Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5 0 and 3 0 regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like kinase 3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3 0 region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

One clear hallmark of mammalian promoters is the presence of CpG islands (CGIs) at more than two-thirds of genes, whereas TATA boxes are only present at a minority of promoters. Using genome-wide approaches, we show that GC content and CGIs are major promoter elements in mammalian cells, able to govern open chromatin conformation and support paused transcription. First, we define three classes of promoters with distinct transcriptional directionality and pausing properties that correlate with their GC content. We further analyze the direct influence of GC content on nucleosome positioning and depletion and show that CpG content and CGI width correlate with nucleosome depletion both in vivo and in vitro. We also show that transcription is not essential for nucleosome exclusion but influences both a weak +1 and a well-positioned nucleosome at CGI borders. Altogether our data support the idea that CGIs have become an essential feature of promoter structure defining novel regulatory properties in mammals.[Supplemental material is available for this article.]How the transcriptional machinery accesses gene promoters is a central question for understanding gene regulation. Because eukaryotic genomes are highly compacted, most of the DNA is not easily accessible to transcription factors, to the notable exception of promoters that tend to be more open chromatin structures. In recent years, with the development of genome-wide approaches, several studies in various organisms have described that nucleosomes are at least partially dependent on the primary sequence for their positioning (Kaplan et al. 2009). In yeast and Drosophila, for example, AT-rich stretches at promoters exclude nucleosomes both in vivo and in vitro, although the correlation of both data sets has been discussed in the literature, as opposed to GC-rich regions, often found in gene bodies, which favor nucleosome occupancy (Kaplan et al. 2009(Kaplan et al. , 2010Zhang et al. 2009;Pugh 2010). In contrast, most mammalian promoters are enriched for GC-rich areas-also called CpG islands (CGIs)-whereas TATA boxes only appear in a minority of genes (Sandelin et al. 2007). CGIs are defined as large genomic areas of over-enriched CpG dinucleotides estimated to amount to between 20,000 and 30,000 in various mammalian genomes lacking counterparts in cold blooded organisms or other eukaryotes (Illingworth and Bird 2009;Sharif et al. 2010). Although recent works suggest a link between nucleosome depletion at promoters and the presence of CGIs, a direct correlation was never established (Li et al. 2011;Valouev et al. 2011). Furthermore, it remains to be determined whether nucleosome depletion is a cause or a consequence of promoter transcription because in vitro experimental approaches describe apparently contradictory results (Ramirez-Carrozzi et al. 2009;Valouev et al. 2011).In this study, we report that GC content and CGIs are major promoter elements in mammals able to govern open chromatin conformation and support paused transcription genome-wide. First, we define t...