Céline Vallot

Regional copy number–independent deregulation of transcription in cancer

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

Characterization of the Recurrent 8p11-12 Amplicon Identifies PPAPDC1B, a Phosphatase Protein, as a New Therapeutic Target in Breast Cancer

The 8p11-12 chromosome region is one of the regions most frequently amplified in breast carcinoma (10-15% of cases). Several genes within this region have been identified as candidate oncogenes, as they are both amplified and overexpressed. However, very few studies have explored the role of these genes in cell transformation, with the aim of identifying valuable therapeutic targets. An analysis of comparative genomic hybridization array and expression profiling data for a series of 152 ductal breast carcinomas and 21 cell lines identified five genes (LSM1, BAG4, DDHD2, PPAPDC1B, and WHSC1L1) within the amplified region as consistently overexpressed due to an increased gene copy number. The use of small interfering RNA to knock down the expression of each of these genes showed the major role played by two genes, PPAPDC1B and WHSC1L1, in regulating the survival and transformation of two different cell lines harboring the 8p amplicon. The role of these two genes in cell survival and cell transformation was also confirmed by long-term knockdown expression studies using short hairpin RNAs. The potential of PPAPDC1B, which encodes a transmembrane phosphatase, as a therapeutic target was further shown by the strong inhibition of growth of breast tumor xenografts displaying 8p11-12 amplification induced by the silencing of PPAPDC1B. The oncogenic properties of PPAPDC1B were further shown by its ability to transform NIH-3T3 fibroblasts, inducing their anchorage-independent growth. Finally, microarray experiments on PPAPDC1B knockdown indicated that this gene interfered with multiple cell signaling pathways, including the Janus-activated kinase-signal transducer and activator of transcription, mitogen-activated protein kinase, and protein kinase C pathways. PPAPDC1B may also potentiate the estrogen receptor pathway by down-regulating DUSP22.

XACT, a long noncoding transcript coating the active X chromosome in human pluripotent cells

X-chromosome inactivation (XCI) in mammals relies on XIST, a long noncoding transcript that coats and silences the X chromosome in cis. Here we report the discovery of a long noncoding RNA, XACT, that is expressed from and coats the active X chromosome specifically in human pluripotent cells. In the absence of XIST, XACT is expressed from both X chromosomes in humans but not in mice, suggesting a unique role for XACT in the control of human XCI initiation.

Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

A Novel Epigenetic Phenotype Associated With the Most Aggressive Pathway of Bladder Tumor Progression

Background Epigenetic silencing can extend to whole chromosomal regions in cancer. There have been few genome-wide studies exploring its involvement in tumorigenesis. Methods We searched for chromosomal regions affected by epigenetic silencing in cancer by using Affymetrix microarrays and real-time quantitative polymerase chain reaction to analyze RNA from 57 bladder tumors compared with normal urothelium. Epigenetic silencing was verified by gene re-expression following treatment of bladder cell lines with 5-aza-deoxycytidine, a DNA demethylating agent, and trichostatin A, a histone deacetylase inhibitor. DNA methylation was studied by bisulfite sequencing and histone methylation and acetylation by chromatin immunoprecipitation. Clustering was used to distinguish tumors with multiple regional epigenetic silencing (MRES) from those without and to analyze the association of this phenotype with histopathologic and molecular types of bladder cancer. The results were confirmed with a second panel of 40 tumor samples and extended in vitro with seven bladder cancer cell lines. All statistical tests were two-sided. Results We identified seven chromosomal regions of contiguous genes that were silenced by an epigenetic mechanism. Epigenetic silencing was not associated with DNA methylation but was associated with histone H3K9 and H3K27 methylation and histone H3K9 hypoacetylation. All seven regions were concordantly silenced in a subgroup of 26 tumors, defining an MRES phenotype. MRES tumors exhibited a carcinoma in situ–associated gene expression signature (25 of 26 MRES tumors vs 0 of 31 non-MRES tumors, P < 10−14), rarely carried FGFR3 mutations (one of 26 vs 22 of 31 non-MRES tumors, P < 10−6), and contained 25 of 33 (76%) of the muscle-invasive tumors. Cell lines derived from aggressive bladder tumors presented epigenetic silencing of the same regions. Conclusions We have identified an MRES phenotype characterized by the concomitant epigenetic silencing of several chromosomal regions, which, in bladder cancer, is specifically associated with the carcinoma in situ gene expression signature.

Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape.

Human pluripotent stem cells (hPSCs) display extensive epigenetic instability, particularly on the X chromosome. In this study, we show that, in hPSCs, the inactive X chromosome has a specific heterochromatin landscape that predisposes it to erosion of X chromosome inactivation (XCI), a process that occurs spontaneously in hPSCs. Heterochromatin remodeling and gene reactivation occur in a non-random fashion and are confined to specific H3K27me3-enriched domains, leaving H3K9me3-marked regions unaffected. Using single-cell monitoring of XCI erosion, we show that this instability only occurs in pluripotent cells. We also provide evidence that loss of XIST expression is not the primary cause of XCI instability and that gene reactivation from the inactive X (Xi) precedes loss of XIST coating. Notably, expression and coating by the long non-coding RNA XACT are early events in XCI erosion and, therefore, may play a role in mediating this process.

XACT Noncoding RNA Competes with XIST in the Control of X Chromosome Activity during Human Early Development

Summary Sex chromosome dosage compensation is essential in most metazoans, but the developmental timing and underlying mechanisms vary significantly, even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACT and XIST on active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts, the XIST RNA adopts an unusual, highly dispersed organization, which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACT influences XIST accumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.

A vlincRNA participates in senescence maintenance by relieving H2AZ-mediated repression at the INK4 locus

Non-coding RNAs (ncRNAs) play major roles in proper chromatin organization and function. Senescence, a strong anti-proliferative process and a major anticancer barrier, is associated with dramatic chromatin reorganization in heterochromatin foci. Here we analyze strand-specific transcriptome changes during oncogene-induced human senescence. Strikingly, while differentially expressed RNAs are mostly repressed during senescence, ncRNAs belonging to the recently described vlincRNA (very long intergenic ncRNA) class are mainly activated. We show that VAD, a novel antisense vlincRNA strongly induced during senescence, is required for the maintenance of senescence features. VAD modulates chromatin structure in cis and activates gene expression in trans at the INK4 locus, which encodes cell cycle inhibitors important for senescence-associated cell proliferation arrest. Importantly, VAD inhibits the incorporation of the repressive histone variant H2A.Z at INK4 gene promoters in senescent cells. Our data underline the importance of vlincRNAs as sensors of cellular environment changes and as mediators of the correct transcriptional response.

XACT, a long non-coding transcript coating the active X chromosome in human pluripotent cells

X-chromosome inactivation (XCI) in mammals relies on XIST, a long noncoding transcript that coats and silences the X chromosome in cis. Here we report the discovery of a long noncoding RNA, XACT, that is expressed from and coats the active X chromosome specifically in human pluripotent cells. In the absence of XIST, XACT is expressed from both X chromosomes in humans but not in mice, suggesting a unique role for XACT in the control of human XCI initiation.

Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.