The 'Alu-ome' shapes the epigenetic environment of regulatory elements controlling cellular defense.

Promoters 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.

HP1γ binding pre-mRNA intronic repeats modulates RNA splicing decisions.

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1γ/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1γ in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1γ 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 HP1γ 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.

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.

Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest.

Mammalian female meiosis is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that a gradual decline of BubR1 contributes to age-related aneuploidization. Here we employ a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I and the establishment of robust kinetochore-microtubule attachments in a meiosis-specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multifaceted role in chromosome segregation during the first meiotic division and suggest that age-related decline of BubR1 is a key determinant of the formation of aneuploid oocytes as women approach menopause.

Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF.

HP1 proteins are transcriptional regulators that, like histones, are targets for post-translational modifications defining an HP1-mediated subcode. HP1γ has multiple phosphorylation sites, including serine 83 (S83) that marks it to sites of active transcription. In a guinea pig model for Shigella enterocolitis, we observed that the defective type III secretion mxiD Shigella flexneri strain caused more HP1γ phosphorylation in the colon than the wild-type strain. Shigella interferes with HP1 phosphorylation by injecting the phospholyase OspF. This effector interacts with HP1γ and alters its phosphorylation at S83 by inactivating ERK and consequently MSK1, a downstream kinase. MSK1 that here arises as a novel HP1γ kinase, phosphorylates HP1γ at S83 in the context of an MSK1-HP1γ complex, and thereby favors its accumulation on its target genes. Genome-wide transcriptome analysis reveals that this mechanism is linked to up-regulation of proliferative gene and fine-tuning of immune gene expression. Thus, in addition to histones, bacteria control host transcription by modulating the activity of HP1 proteins, with potential implications in transcriptional reprogramming at the mucosal barrier.

Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk.

The nuclear bile acid receptor farnesoid X receptor (FXR) is an important transcriptional regulator of bile acid, lipid, and glucose metabolism. FXR is highly expressed in the liver and intestine and controls the synthesis and enterohepatic circulation of bile acids. However, little is known about FXR-associated proteins that contribute to metabolic regulation. Here, we performed a mass spectrometry-based search for FXR-interacting proteins in human hepatoma cells and identified AMPK as a coregulator of FXR. FXR interacted with the nutrient-sensitive kinase AMPK in the cytoplasm of target cells and was phosphorylated in its hinge domain. In cultured human and murine hepatocytes and enterocytes, pharmacological activation of AMPK inhibited FXR transcriptional activity and prevented FXR coactivator recruitment to promoters of FXR-regulated genes. Furthermore, treatment with AMPK activators, including the antidiabetic biguanide metformin, inhibited FXR agonist induction of FXR target genes in mouse liver and intestine. In a mouse model of intrahepatic cholestasis, metformin treatment induced FXR phosphorylation, perturbed bile acid homeostasis, and worsened liver injury. Together, our data indicate that AMPK directly phosphorylates and regulates FXR transcriptional activity to precipitate liver injury under conditions favoring cholestasis.

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.

The elongation complex components BRD4 and MLLT3/AF9 are transcriptional coactivators of nuclear retinoid receptors.

Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.

10-million-years AGO: argonaute on chromatin in yeast and human, a conserved mode of action?

Whereas in yeast the function and mode of action of nuclear RNAi are well documented, mammalian nuclear RNAi is a matter of debates. Several papers support a role for nuclear Argonaute in alternative splicing. However, the molecular mechanism remains elusive. Here, we discuss the human nuclear RNAi mechanism in light of what is known of the yeast process.

Nucleosome assembly proteins and their interacting proteins in neuronal differentiation.

Neuronal differentiation from neural stem cells into mature neurons is guided by the concerted action of specific transcription factors that stepwise exercise their role in the context of defined chromatin states. Amongst the classes of proteins that influence chromatin compaction and modification are nucleosome assembly proteins (NAPs). Mammals possess several nucleosome assembly protein 1 like proteins (NAP1L) that show either ubiquitous or neuron-restricted expression. The latter group is presumably involved in the process of neuronal differentiation. Mammalian NAP1Ls can potentially form both homo- and hetero-dimers and octamers, in theory allowing thousands of different combinations to be formed. Detailed studies have been performed on several of the NAP1Ls that point to a range of molecular roles, including transcriptional regulation, nuclear import, and control of cell division. This article aims at summarizing current knowledge of the mammalian NAP1L family and its interactions.

Argonaute proteins couple chromatin silencing to alternative splicing.

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.

Mps1 at kinetochores is essential for female mouse meiosis I.

In female meiosis, chromosome missegregations lead to the generation of aneuploid oocytes and can cause the development of trisomies or infertility. Because mammalian female meiosis I is error prone, the full functionality of control mechanisms, such as the spindle assembly checkpoint (SAC), has been put into question. The SAC monitors the correct orientation, microtubule occupancy and tension on proteinaceous structures named kinetochores. Although it has been shown previously that the SAC exists in meiosis I, where attachments are monopolar, the role of microtubule occupancy for silencing the SAC and the importance of certain essential SAC components, such as the kinase Mps1, are unknown in mammalian oocytes. Using a conditional loss-of-function approach, we address the role of Mps1 in meiotic progression and checkpoint control in meiosis I. Our data demonstrate that kinetochore localization of Mps1 is required for the proper timing of prometaphase and is essential for SAC control, chromosome alignment and aurora C localization in meiosis I. The absence of Mps1 from kinetochores severely impairs chromosome segregation in oocyte meiosis I and, therefore, fertility in mice. In addition, we settle a long-standing question in showing that kinetochore-microtubule attachments are present in prometaphase I at a time when most of the SAC protein Mad2 disappears from kinetochores.

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

Pre-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.

Interaction between nucleosome assembly protein 1-like family members.

Mammals possess five nucleosome assembly protein 1-like (NAP1L) proteins, with three of them being expressed exclusively in the nervous system. The biological importance of the neuron-specific NAP1L2 protein is demonstrated by the neural tube defects occurring during the embryonic development of Nap1l2 mutant mice, which are associated with an overproliferation of neural stem cells and decreased neuronal differentiation. NAP1L2 controls the expression of its target genes, such as the cell cycle regulator Cdkn1c, at least in part via an effect on histone acetylation. Using a two-hybrid analysis, we have identified several proteins interacting with NAP1L2, including the ubiquitously expressed members of the nucleosome assembly protein family, NAP1L1 and NAP1L4. Structural studies further predict that all five NAP1-like proteins are able to interact directly via their highly conserved α-helices. These elements, in conjunction with the coexpression of all the NAP1-like proteins in neurons and the finding that deletion of Nap1l2 affects the cytoplasmic-nuclear distribution patterns of both NAP1L1 and NAP1L4 and their recruitment to target genes, suggest that combinatorial variation within the NAP family may ensure adaptation to the specific requirements for neuronal differentiation such as intercellular repartition, chromatin modification, transcriptional regulation, or the recruitment of specific transcription factors.

Regulation of an inducible promoter by an HP1beta-HP1gamma 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 HP1alpha, HP1beta and HP1gamma with RNAPII. We find that, in the absence of stimulation, HP1beta is present on the promoter together with the non-processive RNAPII and functions as a negative regulator. On activation, HP1beta bound to methylated H3K9 is rapidly released concurrent with histone H3 phospho-acetylation, and is replaced by HP1gamma. 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.

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.

Distinct roles of the steroid receptor coactivator 1 and of MED1 in retinoid-induced transcription and cellular differentiation.

Retinoic acid receptors (RARs) are the molecular relays of retinoid action on transcription, cellular differentiation and apoptosis. Transcriptional activation of retinoid-regulated promoters requires the dismissal of corepressors and the recruitment of coactivators to promoter-bound RAR. RARs recruit in vitro a plethora of coactivators whose actual contribution to retinoid-induced transcription is poorly characterized in vivo. Embryonal carcinoma P19 cells, which are highly sensitive to retinoids, were depleted from archetypical coactivators by RNAi. SRC1-deficient P19 cells showed severely compromised retinoid-induced responses, in agreement with the supposed role of SRC1 as a RAR coactivator. Unexpectedly, Med1/TRAP220/DRIP205-depleted cells exhibited an exacerbated response to retinoids, both in terms transcriptional responses and of cellular differentiation. Med1 depletion affected TFIIH and cdk9 detection at the prototypical retinoid-regulated RARbeta2 promoter, and favored a higher RNA polymerase II detection in transcribed regions of the RARbeta2 gene. Furthermore, the nature of the ligand impacted strongly on the ability of RARs to interact with a given coactivator and to activate transcription in intact cells. Thus RAR accomplishes transcriptional activation as a function of the ligand structure, by recruiting regulatory complexes which control distinct molecular events at retinoid-regulated promoters.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Requirements for heterodimerization between the orphan nuclear receptor Nurr1 and retinoid X receptors.

The nuclear receptor nurr1 is a transcription factor involved in the development and maintenance of neurons synthesizing the neurotransmitter dopamine. Although the lack of nurr1 expression has dramatic consequences for these cells either in terms of differentiation or survival, the mechanisms by which nurr1 controls gene transcription still remain unclear. In the intent to understand better the modalities of action of this nuclear receptor, we have undertaken a systematic analysis of the transcriptional effects and DNA binding properties of nurr1 as a monomer or when forming dimers with the different isotypes of the retinoic X receptor (RXR). Here, we show that nurr1 acts as a gene activator independently of RXR and through an AF2-independent mechanism. In addition, heterodimerization with RXR is isotype-specific, involves multiple domains in the C-terminal region of nurr1, and requires RXR binding to DNA. RXR(alpha)-nurr1 and RXRgamma-nurr1 heterodimers bind direct repeat response elements and display no specific requirements with respect to half-site spacing. However, the retinoid responsiveness of DNA-bound heterodimers requires the reiteration of at least three nurr1 binding sites, thereby limiting retinoid-induced nurr1 transcriptional activity to specific direct response elements.