Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Bromodomain-dependent stage-specific male genome programming by Brdt.

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a 'progenitor cells' gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

Genomic binding of Pol III transcription machinery and relationship with TFIIS transcription factor distribution in mouse embryonic stem cells.

RNA polymerase (Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of Pol III and its associated transcription factors on the genome of mouse embryonic stem cells using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class III genes was bound and thus transcribed. A few hundred SINEs were associated with the Pol III transcription machinery. We observed that Pol III and its transcription factors were present at 30 unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with >80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of Pol III. These sites were associated with cohesins and often located close to CTCF-binding sites, suggesting that TFIIIC might cooperate with these factors to organize the chromatin. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a Pol III transcription factor in mammals.

Collecting duct is a site of sodium retention in PAN nephrosis: a rationale for amiloride therapy.

Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na(+) retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na(+) excretion, Na(+) balance, or ascites formation in PAN nephrotic rats.