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Marking of DNA, histones, and RNA is central to gene expression regulation in development and disease. On mRNA, m6A is installed by the METTL3-METTL14 methyltransferase complex. Recent evidence links m6A to histone modifications, but the crosstalk between m6A and DNA methylation remains scarcely explored. Our findings indicate co-occurrence of m6A with gene-body 5mC, this being associated with increased gene expression. Accordingly, METTL3-depleted cells display intragenic DNA hypomethylation and decreased expression of the corresponding genes. Mechanistically, we evidence direct binding of METTL3-METTL14 to DNA methyltransferase DNMT1 and METTL14-mediated recruitment of DNMT1 to chromatin. We show that gene-body 5mC and the mRNA-stability-enhancing effect of coding-sequence m6A both contribute to increased gene expression. We substantiate our findings on embryonic stem cells, where METTL3 regulates gene-body methylation and expression of key differentiation markers. Our results highlight a previously unrecognized layer of gene expression regulation, involving direct mechanistic links between RNA modification and DNA methylation., Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/published

The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation., info:eu-repo/semantics/published

The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodelling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation requires, at least partially, RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits do not define alternate versions of the complex.

In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis. In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation (Materne et al., 2015) but the regulatory steps remain poorly understood. Here we report a genetic screen that highlighted H2B deubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2Bubi at the promoter where it represses expression, and over the transcribed region where it sustains it. By promoting H3K4 methylation at the promoter, H2Bubi initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR (nucleosome depleted region) formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.

The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The earlyincrease of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.

The RNA Polymerase II is a macromolecular complex composed of 12 subunits. Its biggest subunit, Rpb1, exhibits a peculiar domain on its carboxy-terminal region named CTD. It is composed of the highly conserved repeat of the heptapeptid Y1S2P3T4S5P6S7 repeated 26, 29 and 52 times in S. cerevisiae, S. pombe and mammals respectively. The CTD follows a pattern of phosphorylation-dephosphorylation in the course of gene transcription. The serine in position 5 (of every heptapeptid repetition) is phosphorylated (S5P) close to the promoter region while the serine in position 2 is phosphorylated (S2P) next to the 3’ of transcribed regions. This phosphorylation combination both reinforces and restricts the recruitment of proteins required for mRNA maturation. Indeed, under certain states of phosphorylation, the CTD serves as a docking platform for capping and splicing enzymes or poly-adenylation factors. It was also later shown that the phosphorylated CTD recruits chromatin modifiers. Transcription occurs through nucleosomes, the base unit of chromatin. One nucleosome is formed of a heterodimer of histones H2A and H2B and a tetramer of histones H3 and H4 surrounded by 147 base pairs of DNA. The histones can be moved, replaced, evicted or modified in a covalent way. Many enzymes are involved in such active processes. The nucleosome occupancy is not homogeneous across the genome. Some regions are depleted in nucleosomes and hence called Nucleosome Depleted Region (NDR). Their formation is an ATP-consuming active process and requires chromatin remodelers. Those NDRs are often located in genes promoter regions and make the DNA sequence accessible to initiate transcription. Their presence/absence is thus an important layer of transcriptional regulation. Surprisingly, in S. pombe, a subset of genes shows an unexpected pattern of CTD phosphorylation: S2P reaches high level in 5' regions where it is usually low. Strikingly, the complete absence of S2P has only a minor impact on genom, (DOCSC03) -- UNamur, 2014

The process of specific gene transcription by RNA polymerase II (Pol II) is initiated by thebinding of specific transcription factors to DNA. A global understanding of the mechanisms of genetranscriptional regulation of Saccharomyces cerevisiae goes through the description of the targets andthe behavior of those transcription factors.The GATA factors are specific transcription factors intervening in the regulation of NitrogenCatabolite Repression (NCR)-sensitive genes, a mechanism encompassing the transcriptionalregulations leading to the preferential use of good nitrogen sources of the growth medium of yeast inthe presence of less good nitrogen sources. Those 4 GATA factors involved in NCR comprise 2activators (Gat1 and Gln3) and 2 repressors (Gzf3 and Dal80).Generally speaking, the promoters of genes have always been described like the main place forthe integration of the transcription regulation signals relayed by the general and specific transcriptionfactors and the chromatin remodeling factors. Furthermore, the GATA factors have been described asintegrating the external signals of nitrogen availability thanks to their specific DNA binding toconsensus GATA sequences in the promoter of NCR-sensitive genes. The results presented hereintroduce many nuances to the model, notably implying new proteins but also new regions in theregulation process of the NCR-sensitive gene regulation. Indeed, the first goal of this work is todiscover and understand the mechanisms of NCR-sensitive gene regulation that will explain thevariations in their expression levels in the presence of various nitrogen sources and their dependencytowards the GATA factors.Strikingly, it appeared that GATA factor positioning was not limited to the promoter, butoccurred also in the transcribed region. It seems that the transcription factors may have been drivenby the general transcription machinery (Pol II). The binding of a chromatin remo, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/nonPublished

Sexual reproduction is a fundamental aspect of eukaryotic cells, and a conserved feature of gametogenesis is its dependency on a master regulator. The ste11 gene was isolated more than 20 years ago by the Yamamoto laboratory as a suppressor of the uncontrolled meiosis driven by a pat1 mutant. Numerous studies from this laboratory and others have established the role of the Ste11 transcription factor as the master regulator of the switch between proliferation and differentiation in fission yeast. The transcriptional and post-transcriptional controls of ste11 expression are intricate, but most are not redundant. Whereas the transcriptional controls ensure that the gene is transcribed at a high level only when nutrients are rare, the post-transcriptional controls restrict the ability of Ste11 to function as a transcription factor to the G1-phase of the cell cycle from where the differentiation programme is initiated. Several feedback loops ensure that the cell fate decision is irreversible. The complete panel of molecular mechanisms operating to warrant the timely expression of the ste11 gene and its encoded protein basically mirrors the advances in the understanding of the numerous ways by which gene expression can be modulated., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNALys UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.