Mécanismes de régulation de la transcription par les insulateurs impliquant leur rôle dans l'organisation de la chromatine

L'organisation des chromosomes est essentielle à la régulation de nombreux processus incluant l'activité des gènes, leur transmission fidèle au cours de la division cellulaire et la prolifération des cellules. Les insulateurs sont des séquences d'ADN particulières qui marquent des zones frontières dans la chromatine, subdivisant ainsi le génome en sous-domaines fonctionnellement indépendants. Bien que des insulateurs aient été identifiés chez la plupart des eucaryotes, les mécanismes qui leur permettent de définir des frontières et/ou de réguler les gènes voisins restent à préciser. Mon travail de thèse a porté sur l'étude des insulateurs dont la fonction est assurée par la protéine BEAF chez Drosophila melanogaster. Par une approche de déplétion de BEAF par interférence à l'ARN dans des cellules en culture, j'ai étudié l'influence de cette protéine sur l'organisation d'une frontière putative entre euchromatine et hétérochromatine, ainsi que sur l'expression des gènes environnants. Je me suis ensuite intéressée aux mécanismes de régulation transcriptionnelle impliquant BEAF. Proches de nombreux promoteurs, les insulateurs BEAF régulent la transcription de gènes dont l'expression est essentielle pour la cellule. En combinant plusieurs approches à l'échelle du génome, nous avons montré que BEAF est impliqué dans le positionnement précis des nucléosomes au niveau du site de démarrage de la transcription. Ce positionnement permet la régulation d'une étape précoce de la transcription par la polymérase II, à savoir l'étape de pause à proximité du promoteur qui apparaît comme primordiale pour l'expression normale d'un grand nombre de gènes.Chromosomes organization is essential for many processes including genes activity, faithful genetic transmission during cell division and cell proliferation. Insulators are specific DNA sequences that specify chromatin borders, dividing the genome into functionally independent sub-domains. Insulators have been identified in most eukaryotes but the mechanisms that enable them to define borders and/or regulate nearby genes are still unclear. During my PhD studies, I focused on specific insulators whose function is carried out by the BEAF protein in Drosophila melanogaster. By depleting BEAF by RNA interference in cultured cells, I've studied the effect of this protein on chromatin organization at a putative border between euchromatin and heterochromatin, as well as on the expression of surrounding genes. I then extended these studies to the mechanisms of transcriptional regulation that involve BEAF. Localized near many promoters, BEAF insulators regulate the transcription of genes whose expression is essential for cells. Combining several genome-wide approaches, we have shown that BEAF is involved in the precise positioning of nucleosomes at transcription start sites. This positioning enables the regulation of an early step of polymerase II transcription, namely the pause near promoters which appears to be essential for the normal expression of many genes

BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites.

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle

RNF40 regulates gene expression in an epigenetic context-dependent manner

Background Monoubiquitination of H2B (H2Bub1) is a largely enigmatic histone modification that has been linked to transcriptional elongation. Because of this association, it has been commonly assumed that H2Bub1 is an exclusively positively acting histone modification and that increased H2Bub1 occupancy correlates with increased gene expression. In contrast, depletion of the H2B ubiquitin ligases RNF20 or RNF40 alters the expression of only a subset of genes. Results Using conditional Rnf40 knockout mouse embryo fibroblasts, we show that genes occupied by low to moderate amounts of H2Bub1 are selectively regulated in response to Rnf40 deletion, whereas genes marked by high levels of H2Bub1 are mostly unaffected by Rnf40 loss. Furthermore, we find that decreased expression of RNF40-dependent genes is highly associated with widespread narrowing of H3K4me3 peaks. H2Bub1 promotes the broadening of H3K4me3 to increase transcriptional elongation, which together lead to increased tissue-specific gene transcription. Notably, genes upregulated following Rnf40 deletion, including Foxl2, are enriched for H3K27me3, which is decreased following Rnf40 deletion due to decreased expression of the Ezh2 gene. As a consequence, increased expression of some RNF40-“suppressed” genes is associated with enhancer activation via FOXL2. Conclusion Together these findings reveal the complexity and context-dependency whereby one histone modification can have divergent effects on gene transcription. Furthermore, we show that these effects are dependent upon the activity of other epigenetic regulatory proteins and histone modifications

Pengembangan Kurikulum Pendidikan Lingkungan di Kota Tangerang Selatan: Bagaimana Mengintegrasikan Deklarasi Tbilisi dalam Kurikulum

This study aims to develop environmental education curriculum based on Tbilisi Declaration. There were five environmental education goals of Tbilisi Declaration namely knowledge, awareness, attitudes, skills, and participation. Curriculum was developed for supporting education policy in South Tangerang, Banten Province, Indonesia. Letter of Education Division South Tangerang City nomor 800/KEP 1222-dikdas/2014 stated environmental education as local curriculum in South Tangerang Elementary School. Curriculum was developed by developmental research. There was four steps to develop curriculum, every steps had three activities, i.e. making, reviewing, and fixing. This study result some standard and basic competences. Research developed environmental education was integrated with thematic subject matter in 1st and 2nd grade, but as monolithic subject matter in 3rd – 6rd grade. Using exiting competence standard from 2014 curriculum and format standard competence 2006 made this curriculum have flexibility to implement for the school that used 2006 or 2013 curriculum

Mécanismes de régulation de la transcription par les insulateurs impliquant leur rôle dans l'organisation de la chromatine

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

Interstrand crosslink (ICL)-inducing agents block the separation of the two DNA strands. They prevent transcription and replication and are used in clinics for the treatment of cancer and skin diseases. Here, we have introduced a single psoralen ICL at a specific site in plasmid DNA using a triplex-forming-oligonucleotide (TFO)-psoralen conjugate and studied its repair in Xenopus egg extracts that support nuclear assembly and replication of plasmid DNA. Replication forks arriving from either side stalled at the psoralen ICL. In contrast to previous observations with other ICL-inducing agents, the leading strands advanced up to the lesion without any prior pausing. Subsequently, incisions were introduced on one parental strand on both sides of the ICL. These incisions could be detected whether one or both forks reached the ICL. Using small molecule inhibitors, we found that the ATR-Chk1 pathway, but not the ATM-Chk2 pathway, stimulated both the incision step and the subsequent processing of the broken replication intermediates. Our results highlight both similarities and differences in fork stalling and repair induced by psoralen and by other ICL-forming agents

FORK-seq: single molecule profiling of DNA replication

International audienc

TFO and purification of monomodified plasmid.

<p>(A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018554#s4" target="_blank">Material and Methods</a>. (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.</p

Model for psoralen ICL repair in Xenopus egg extract.

<p>During replication of plasmid DNA containing a single psoralen ICL, replication forks stall at the ICL advancing up to 1 nt from the lesion. Coordinated incisions of one parental strand around the lesion can occur whether one (right) or both (left) forks have stalled, resulting in unhooking of the lesion. Next, translesion synthesis occurs across the unhooked psoralen adduct and other events implying HR and/or NER regenerate two repaired duplexes as proposed elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018554#pone.0018554-Muniandy1" target="_blank">[5]</a>. Both incision events and subsequent processing of the broken replication intermediates are stimulated by the ATR-Chk1 pathway.</p

Effect of ATR and Chk1 inhibition on processing of stalled replication forks at the psoralen ICL.

<p>(A) pTUC-PSO was incubated for 95 min in a Xenopus egg extract in absence (NT; not treated) or presence of 5 mM caffeine or 10 µM UCN-01 and [α-³²P]-dATP and analyzed on 2D gels. Total signal and spots 1, 2 and 3 (as labeled on the interpretative diagram) were quantified in the presence of caffeine (B), UCN-01 (C), KU55933 (D) or C3742 (E). Results were normalized to the signals obtained with the non-treated plasmid. Exp1, Exp2, Exp3, Exp4, Exp5 and Exp6 are six experiments in six independently prepared egg extracts.</p