Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation.

Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

Unraveling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

Aurora kinases are critical mitotic serine/threonine kinases and are often implicated in tumorigenesis. Recent studies of the interphase functions for aurora kinase (Aurk)A have considerably expanded our understanding of its role beyond mitosis. To identify the unknown targets of AurkA, we used peptide array-based screening and found E2F4 to be a novel substrate. Phosphorylation of E2F4 by AurkA at Ser75 regulates its DNA binding and subcellular localization. Because E2F4 plays an important role in skeletal muscle differentiation, we attempted to gain insight into E2F4 phosphorylation in this context. We observed that a block in E2F4 phosphorylation retained it better within the nucleus and inhibited muscle differentiation. RNA sequencing analysis revealed a perturbation of the gene network involved in the process of muscle differentiation and mitochondrial biogenesis. Collectively, our findings establish a novel role of AurkA in the process of skeletal muscle differentiation.-Dhanasekaran, K., Bose, A., Rao, V. J., Boopathi, R., Shankar, S. R., Rao, V. K., Swaminathan, A., Vasudevan, M., Taneja, R., Kundu, T. K. Unravelling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

G9a promotes proliferation and inhibits cell cycle exit during myogenic differentiation.

Differentiation of skeletal muscle cells, like most other cell types, requires a permanent exit from the cell cycle. The epigenetic programming underlying these distinct cellular states is not fully understood. In this study, we provide evidence that the lysine methyltransferase G9a functions as a central axis to regulate proliferation and differentiation of skeletal muscle cells. Transcriptome analysis of G9a knockdown cells revealed deregulation of many cell cycle regulatory genes. We demonstrate that G9a enhances cellular proliferation by two distinct mechanisms. G9a blocks cell cycle exit via methylation-dependent transcriptional repression of the MyoD target genes p21(Cip/Waf1) and Rb1. In addition, it activates E2F1-target genes in a methyltransferase activity-independent manner. We show that G9a is present in the E2F1/PCAF complex, and enhances PCAF occupancy and histone acetylation marks at E2F1-target promoters. Interestingly, G9a preferentially associates with E2F1 at the G1/S phase and with MyoD at the G2/M phase. Our results provide evidence that G9a functions both as a co-activator and a co-repressor to enhance cellular proliferation and inhibit myogenic differentiation.

P/CAF mediates PAX3-FOXO1-dependent oncogenesis in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric cancer of skeletal muscle with poor prognosis. A PAX3-FOXO1 fusion protein acts as a driver of malignancy in ARMS by disrupting tightly coupled but mutually exclusive pathways of proliferation and differentiation. While PAX3-FOXO1 is an attractive therapeutic target, no current treatments are designed to block its oncogenic activity. The present work shows that the histone acetyltransferase P/CAF (KAT2B) is overexpressed in primary tumours from ARMS patients. Interestingly, in fusion-positive ARMS cell lines, P/CAF acetylates and stabilizes PAX3-FOXO1 rather than MyoD, a master regulator of muscle differentiation. Silencing P/CAF, or pharmacological inhibition of its acetyltransferase activity, down-regulates PAX3-FOXO1 levels concomitant with reduced proliferation and tumour burden in xenograft mouse models. Our studies identify a P/CAF-PAX3-FOXO1 signalling node that promotes oncogenesis and may contribute to MyoD dysfunction in ARMS. This work exemplifies the therapeutic potential of targeting chromatin-modifying enzymes to inhibit fusion oncoproteins that are a frequent event in sarcomas. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation.

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.

ApoE maintains neuronal integrity via microRNA and H3K27me3-mediated repression.

ApoE regulates neurogenesis, although how it influences genetic programs remains elusive. Cortical neurons induced from isogenic control and ApoE-/- human neural stem cells (NSCs) recapitulated key transcriptomic signatures of in vivo counterparts identified from single-cell human midbrain. Surprisingly, ApoE expression in NSC and neural progenitor cells (NPCs) is not required for differentiation. Instead, ApoE prevents the over-proliferation of non-neuronal cells during extended neuronal culture when it is not expressed. Elevated miR-199a-5p level in ApoE-/- cells lowers the EZH1 protein and the repressive H3K27me3 mark, a phenotype rescued by miR-199a-5p steric inhibitor. Reduced H3K27me3 at genes linked to extracellular matrix organization and angiogenesis in ApoE-/- NPC correlates with their aberrant expression and phenotypes in neurons. Interestingly, the ApoE coding sequence, which contains many predicted miR-199a-5p binding sites, can repress miR-199a-5p without translating into protein. This suggests that ApoE maintains neurons integrity through the target-directed miRNA degradation of miR-199a-5p, imparting the H3K27me3-mediated repression of non-neuronal genes during differentiation.

Cancer Cachexia: Signaling and Transcriptional Regulation of Muscle Catabolic Genes.

Cancer cachexia (CC) is a multifactorial syndrome characterized by a significant reduction in body weight that is predominantly caused by the loss of skeletal muscle and adipose tissue. Although the ill effects of cachexia are well known, the condition has been largely overlooked, in part due to its complex etiology, heterogeneity in mediators, and the involvement of diverse signaling pathways. For a long time, inflammatory factors have been the focus when developing therapeutics for the treatment of CC. Despite promising pre-clinical results, they have not yet advanced to the clinic. Developing new therapies requires a comprehensive understanding of how deregulated signaling leads to catabolic gene expression that underlies muscle wasting. Here, we review CC-associated signaling pathways and the transcriptional cascade triggered by inflammatory cytokines. Further, we highlight epigenetic factors involved in the transcription of catabolic genes in muscle wasting. We conclude with reflections on the directions that might pave the way for new therapeutic approaches to treat CC.

EHMT2 epigenetically suppresses Wnt signaling and is a potential target in embryonal rhabdomyosarcoma.

Wnt signaling is downregulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way toward novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the DKK1 promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among 13 drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-ß-catenin node holds promise for differentiation therapy.

Chromatin Immunoprecipitation in Skeletal Myoblasts.

Chromatin immunoprecipitation (ChIP) is a powerful and sensitive technique that is widely used to study DNA-protein interactions. It enables an unbiased genome-wide analysis of transcriptional changes during several biological processes including cellular differentiation. Here, we describe a step-by-step protocol to identify histone modifications, transcription factor, and co-factor binding to chromatin in skeletal myoblasts. We discuss critical steps during cell harvesting, sonication, and immunoprecipitation and provide notes to evade common pitfalls.

Epigenetic Regulation of the PTEN-AKT-RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation-sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activity-dependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9a-deficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. SIGNIFICANCE: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.

GLP inhibits heterochromatin clustering and myogenic differentiation by repressing MeCP2.

Myogenic differentiation is accompanied by alterations in the chromatin states, which permit or restrict the transcriptional machinery and thus impact distinctive gene expression profiles. The mechanisms by which higher-order chromatin remodeling is associated with gene activation and silencing during differentiation is not fully understood. In this study, we provide evidence that the euchromatic lysine methyltransferase GLP regulates heterochromatin organization and myogenic differentiation. Interestingly, GLP represses expression of the methyl-binding protein MeCP2 that induces heterochromatin clustering during differentiation. Consequently, MeCP2 and HP1γ localization at major satellites are altered upon modulation of GLP expression. In GLP knockdown cells, depletion of MeCP2 restored both chromatin organization and myogenic differentiation. These results identify a novel regulatory axis between a histone methylation writer and DNA methylation reader, which is important for heterochromatin organization during differentiation.

A drive in SUVs: From development to disease.

Progression of cells through distinct phases of the cell cycle, and transition into out-of-cycling states, such as terminal differentiation and senescence, is accompanied by specific patterns of gene expression. These cell fate decisions are mediated not only by distinct transcription factors, but also chromatin modifiers that establish heritable epigenetic patterns. Lysine methyltransferases (KMTs) that mediate methylation marks on histone and non-histone proteins are now recognized as important regulators of gene expression in cycling and non-cycling cells. Among these, the SUV39 sub-family of KMTs, which includes SUV39H1, SUV39H2, G9a, GLP, SETDB1, and SETDB2, play a prominent role. In this review, we discuss their biochemical properties, sub-cellular localization and function in cell cycle, differentiation programs, and cellular senescence. We also discuss their aberrant expression in cancers, which exhibit de-regulation of cell cycle and differentiation.

G9a inhibits MEF2C activity to control sarcomere assembly.

In this study, we demonstrate that the lysine methyltransferase G9a inhibits sarcomere organization through regulation of the MEF2C-HDAC5 regulatory axis. Sarcomeres are essential for muscle contractile function. Presently, skeletal muscle disease and dysfunction at the sarcomere level has been associated with mutations of sarcomere proteins. This study provides evidence that G9a represses expression of several sarcomere genes and its over-expression disrupts sarcomere integrity of skeletal muscle cells. G9a inhibits MEF2C transcriptional activity that is essential for expression of sarcomere genes. Through protein interaction assays, we demonstrate that G9a interacts with MEF2C and its co-repressor HDAC5. In the presence of G9a, calcium signaling-dependent phosphorylation and export of HDAC5 to the cytoplasm is blocked which likely results in enhanced MEF2C-HDAC5 association. Activation of calcium signaling or expression of constitutively active CaMK rescues G9a-mediated repression of HDAC5 shuttling as well as sarcomere gene expression. Our results demonstrate a novel epigenetic control of sarcomere assembly and identifies new therapeutic avenues to treat skeletal and cardiac myopathies arising from compromised muscle function.

G9a, a multipotent regulator of gene expression.

Lysine methylation of histone and non-histone substrates by the methyltransferase G9a is mostly associated with transcriptional repression. Recent studies, however, have highlighted its role as an activator of gene expression through mechanisms that are independent of its methyltransferase activity. Here we review the growing repertoire of molecular mechanisms and substrates through which G9a regulates gene expression. We also discuss emerging evidence for its wide-ranging functions in development, pluripotency, cellular differentiation and cell cycle regulation that underscore the complexity of its functions. The deregulated expression of G9a in cancers and other human pathologies suggests that it may be a viable therapeutic target in various diseases.

Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor.

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1) cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1)/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation.