Remodelage de la chromatine | EPFL Graph Search

Le remodelage de la chromatine est l'un des trois mécanismes de modification de la structure de la chromatine. Intervenant d'abord au cours de l'étape de maturation de la chromatine, il permet l'obtention d'un certain état final de sa structure. Plusieurs protéines sont impliquées dans ce processus de remodelage (familles SW1/SNF, ISW1, INO, CHD). Ces protéines, appelées facteurs de remodelage de la chromatine, forment des complexes multi-protéiques et utilisent l'énergie libérée par l'hydrolyse de l'ATP pour induire des changements conformationnels au niveau du nucléosome et des domaines de la chromatine. Les facteurs de remodelages sont incapables de se fixer directement sur l'ADN, c'est pourquoi leur rapprochement avec la chromatine est permis par leur affinité avec des facteurs de transcription, ces derniers étant capables de reconnaitre des séquences cibles (dits séquences cis ou élément de réponse). Le contrôle de la structure chromatinienne via le remodelage permet égalem

The chromatin remodeller Chd7 and the calcium ATPase Serca2 - adding new facets to the role of Notch in Hematopoiesis and T-ALL

Monique Coersmeyer

Project I: Chd7 Deficiency does not affect N1-driven T-ALL Induction and Maintenance nor impair Hematopoiesis Notch1 has been shown to be a key driver in pediatric T-cell acute lymphobastic leukemia (T-ALL). Previous work in the lab identified the chromatin-remodeling enzyme Chd7 to be highly over-expressed in aggressive versus pre-malignant disease. The aim of this project was to study if Chd7 is required during T-ALL initiation and disease maintenance as well as to characterize its role in the hematopoietic system under physiological and stress conditions. Therefore, Chd7 cKO animals were crossed with the MxCre deleter to ablate Chd7 specifically in BM progenitors. Chd7 deficiency did not induce any overt phenotype in the hematopoietic system, when studied under âsteadyâ state or stress conditions. In addition, Chd7 proved to be dispensable in the initiation and maintenance of murine and human Notch1-driven T-ALL. Therefore, we can rule out a role of Chd7 during late-stage leukemogenesis in Notch1-induced murine and human T-ALL. Project II: Serca2, a putative drug target to fight Notch1-driven T cell acute lymphoblastic leukemia, and its role in Hematopoiesis The Notch signalling pathway represents a bona fide drug target. We and Roti et al. performed a chemical compound screen and identified Cyclopiazonic acid (CPA) as potent Notch inhibitor. Biological target of this compound is the Sarco/Endoplasmic Reticulum ATPase2 (Serca2) that is involved in calcium homeostasis within the cell. Recently, it was shown that Serca2 regulates Notch1 (N1) receptor maturation and inhibition of Serca ATPases, using thapsigargin, blocks T-ALL progression in xenografts (Roti et al., 2013). The putative global effects of Serca2 inhibition on hematopoiesis have, however, not been addressed in this study. Aim of this study was to characterize the function of Serca2 in the hematopoietic system. Therefore, we crossed Serca2 cKO with MxCre to ablate Serca2 function in BM progenitors. Serca2 haploinsufficiency did not lead to any overt phenotype under physiological or competitive conditions. In contrast, Serca2ÎMxCre chimeras showed (i) impaired long-term reconstitution capacity upon transplantation, (ii) a severe reduction of total BM, splenic and thymic cellularity and (iii) an enrichment of KLS cells, the cell pool that contains HSCs. Mature lineages were highly apoptotic, while KLS were not majorly affected by apoptosis, resided predominantly in G0 phase of the cell cycle, exhibited slower cycling kinetics and were functionally impaired as they formed less and smaller colony forming units in vitro. Deregulated calcium levels can lead to an accumulation of unfolded proteins in the ER. In order to resolve ER stress, the cell activates the unfolded protein response (UPR). In case of unresolved ER stress, however, the cell undergoes apoptosis. We demonstrate that mature hematopoietic lineages up-regulate UPR-associated and pro-apoptotic genes, while KLS cells, in contrast, show unperturbed expression of these genes. In addition, we performed in vitro experiments, inhibiting Serca function in Lin- BM cells via thapsigargin (TH) treatment. TH induced severe apoptosis in the overall cell population, while KLS accumulated and were blocked in the G0 phase, closely mimicking Serca2 LOF in vivo. We therefore question the therapeutic approach of targeting Serca2 in N1-driven T-ALL due to severe side effects induced by Serca2 inactivation in the BM.

EPFL2015

The human SPT20-containing SAGA complex plays a direct role in the regulation of endoplasmic reticulum stress-induced genes

One of the central questions in eukaryotic transcription is how activators can transmit their signal to stimulate gene expression in the context of chromatin. The multisubunit SAGA coactivator complex has both histone acetyltransferase and deubiquitination activities and remodels chromatin to allow transcription. Whether and how SAGA is able to regulate transcription at specific loci is poorly understood. Using mass spectrometry, immunoprecipitation, and Western blot analysis, we have identified human SPT20 (hSPT20) as the human homologue of the yeast Spt20 and show that hSPT20 is a bona fide subunit of the human SAGA (hSAGA; previously called TFTC/STAGA/PCAF) complex and that hSPT20 is required for the integrity of the hSAGA complex. We demonstrate that hSPT20 and other hSAGA subunits, together with RNA polymerase II, are specifically recruited to genes induced by endoplasmic reticulum (ER) stress. In good agreement with the recruitment of hSAGA to the ER stress-regulated genes, knockdown of hSTP20 hampers ER stress response. Surprisingly, hSPT20 recruitment was not observed for genes induced by another type of stress. These results provide evidence for a direct and specific role of the hSPT20-containing SAGA complex in transcriptional induction of ER stress-responsive genes. Thus, hSAGA regulates the transcription of stress-responsive genes in a stress type-dependent manner.

2008

Analysis of long-range gene regulation at the HoxD locus

Ana Rita De Carvalho Amândio Lhopitallier

Hoxd genes are essential for the development of the various body axes in vertebrates and hence the underlying regulatory mechanisms are of paramount importance. Among these various mechanisms are long-range acting enhancers, which are located in the two adjacent regulatory landscapes. Analyses of chromatin architecture at this gene cluster has revealed the existence of two topologically associating domains (TADs) flanking the cluster and encompassing these regulatory landscapes. However, the dynamics of such regulatory regions as well as the stability and functional contribution of specific enhancer-promoter interactions during development remains to be established. In this work, we analysed the 3D chromatin organization and transcription profile at the HoxD locus, at different time points during genital tubercle (GT) development, and observe that the 3D conformation of this regulatory region predates the embryonic emergence of the GT. Along with this tissue development, we observe a reduction in transcript levels correlating with a decrease in enhancer-promoter chromatin loops within the adjacent gene desert. This decrease occurs while maintaining a subset of CTCF/Cohesin associated contacts, which are preserved independently from the transcriptional status of the gene cluster. To further explore the functional contribution of this regulatory landscape, we used CRISPR-Cas9 technology to generate mice carrying partial deletions of this region, as well as targeted deletions of both transient (enhancer associated) and constitutive (CTCF/Cohesin associated) contacts. We observe that single deletions of both transient and constitutive contacts displayed little if any effect on Hoxd genes expression in the GT. On the contrary, the single deletion of a previous characterized Hoxd enhancer, the Prox element, or deletions comprising several enhancers, result in the reduction of Hoxd genes expression levels. Overall our results suggest that not all enhancer elements within a complex regulatory landscape have the same functional strength, and highlight the existence of a dynamic yet robust system to tightly regulate Hoxd genes expression.

EPFL2019