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Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme. Pyruvate kinase M2 (PKM2) activity is regulated by the availability of metabolic intermediates for nucleotide and amino acid synthesis. Here, the authors show that PKM2 citrullination by PADI1 and PADI3 lowers its sensitivity to metabolic inhibitors leading to increased glycolysis and reduced cancer cell proliferation.

A known oncogene, MITF , resides in a region of chromosome 3 that is amplified in melanomas and associated with poor prognosis; now, a long non-coding RNA gene, SAMMSON , is shown to also lie in this region, to also act as a melanoma-specific survival oncogene, and to be a promising therapeutic target for anti-melanoma therapy. An oncogenic non-coding RNA The known oncogene MITF is found in the 3p13–3p14 region of chromosome 3 that is amplified in melanomas and associated with poor prognosis. This study shows that a long non-coding RNA, SAMMSON , also lies in this region and is co-gained with MITF . SAMMSON interacts with p32 and thereby affects mitochondrial function in a pro-oncogenic manner. SAMMSON depletion sensitizes melanoma cells to MAPK-targeting therapeutics in vivo and in patient-derived xenograft models. These results point to SAMMSON as a potentially useful biomarker for malignancy and as an anti-melanoma therapeutic target. Focal amplifications of chromosome 3p13–3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon 1 . However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF . In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans , SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF , NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses.

The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

ObjectiveHepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention.DesignLiver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention.ResultsIn patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients.ConclusionOur results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

RNA polymerase II transcription can efficiently occur when mouse embryonic stem cells lack TBP and TBP-like proteins, confirming that this initiation factor may not be as essential as once thought.RNA polymerase II transcription can efficiently occur when mouse embryonic stem cells lack TBP and TBP-like proteins, confirming that this initiation factor may not be as essential as once thought.

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1 , HOXB9 and MITF and gain of MYC and NFE2L2 -associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells. The molecular mechanisms involved in the development of SMARCB1-deficient renal medullary carcinomas (RMCs) remain to be characterised. Here, the authors integrated RMC omics data to show that ferroptosis resistance contributes to transformation of renal thick ascending limb cells into several RMC cell states.

Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8 . Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

Regulation of gene expression involves a complex and dynamic dialogue between transcription factors, chromatin remodelling and modification complexes and the basal transcription machinery. To address the function of the Taf4 subunit of general transcription factor TFIID in the regulation of insulin signalling, it was inactivated in adult murine pancreatic beta cells. Taf4 inactivation impacted the expression of critical genes involved in beta-cell function leading to increased glycaemia, lowered plasma insulin levels and defective glucose-stimulated insulin secretion. One week after Taf4-loss, single-cell RNA-seq revealed cells with mixed beta cell, alpha and/or delta cell identities as well as a beta cell population trans-differentiating into alpha-like cells. Computational analysis of single-cell RNA-seq defines how known critical beta cell and alpha cell determinants may act in combination with additional transcription factors and the NuRF chromatin remodelling complex to promote beta cell trans-differentiation.

Background Crosstalk between genetic, epigenetic, and immune alterations in upper tract urothelial carcinomas and their role in shaping muscle invasiveness and patient outcome are poorly understood. Results We perform an integrative genome- and methylome-wide profiling of diverse non-muscle-invasive and muscle-invasive upper tract urothelial carcinomas. In addition to mutations of FGFR3 and KDM6A , we identify ZFP36L1 as a novel, significantly mutated tumor suppressor gene. Overall, mutations of ZFP36 family genes ( ZFP36 , ZFP36L1 , and ZFP36L2 ) are identified in 26.7% of cases, which display a high mutational load. Unsupervised DNA methylation subtype classification identifies two epi-clusters associated with distinct muscle-invasive status and patient outcome, namely, EpiC-low and EpiC-high. While the former is hypomethylated, immune-depleted, and enriched for FGFR3 -mutated, the latter is hypermethylated, immune-infiltrated, and tightly associated with somatic mutations of SWI/SNF genes. Conclusions Our study delineates for the first time the key role for convergence between genetic and epigenetic alterations in shaping clinicopathological and immune upper tract urothelial carcinoma features.

The melanocyte lineage-determining Microphthalmia-associated transcription factor (MITF) drives proliferation and survival of melanocytic melanoma cells through regulation of both coding genes and long non-coding RNAs (LncRNAs). Here we characterize LINC00520 (hereafter called L ncRNA ENhancer of T ranslation, LENT) regulated by MITF and strongly expressed in melanocytic melanoma cells. LENT is essential for the proliferation and survival of cultured melanocytic melanoma cells and xenograft tumors. LENT interacts with the G4 quadruplex resolvase DHX36, and both associate with the ribosome in the 80S and light polysome fractions. LENT modulates DHX36 association with a collection of mRNAs regulating their engagement with polysomes and fine-tuning their subsequent translation. These mRNAs encode proteins involved in endoplasmic reticulum (ER) and mitochondrial homeostasis as well as autophagy. Consequently, LENT silencing leads to extensive autophagy and mitophagy, compromised oxidative metabolic capacity, accompanied by an accumulation and mis-localization of mitochondrial proteins leading to proteotoxic stress and apoptosis. The LENT-DHX36 axis therefore fine-tunes translation of proteins involved in ER and mitochondrial homeostasis, suppressing autophagy and promoting survival and proliferation of melanoma cells.