| Chromatin is more than just the eucaryotic DNA packaging system; it is the substrate of all reactions involving DNA in eukaryotic cells and actively regulates RNA Polymerase II (RNAPolII) access to DNA. Responsible for all mRNA transcription in eucaryotes, the RNAPolII must, following its recruitment to the preinitiation complex, overcome the chromatin barrier in order to transcribe genes. The RNAPolII CTD allows for the co-transcriptional coordination of mRNA maturation and chromatin modifications. The work covered in this thesis addresses two aspects of transcription: the chromatin substrate, with the localization of H2A variant, H2A.Z, and the transcription complex with the phosphorylation cycle of the RNAPolII CTD.;Following the introduction, chapter 2 constitutes a detailed and annotated Saccharomyces cerevisiae ChIP-chip protocol, from the culture to the hybridization of the array, with an emphasis on the proper controls required for chromatin study. This technique, extremely powerful for the in vivo study of all DNA transactions, leads to a better understanding of chromatin function in nuclear phenomena, thanks to the localization of histone variants and modifications.;The third chapter maps the H2A.Z variant across the yeast genome at ∼300 base pairs resolution using ChIP-chip. Our data shows that H2A.Z is incorporated into one or two promoter-bound nucleosomes at the majority of genes. H2A.Z enrichment is anticorrelated with transcription, and the results suggest that it configures chromatin structure to poise genes for transcriptional activation. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning.;The next chapter focuses on the transcription machinery and, more precisely, on the phosphorylation cycle of RNAPolII. The CTD contains repetitions of a heptapeptide (Y1S2P3T4S 5P6S7) on which all serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Here, we systematically profiled the location of the RNAPII phospho-isoforms in wild-type cells and mutants for most CTD modifying enzymes. The results provide evidence for a uniform CTD cycle across genes. Together with results from in vitro assays, these data reveal a complex interplay between the modifying enzymes, identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. Moreover, it reinforces the notion of variants of the phosphorylation marks, even though the exact nature of the variant is still difficult to identify.;The discussion introduces the studies that followed this work, including new projects conceived in our lab. Key. |
