Reduced RNA turnover as a driver of cellular senescence

Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3' gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence

The ‘Alu-ome’ shapes the epigenetic environment of regulatory elements controlling cellular defense

International audiencePromoters and enhancers are sites of transcription initiation (TSSs) and carry specific histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval ranging from TSS to first upstream Alu, accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of these intervals greatly varies in-between tissues, being longer in stem-and shorter in immune-cells. The very shortest TSS-to-first-Alu intervals were observed at promoters active in T-cells, particularly at immune genes, where first-Alus were traversed by RNA polymerase II transcription, while accumulating H3K4me1 signal. Finally, DNA methylation at first-Alus was found to evolve with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes

Structural Determinants of the Ligand-Binding Site of the Human Retinoic Acid Receptor .alpha.

International audienceThe ligand-dependent transactivating properties of retinoic acid receptors are controlled through a complex structure at the C-terminus of these proteins, commonly referred to as the hormone binding domain. This domain is involved not only in ligand recognition but also in protein-protein interactions such as homo- and heterodimerization processes. To identify more precisely regions of the human all-trans-retinoic acid receptor alpha (hRAR alpha) that are involved in ligand binding, we constructed a series of deletion mutants of this molecule and overexpressed them in bacteria. We found that the C-terminal part of the D domain (amino acids 186-198) was necessary for ligand binding. The F domain and the 10 C-terminal amino acids of the E domain were dispensable for high-affinity binding of various natural and synthetic retinoids. A further deletion to position 403 resulted in a moderate decrease in affinity for all-trans-(ATRA) and 9-cis-retinoic acids, whereas the binding of two RAR alpha-specific ligands (Am80 and Am580) was abolished. In addition, hRAR alpha and the minimal hormone binding domain (amino acids 186-410) bound ATRA with a positive, cooperative mechanism. This behavior was not observed with CD367, a conformationally restricted synthetic retinoid. The positive cooperativity could be correlated with stable ATRA binding to RAR homodimers, whose formation was triggered by ligand. In the same conditions, only monomeric CD367-RAR alpha complexes were detected. These data indicate that ligand binding to hRAR alpha requires the presence of part of the D domain, whereas the C-terminal end of the E domain is involved in more subtle ligand recognition processes.(ABSTRACT TRUNCATED AT 250 WORDS

Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons

International audiencePre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here we find that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene the chromodomain protein HP1γ, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1γ on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1γ as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions

Targeting of chromatin readers: a novel strategy used by the Shigella flexneri virulence effector OspF to reprogram transcription

Shigella flexneri, a gram-negative bacterium responsible of bacillary dysentery, uses multiple strategies to overcome host immune defense. We have decrypted how this bacterium manipulates host-cell chromatin binders to take control of immune gene expression. We found that OspF, an injected virulence factor previously identified as a repressor of immune gene expression, targets the chromatin reader HP1γ. Heterochromatin Protein 1 family members specifically recognize and bind histone H3 methylated at Lys 9. Although initially identified as chromatin-associated transcriptional silencers in heterochromatin, their location in euchromatin indicates an active role in gene expression. Notably, HP1γ phosphorylation at Serine 83 defines a subpopulation exclusively located to euchromatin, targeted to the site of transcriptional elongation. We showed that OspF directly interacts with HP1γ, and causes HP1 dephosphorylation, suggesting a model in which this virulence effector “uses” HP1 proteins as beacons to target and repress immune gene expression (Harouz, et al. EMBO J (2014)). OspF alters HP1γ phosphorylation mainly by inactivating the Erk-activated kinase MSK1, spotlighting it as a new HP1 kinase. In vivo, infectious stresses trigger HP1γ phosphorylation in the colon, principally in the lamina propria and the intestinal crypts. Several lines of evidence suggest that HP1 proteins are modified as extensively as histones, and decrypting the impact of these HP1 post-translational modifications on their transcriptional activities in vivo will be the next challenges to be taken up

Nap1l2 Promotes Histone Acetylation Activity during Neuronal Differentiation▿

The deletion of the neuronal Nap1l2 (nucleosome assembly protein 1-like 2) gene in mice causes neural tube defects. We demonstrate here that this phenotype correlates with deficiencies in differentiation and increased maintenance of the neural stem cell stage. Nap1l2 associates with chromatin and interacts with histones H3 and H4. Loss of Nap1l2 results in decreased histone acetylation activity, leading to transcriptional changes in differentiating neurons, which include the marked downregulation of the Cdkn1c (cyclin-dependent kinase inhibitor 1c) gene. Cdkn1c expression normally increases during neuronal differentiation, and this correlates with the specific recruitment of the Nap1l2 protein and an increase in acetylated histone H3K9/14 at the site of Cdkn1c transcription. These results lead us to suggest that the Nap1l2 protein plays an important role in regulating transcription in developing neurons via the control of histone acetylation. Our data support the idea that neuronal nucleosome assembly proteins mediate cell-type-specific mechanisms of establishment/modification of a chromatin-permissive state that can affect neurogenesis and neuronal survival

HP1γ binding pre‐mRNA intronic repeats modulates RNA splicing decisions

International audienceHP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1c/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates cotranscriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1c in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1c is specifically targeted to hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1c RNA association tethers unspliced pre-mRNA to chromatin via the intronic regions and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing

Regulation of an inducible promoter by an HP1β–HP1γ switch

The mammalian heterochromatin protein 1 (HP1) family of proteins was recently shown to be involved in transient repression of inducible promoters. One of these promoters is the HIV1 long terminal repeat, which, during viral latency, recruits a non-processive RNA polymerase II (RNAPII) that synthesizes a short regulatory transcript. Here, we have used this promoter to examine the interplay of HP1α, HP1β and HP1γ with RNAPII. We find that, in the absence of stimulation, HP1β is present on the promoter together with the non-processive RNAPII and functions as a negative regulator. On activation, HP1β bound to methylated H3K9 is rapidly released concurrent with histone H3 phospho-acetylation, and is replaced by HP1γ. This isoform localizes to the promoter but also inside the coding region, together with the processive RNAPII. Our data show that HP1 recruitment–release is a sequential mechanism that is precisely regulated and highly dependent on transcription