor A (NF-A), binds at a single site in the intergenic promoter region and recognizes an 8-bp sequence, GCCAAGCC. Penicillin production is sensitive to nitrogen catabolite repression. The global-acting nitrogen regulatory protein NIT2 of Neurospora crassa binds strongly to the intergenic promoter region of the pcbAB and pcbC genes at a single site that contains two closely spaced GATA sequences.

A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression

Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.

Nucleotide sequences of the trpI, trpB, and trpA genes of Pseudomonas syringae: positive control unique to fluorescent pseudomonads

A 904-bp probe from Pseudomonas aeruginosa was used to identify the trpB, trpA and trpI genes of Pseudomonas syringae. Transcription initiation at the P. syringae trpBA promoter in vitro was activated by the P. aeruginosa TrpI protein in the presence of indoleglycerol phosphate. Thus, trpB and trpA are regulated positively in three species of fluorescent pseudomonads, P. aeruginosa P. putida, and P. syringae, but in no other eubacteria so far investigated [Crawford, Annu. Rev. Microbiol. 43 (1989) 567–600]. In addition to conservation of protein-coding sequences, there is a high degree of nucleotide sequence identity in the intergenic control region that includes the divergent trpI and trpBA promoters, especially in the binding sites for TrpI protein. Differences in patterns of codon usage distinguish the trpI genes of P. syringae and P. putida from P. aeruginosa trpI and from the trpB and trpA genes of all three species.

Gene regulation in two dimensions: the proneural achaete and scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region.

The mechanisms that generate precise patterns of discrete cell types within developing fields are not well understood. One model for analyzing how cells interpret positional information in two dimensions is the regulation of proneural cluster formation within insect segments. Two adjacent proneural regulatory genes, achaete and scute, are expressed coincidently in cell clusters at reproducible anteroposterior (AP) and dorsoventral (DV) coordinates within the Drosophila embryo from which single neuroblasts later arise. Here, we show that the AP and DV position of these clusters is regulated through a common cis-acting region between the genes under the initial control of the products of the pair-rule and DV polarity genes and is later maintained by selected segment polarity genes. The combination of proneural gene activation/repression in AP stripes and repression within specific DV domains positions each cluster of achaete/scute expressing cells within segments; interactions between these cells then determine individual cell fates.