Mapping separase-mediated cleavage in situ.

Separase is a protease that performs critical functions in the maintenance of genetic homeostasis. Among them, the cleavage of the meiotic cohesin during meiosis is a key step in producing gametes in eukaryotes. However, the exact chromosomal localization of this proteolytic cleavage was not addressed due to the lack of experimental tools.

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line.

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1.

The downregulation of putative anticancer target BORIS/CTCFL in an addicted myeloid cancer cell line modulates the expression of multiple protein coding and ncRNA genes.

The gene, is a testis-specific paralog frequently erroneously activated in cancer, although its exact role in cancer remains unclear. BORIS is both a transcription factor and an architectural chromatin protein. BORIS' normal role is to establish a germline-like gene expression and remodel the epigenetic landscape in testis; it similarly remodels chromatin when activated in human cancer.

The cancer-associated CTCFL/BORIS protein targets multiple classes of genomic repeats, with a distinct binding and functional preference for humanoid-specific SVA transposable elements.

Background: A common aberration in cancer is the activation of germline-specific proteins. The DNA-binding proteins among them could generate novel chromatin states, not found in normal cells. The germline-specific transcription factor BORIS/CTCFL, a paralog of chromatin architecture protein CTCF, is often erroneously activated in cancers and rewires the epigenome for the germline-like transcription program.

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions.

Background: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation.

Results: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin.

Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA.

Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast.

RNA interference (RNAi) is widespread in eukaryotes and regulates gene expression transcriptionally or post-transcriptionally. In fission yeast, RNAi is tightly coupled to template transcription and chromatin modifications that establish heterochromatin in cis. Exogenous double-stranded RNA (dsRNA) triggers seem to induce heterochromatin formation in trans only when certain silencing proteins are overexpressed.

Synthetic heterochromatin bypasses RNAi and centromeric repeats to establish functional centromeres.

In the central domain of fission yeast centromeres, the kinetochore is assembled on CENP-A(Cnp1) nucleosomes. Normally, small interfering RNAs generated from flanking outer repeat transcripts direct histone H3 lysine 9 methyltransferase Clr4 to homologous loci to form heterochromatin. Outer repeats, RNA interference (RNAi), and centromeric heterochromatin are required to establish CENP-A(Cnp1) chromatin.

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1.

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex.

Transcription of the MAT2A gene, coding for methionine adenosyltransferase, is up-regulated by E2F and Sp1 at a chromatin level during proliferation of liver cells.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes.

RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.

We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter.

A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene.

Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region.

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet). In mammals MAT activity derives from two separate genes which display a tissue-specific pattern of expression. While MAT1A is expressed only in the adult liver, MAT2A is expressed in non-hepatic tissues.

Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.

Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A.