robust-3d-dna-fish-using-directly-labeled-probes

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

The Babraham Institute

The subtelomeric region of human chromosome 4q contains the locus for facioscapulohumeral muscular dystrophy (FSHD). The FSHD mutation is a deletion within an array of 3.3-kb tandem repeats (D4Z4). The disease mechanism is unknown but is postulated to involve position effect. A closely related 3.3-kb array on chromosome 10qter, in contrast, is not associated with a disease phenotype. We show here that the 4q homology on chromosome 10 is not confined to the 3.3-kb repeats but extends both proximally (42 kb) and distally to include the telomere. We have also identified the most distal expressed gene on 10q known so far, mapping only 96 kb from the 3.3-kb repeat array. A 4q variant has also been identified; there is 92%nucleotide identity between the two 4q forms, 4qA and 4qB. The 4qter and 10qter forms show homology to other chromosome ends, including 4p, 21q, and 22q, and these regions may represent a relatively common subtelomeric domain.

Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin.

The possibility that immune responses to autoantigens may contribute to the development of atopic disease has been largely ignored. In this paper, we describe the chromosomal localization of the gene for squamous cell carcinoma-associated reactive antigen for cytotoxic T cells (SART-1). The SART-1 gene localized to a region of 11q12-13 showing strong linkage to atopy in previous studies. Further analysis of this gene revealed the presence of at least 20 exons of varying lengths and four novel single-nucleotide polymorphisms, one of which resulted in an amino acid substitution. Association analysis in families recruited on the basis of affected sib pairs for asthma reveal significant association for both coding region polymorphisms with atopy. We therefore hypothesize that polymorphic variation within the SART-1 gene may account for individuals developing atopy.

Identification of the autoantigen SART-1 as a candidate gene for the development of atopy.

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJ(H) to VDJ(H) recombination and terminates concomitantly with VDJ(H) recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate V(H)-to-DJ(H) recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

Antisense intergenic transcription in V(D)J recombination.

The genomic basis of facioscapulohumeral muscular dystrophy (FSHD) is of considerable interest because of the unique nature of the molecular mutation, which is a deletion within a large, complex DNA tandem array (D4Z4). This repeat maps within 30 kb of the 4q telomere. Although D4Z4 repeat units each contain an open reading frame that could encode a homeodomain protein, there is no evidence that the repeat is transcribed, and the underlying disease mechanism probably involves a position effect. A recent study has identified a protein complex bound to D4Z4 that contains YY1 and HMGB2, implicating a role for D4Z4 as a repressor. The 4q telomere has two variants, 4qA and 4qB. Although these alleles are present at almost equal frequencies in the general population, FSHD is associated only with the 4qA allele and never with 4qB. This suggests a functional difference between the telomere variants, either in predisposition to deletions within D4Z4 or in the pathological consequence of the deletion. Comparative mapping studies of the FSHD region in primates, mouse and Fugu rubripes have given insights into the evolutionary history of the D4Z4 repeat and of 4qter, although as yet they have not provided any solutions to the FSHD puzzle.

Genomic analysis of facioscapulohumeral muscular dystrophy.

The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Emu) only modestly reduce D(H)-->J(H) rearrangements when assessed in peripheral B cells. However, deletion of Emu severely impairs recombination of V(H) gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Emu deletions on primary D(H)-->J(H) rearrangement: 1) Accessibility at the D(H)J(H) cluster is controlled by a redundant cis-element in the absence of Emu. One candidate for this element lies 5' to D(Q52) (PD(Q52)) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, D(H)-->J(H) recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PD(Q52) and Emu in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PD(Q52) promoter is dispensable for germline transcription and recombination of the D(H)J(H) cluster. In contrast, we demonstrate that Emu directly regulates accessibility of the D(H)J(H) region. These findings reveal a significant role for Emu in the control mechanisms that activate IgH gene assembly and suggest that impaired V(H)-->D(H)J(H) rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in D(H)-->J(H) recombination.

Regulation of IgH gene assembly: role of the intronic enhancer and 5'DQ52 region in targeting DHJH recombination.

The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5' end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3' end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.

Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region.

V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Emu and is abrogated in mice lacking this enhancer, indicating that Emu regulates DH antisense transcription. Emu was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Emu controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Emu can regulate chromatin accessibility throughout the DH region. In contrast, Emu deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.

Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Emu.

Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.

Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed "DUX4," containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism.

Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice.

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations.

V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.

Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igalpha and Igbeta chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110delta isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or "tonic" BCR signaling, are not substantially affected by a deficiency in p110delta. Here, we show that p110alpha, but not p110beta, compensated in the absence of p110delta to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110alpha and p110delta activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110delta, however, p110alpha did not contribute to agonist-induced BCR signaling. These studies indicate that either p110alpha or p110delta can mediate tonic signaling from the BCR, but only p110delta can contribute to antigen-dependent activation of B cells.

The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development.

Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMIN(Î”B/Î”B)) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMIN(Î”B/Î”B) cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

Article	Year
Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin. Genomics\| PubMed ID: 11829491	2002
Identification of the autoantigen SART-1 as a candidate gene for the development of atopy. Human molecular genetics\| PubMed ID: 12189166	2002
Antisense intergenic transcription in V(D)J recombination. Nature immunology\| PubMed ID: 15107847	2004
Genomic analysis of facioscapulohumeral muscular dystrophy. Briefings in functional genomics & proteomics\| PubMed ID: 15239924	2003
Regulation of IgH gene assembly: role of the intronic enhancer and 5'DQ52 region in targeting DHJH recombination. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16456003	2006
Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16547259	2006
Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Emu. Molecular and cellular biology\| PubMed ID: 17526723	2007
Myc dynamically and preferentially relocates to a transcription factory occupied by Igh. PLoS biology\| PubMed ID: 17622196	2007
Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy. American journal of human genetics\| PubMed ID: 17668377	2007
Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice. The Journal of experimental medicine\| PubMed ID: 18086860	2007
AID is required for the chromosomal breaks in c-myc that lead to c-myc/IgH translocations. Cell\| PubMed ID: 19070574	2008
Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation. Advances in experimental medicine and biology\| PubMed ID: 19731801	2009
The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development. Science signaling\| PubMed ID: 20699475	2010
ATMIN is required for maintenance of genomic stability and suppression of B cell lymphoma. Cancer cell\| PubMed ID: 21575860	2011

Daniel J. Bolland

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