A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells

Although there is now evidence that the expression of centromeric (CT) and pericentric (PCT) sequences are key players in major genomic functions, their transcriptional status in human cells is still poorly known. The main reason for this lack of data is the complexity and high level of polymorphism of these repeated sequences, which hampers straightforward analyses by available transcriptomic approaches. Here a transcriptomic macro-array dedicated to the analysis of CT and PCT expression is developed and validated in heat-shocked (HS) HeLa cells. For the first time, the expression status of CT and PCT sequences is analyzed in a series of normal and cancer human cells and tissues demonstrating that they are repressed in all normal tissues except in the testis, where PCT transcripts are found. Moreover, PCT sequences are specifically expressed in HS cells in a Heat-Shock Factor 1 (HSF1)-dependent fashion, and we show here that another independent pathway, involving DNA hypo-methylation, can also trigger their expression. Interestingly, CT and PCT were found illegitimately expressed in somatic cancer samples, whereas PCT were repressed in testis cancer, suggesting that the expression of CT and PCT sequences may represent a good indicator of epigenetic deregulations occurring in response to environmental changes or in cell transformation

Nuclear Stress Bodies

Nuclear stress bodies (nSBs) are unique subnuclear organelles which form in response to heat shock. They are initiated through a direct interaction between heat shock transcription factor 1 (HSF1) and pericentric tandem repeats of satellite III sequences and correspond to active transcription sites for noncoding satellite III transcripts. Given their unusual features, nSBs are distinct from other known transcription sites. In stressed cells, they are thought to participate in rapid, transient, and global reprogramming of gene expression through different types of mechanisms including chromatin remodeling and trapping of transcription and splicing factors. The analysis of these atypical and intriguing structures uncovers new facets of the relationship between nuclear organization and nuclear function

Différenciation du nucléosome: le rôle des variants de l’histone H2A

Les histones conventionnelles H2A, H2B, H3 et H4 sont des protéines basiques très conservées autour desquelles s’enroule l’ADN pour former l’unité de base de la chromatine: le nucléosome. Ces histones du coeur du nucléosome peuvent être remplacées par des variants qui sont présents chez tous les organismes eucaryotes. Les variants d’histones participent, avec les autres voies de modification du nucléosome, à la spécialisation fonctionnelle de la chromatine. Dans cette synthèse, nous nous focalisons sur trois variants majeurs de l’histone H2A (H2A.X, H2A.Z et macroH2A). Les recherches récentes mettent en lumière leur implication dans des évènements cruciaux comme la réparation de l’ADN ou la régulation de la transcription.The histones H2A, H2B, H3 and H4 are very conserved basic proteins that wrap almost two turns of DNA to form the nucleosome core. Conventional histones can be replaced with histone variants that are found in all eukaryotic organisms. Together with other nucleosome modification pathways, histone variants participate in the functional specialization of chromatin. In this review, we focus on three major H2A histone variants: H2A.X, H2A.Z and macroH2A. Recent discoveries highlight their involvement in crucial events such as DNA repair and transcriptional regulation

HSF1-Activated Non-Coding Stress Response: Satellite lncRNAs and Beyond, an Emerging Story with a Complex Scenario

International audienceIn eukaryotes, the heat shock response is orchestrated by a transcription factor named Heat Shock Factor 1 (HSF1). HSF1 is mostly characterized for its role in activating the expression of a repertoire of protein-coding genes, including the heat shock protein (HSP) genes. Remarkably, a growing set of reports indicate that, upon heat shock, HSF1 also targets various non-coding regions of the genome. Focusing primarily on mammals, this review aims at reporting the identity of the non-coding genomic sites directly bound by HSF1, and at describing the molecular function of the long non-coding RNAs (lncRNAs) produced in response to HSF1 binding. The described non-coding genomic targets of HSF1 are pericentric Satellite DNA repeats, (sub)telomeric DNA repeats, Short Interspersed Nuclear Element (SINE) repeats, transcriptionally active enhancers and the NEAT1 gene. This diverse set of non-coding genomic sites, which already appears to be an integral part of the cellular response to stress, may only represent the first of many. Thus, the study of the evolutionary conserved heat stress response has the potential to emerge as a powerful cellular context to study lncRNAs, produced from repeated or unique DNA regions, with a regulatory function that is often well-documented but a mode of action that remains largely unknow

Chromosome Y pericentric heterochromatin is a primary target of HSF1 in male cells

International audienceAbstract The heat shock factor 1 (HSF1)-dependent transcriptional activation of human pericentric heterochromatin in heat-shocked cells is the most striking example of transcriptional activation of heterochromatin. Until now, pericentric heterochromatin of chromosome 9 has been identified as the primary target of HSF1, in both normal and tumor heat-shocked cells. Transcriptional awakening of this large genomic region results in the nuclear accumulation of satellite III ( SATIII ) noncoding RNAs (ncRNAs) and the formation in cis of specific structures known as nuclear stress bodies (nSBs). Here, we show that, in four different male cell lines, including primary human fibroblasts and amniocytes, pericentric heterochromatin of chromosome Y can also serve as a unique primary site of HSF1-dependent heterochromatin transcriptional activation, production of SATIII ncRNA, and nucleation of nuclear stress bodies (nSBs) upon heat shock. Our observation suggests that the chromosomal origin of SATIII transcripts in cells submitted to heat shock is not a determinant factor as such, but that transcription of SATIII repetitive units or the SATIII ncRNA molecules is the critical element of HSF1-dependent transcription activation of constitutive heterochromatin

Sizes of HSF1 complex in unstressed and heat shocked HeLa cells using glycerol gradient fractionation.

<p>HSF1-eGFP and HSF1-K80Q-eGFP were estimated to 150 kD in NHS and superior to 669 kDa in HS. The complex size of HSF1- ΔTRIM-eGFP in NHS was estimated to 66–150 kDa in NHS and about 200 kDa in HS. The complex size of HSF1-ΔDBD-eGFP in NHS was estimated to 66–150 kDa in NHS and about 450 kDa in HS. TE: total extract before ultra-centrifugation. The fraction corresponding to standard proteins of masses 66, 200 and 669 kDa are indicated with arrows.</p

FRAP analysis of the WT HSF1 and mutants in unstressed and stressed cells.

<p>Fluorescence recovery curves after photobleaching of HSF1 full length-eGFP before (NHS) and after heat shock (HS) in the nucleus. WT HSF1-eGFP (red), HSF1-ΔTRIM-eGFP (green), HSF1-K80Q-eGFP (blue) and HSF1-ΔDBD-eGFP (purple).</p