Évaluation du rôle de la protéine BAG-1 dans le cancer ovarien

La protéine BAG-1 est une protéine multifonctionnelle qui interagit avec de nombreuses protéines incluant la protéine anti-apoptotique Bcl-2, les protéines heat shock et les récepteurs de facteurs de croissance HGF-R et PDGF-R. La surexpression des protéines BAG-1 et Bcl-2 permet une augmentation du potentiel anti-apoptotique de Bcl-2. Quatre isoformes de la protéine humaine BAG-1 sont issus de la traduction alternative d'un ARNm. Ces quatre isoformes possèdent un domaine commun en C-terminal nommé"domaine BAG". Ce domaine serait le site de liaison de protéines impliquées dans la régulation des mécanismes de l'apoptose tel que Bcl-2, Hsc/Hsp70 et le récepteur androgénique. La présente étude vise à évaluer l'implication de la protéine BAG-1 dans la pathogénèse du cancer ovarien. Pour ce faire, deux approches ont été utilisées soit, dans un premier temps, l'inhibition fonctionnelle de la protéine (par un scFv anti-BAG-1) ou de l'expression de l'ARNm (par un siRNA) et, dans un deuxième temps, la surexpression exogène de la protéine (gain de fonction)."--Résumé abrégé par UMI

Évaluation du rôle de la protéine BAG-1 dans le cancer ovarien

La protéine BAG-1 est une protéine multifonctionnelle qui interagit avec de nombreuses protéines incluant la protéine anti-apoptotique Bcl-2, les protéines heat shock et les récepteurs de facteurs de croissance HGF-R et PDGF-R. La surexpression des protéines BAG-1 et Bcl-2 permet une augmentation du potentiel anti-apoptotique de Bcl-2. Quatre isoformes de la protéine humaine BAG-1 sont issus de la traduction alternative d'un ARNm. Ces quatre isoformes possèdent un domaine commun en C-terminal nommé"domaine BAG". Ce domaine serait le site de liaison de protéines impliquées dans la régulation des mécanismes de l'apoptose tel que Bcl-2, Hsc/Hsp70 et le récepteur androgénique. La présente étude vise à évaluer l'implication de la protéine BAG-1 dans la pathogénèse du cancer ovarien. Pour ce faire, deux approches ont été utilisées soit, dans un premier temps, l'inhibition fonctionnelle de la protéine (par un scFv anti-BAG-1) ou de l'expression de l'ARNm (par un siRNA) et, dans un deuxième temps, la surexpression exogène de la protéine (gain de fonction)."--Résumé abrégé par UMI

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription

Regulació gènicaRegulación genéticaGene RegulationMono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.National Institutes of Health [R35GM127289 to R.P.F.]; Canadian Institutes of Health Research [MOP-130362 to J.C.T.]; Natural Sciences and Engineering Council of Canada [RGPIN 03661-15 to J.C.T.]; Wellcome Trust Senior Investigator Award [095598/Z/11/Z to J.B.]; Swedish Research Council [VR2015-02312]; Cancerfonden [CAN2016/576 awards to P.S.]; J.C.T. is supported by a fellowship from Fond de recherche Quebec Santé [33115]. Funding for open access charge: National Institutes of Health (USA); Canadian Institutes of Health Research

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications

The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

<div><p>Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast <i>Schizosaccharomyces pombe</i> that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.</p></div

Prf1 does not stably associate with the PAF complex.

<p>(<b>A</b>) TAP purification was carried out using whole cell extracts from the indicated strains and purified material was analyzed by SDS-PAGE and silver staining. The proteins contained in the labeled bands were identified by mass spectrometry (as indicated on the right). “CBP” denotes the residual fusion to the calmodulin binding peptide resulting from the TAP procedure. Leo1 was detected in two bands that also contained either Paf1-CBP (in the <i>paf1-TAP</i> lane) or Paf1 (in the <i>tpr1-TAP</i> lane). Molecular weight standards (in kD) are denoted on the left. (<b>B</b>) Single-step TAP purifications were performed using extracts from the indicated strains. 5% of input fractions (“input”) and 50% of bead-bound fractions (“beads”) were analyzed by SDS-PAGE and western blotting.</p

Model depicting the roles of the Prf1/Rtf1 and PAF pathways in RNAPII elongation.

<p>The Prf1 pathway, involving direct association of Prf1 with phosphorylated Spt5 CTD, is labeled “1.” The PAF pathway, involving multiple Cdk9 targets, is labeled “2.” Potential crosstalk between the two pathways is indicated by the broken double arrow. See text for details.</p

Prf1 and PAF pathways have opposing biological effects.

<p>(<b>A</b>) Quantification of the abnormal septation patterns in the indicated strains. Strains carrying the <i>cdk9^as</i> allele were cultured in DMSO (−) or 2 µM 3-MB-PP1 (+) for 15 hours prior to fixation for microscopy. Error bars denote standard deviations from 3 independent experiments. (<b>B</b>) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the <i>nup189</i>⁺ gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated.</p

Prf1 and PAF have shared roles in histone H2B ubiquitylation but are functionally distinct.

<p>(<b>A</b>) Whole-cell extracts from strains of the indicated genotypes were analyzed by SDS-PAGE and western blotting with the indicated antibodies. (<b>B</b>) Cells of the indicated genotypes were fixed, stained with DAPI/calcofluor, and visualized by microscopy. For each strain fluorescent images (denoting DAPI/calcofluor staining) are shown on the left; bright-field images are shown on the right. (<b>C</b>) Quantification of the abnormal septation patterns in the indicated strains. “Septa” refers to cells with at least one visible division septum, “multi-sep” refers to cells with twinned septa or multiple septa between two nuclei, and “chains” refers to unseparated chains of cells. Error bars denote standard deviations from 3 independent experiments.</p