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Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

ABSTRACT: Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.

SUBMITTER: Pathak RU

PROVIDER: S-EPMC1951503 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

Pathak Rashmi U RU Rangaraj Nandini N Kallappagoudar Satish S Mishra Krishnaveni K Mishra Rakesh K RK

Molecular and cellular biology 20070507 13

Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear ...[more]

PMID: 17485444

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Project description:BACKGROUND:H1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50-60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes. RESULTS:We generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA-PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes. CONCLUSIONS:H1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1?, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

Project description:BackgroundBoundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass.ResultsHere, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a?~?100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7.ConclusionsThe subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.

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Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

Publications

Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

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