4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Multiscale 3D Genome Rewiring during Mouse Neural Development.

Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.

Jmjd2c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation.

Jmjd2 H3K9 demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we reveal that Jmjd2c facilitates this process by stabilising the assembly of mediator-cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a (also known as Ehmt2), independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and further destabilises loading of the mediator and cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c and G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with an impact on timely gene activation.

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

Unexpected Distinct Roles of the Related Histone H3 Lysine 9 Methyltransferases G9a and G9a-Like Protein in Myoblasts.

Lysine methyltransferases G9a and GLP (G9a-like protein) are highly homologous and form functional heterodimeric complexes that establish mono- and dimethylation on histone H3 lysine 9 (H3K9me1, H3K9me2) in euchromatin. Here, we describe unexpected distinct roles for G9a and GLP in skeletal muscle terminal differentiation. Indeed, gain- or loss-of-function assays in myoblasts showed, in agreement with previous reports, that G9a inhibits terminal differentiation. While GLP plays a more intricate role in muscle differentiation,in one hand, both GLP gain and loss of function inhibit late steps of differentiation; on the other hand, in contrast to G9a, GLP overexpression promotes abnormal precocious expression of muscle differentiation-specific genes already in proliferating myoblasts. In agreement, transcriptomic analysis indicates that G9a and GLP regulate different sets of genes. Thus, GLP, but not G9a, inhibits proteasome subunit-encoding genes expression, resulting in an inhibition of the proteasome activities. Subsequently, GLP, but not G9a, overexpression stabilizes MyoD that is likely to be responsible for muscle markers expression in proliferating myoblasts.

An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.

CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing.

G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome.

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

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.

Identification of CAD as an androgen receptor interactant and an early marker of prostate tumor recurrence.

Markers of prostate tumor recurrence after radical prostatectomy are lacking and highly demanded. The androgen receptor (AR) is a nuclear receptor that plays a pivotal role in normal and cancerous prostate tissue. AR interacts with a number of proteins modulating its stability, localization, and activity. To test the hypothesis that an increased expression of AR partners might foster tumor development, we immunopurified AR partners in human tumors xenografted into mice. One of the identified AR partners was the multifunctional enzyme carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the 3 initial steps of pyrimidine biosynthesis. We combined experiments in C4-2, LNCaP, 22RV1, and PC3 human prostate cell lines and analysis of frozen radical prostatectomy samples to study the CAD-AR interaction. We show here that in prostate tumor cells, CAD fosters AR translocation into the nucleus and stimulates its transcriptional activity. Notably, in radical prostatectomy specimens, CAD expression was not correlated with proliferation markers, but a higher CAD mRNA level was associated with local tumor extension (P=0.049) and cancer relapse (P=0.017). These results demonstrate an unsuspected function for a key metabolic enzyme and identify CAD as a potential predictive marker of cancer relapse.

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells.

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response.

Intracellular pathogens such as Listeria monocytogenes subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon (IFN)-stimulated genes (ISGs). IFN-λ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with lntA-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1(+/-) mice or when lntA was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-λ-mediated immune response to control bacterial colonization of the host.

Preferential association of irreversibly silenced E2F-target genes with pericentromeric heterochromatin in differentiated muscle cells.

The heterochromatin-associated H3K9 tri-methylase Suv39h1 is involved in the permanent silencing of E2F target genes in differentiating but not in quiescent cells. Here, we tested the hypothesis that permanent silencing of E2F target genes is associated with their subnuclear positioning close to the pericentromeric heterochromatin compartment, enriched in Suv39h1. Using fluorescence in situ hybridization, we analyzed the subnuclear localization of three E2F target genes relative to the pericentromeric heterochromatin, in cycling fibroblasts or differentiating myoblasts. We observed that all three E2F-target genes have a tendency to relocate closer to the pericentromeric heterochromatin, only when cells differentiate and undergo an irreversible cell cycle withdrawal. These data suggest that repression of E2F target genes in cycling or in differentiating cells is achieved through distinct mechanisms. In differentiating cells, permanent silencing is driven by a Suv39h1-dependent H3K9 tri-methylation and positioning close to the heterochromatin compartment, whereas repression in cycling cells seems independent from subnuclear positioning and requires distinct H3K9 methylation levels.

The core binding factor CBF negatively regulates skeletal muscle terminal differentiation.

BACKGROUND: Core Binding Factor or CBF is a transcription factor composed of two subunits, Runx1/AML-1 and CBF beta or CBFbeta. CBF was originally described as a regulator of hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that CBF is involved in the control of skeletal muscle terminal differentiation. Indeed, downregulation of either Runx1 or CBFbeta protein level accelerates cell cycle exit and muscle terminal differentiation. Conversely, overexpression of CBFbeta in myoblasts slows terminal differentiation. CBF interacts directly with the master myogenic transcription factor MyoD, preferentially in proliferating myoblasts, via Runx1 subunit. In addition, we show a preferential recruitment of Runx1 protein to MyoD target genes in proliferating myoblasts. The MyoD/CBF complex contains several chromatin modifying enzymes that inhibits MyoD activity, such as HDACs, Suv39h1 and HP1beta. When overexpressed, CBFbeta induced an inhibition of activating histone modification marks concomitant with an increase in repressive modifications at MyoD target promoters. CONCLUSIONS/SIGNIFICANCE: Taken together, our data show a new role for Runx1/CBFbeta in the control of the proliferation/differentiation in skeletal myoblasts.

A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.

Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.

Physical and functional interaction between heterochromatin protein 1alpha and the RNA-binding protein heterogeneous nuclear ribonucleoprotein U.

By combining biochemical purification and mass spectrometry, we identified proteins associated with human heterochromatin protein 1alpha (HP1alpha) both in the nucleoplasm and in chromatin. Some of these are RNA-binding proteins, and among them is the protein heterogeneous nuclear ribonucleoprotein U (hnRNP U)/SAF-A, which is linked to chromatin organization and transcriptional regulation. Here, we demonstrate that hnRNP U is a bona fide HP1alpha-interacting molecule. More importantly, hnRNP U depletion reduces HP1alpha-dependent gene silencing and disturbs HP1alpha subcellular localization. Thus, our data demonstrate that hnRNP U is involved in HP1alpha function, shedding new light on the mode of action of HP1alpha and on the function of hnRNP U.