Sound of silence: the properties and functions of repressive Lys methyltransferases.

The methylation of histone Lys residues by Lys methyltransferases (KMTs) regulates chromatin organization and either activates or represses gene expression, depending on the residue that is targeted. KMTs are emerging as key components in several cellular processes, and their deregulation is often associated with pathogenesis. Here, we review the current knowledge on the main KMTs that are associated with gene silencing: namely, those responsible for methylating histone H3 Lys 9 (H3K9), H3K27 and H4K20. We discuss their biochemical properties and the various mechanisms by which they are targeted to the chromatin and regulate gene expression, as well as new data on the interplay between them and other chromatin modifiers.

SETDB1 fuels the lung cancer phenotype by modulating epigenome, 3D genome organization and chromatin mechanical properties.

Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.

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.

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.

The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC. METHODS: hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model. RESULTS: We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo. CONCLUSIONS: Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.

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.

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 Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin.

G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation.

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.

H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1.

The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes--Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)--both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.

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.

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.

Protocol for differential analysis of Trim71-associated protein complexes in mouse embryonic stem cells by mass spectrometry using Perseus.

Characterizing multi-protein complexes using mass spectrometry is crucial for understanding complex biochemical activities that govern cell fate. Investigating dynamic protein-protein interactions requires diverse qualitative and quantitative approaches. We present a protocol for differential analysis of Trim71-associated proteins in mouse embryonic stem cells under two conditions. We describe steps for cytoplasmic extraction, protein immunoprecipitation, sample preparation for mass spectrometry, and data analysis with Perseus. Our versatile tool enables in-depth exploration of protein complex compositional changes, contributing to a deeper understanding of cellular dynamics and making it suitable for various research domains. For complete details on the use and execution of this protocol, please refer to Rapone et al.1.

SETDB1 modulates the TGFß response in Duchenne muscular dystrophy myotubes.

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.

Setdb1 protects genome integrity in murine muscle stem cells to allow for regenerative myogenesis and inflammation.

The histone H3 lysine 9 methyltransferase SETDB1 controls transcriptional repression to direct stem cell fate. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for skeletal muscle regeneration. We find that SETDB1 represses the expression of endogenous retroviruses (ERVs) in MuSCs. ERV de-repression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling shows that chromatin decompaction at ERV loci activates the DNA-sensing cGAS-STING pathway, entailing cytokine expression by Setdb1-null MuSCs. This is followed by aberrant infiltration of inflammatory cells, including pathological macrophages. The ensuing histiocytosis is accompanied by myofiber necrosis, which, in addition to progressive MuSCs depletion, completely abolishes tissue repair. In contrast, loss of Setdb1 in fibro-adipogenic progenitors (FAPs) does not impact immune cells. In conclusion, genome maintenance by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and adequate reparative inflammation.

The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation.

Deciphering the mechanisms underlying skeletal muscle-cell differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20-25 base pair) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. Here, we show that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal-muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype - even when they are not expressed in the corresponding fully differentiated tissue.

The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells.

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.