Papers by Senthilkumar Cinghu
Journal of Biological Chemistry, 2010
RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expr... more RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.
Molecular Cell, 2014
Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity ... more Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y's distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways.
Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone... more Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells (ESCs), bivalency is postulated to poise/prime lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues; however, this hypothesis remains to be directly tested. Most gene promoters DNA-hypermethylated in adult human cancers are bivalently marked in ESCs, and it was speculated that bivalency predisposes them for aberrant de novo DNA methylation and irreversible silencing in cancer, but evidence supporting this model is largely lacking. Here we show that bivalent chromatin does not poise genes for rapid activation but protects promoters from de novo DNA methylation. Genome-wide studies in differentiating ESCs reveal that activation of bivalen...
The development of the heart follows a synergic action of several signaling pathways during gesta... more The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/Bl6/J mice at 4 (28-days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/d...
Free Radical Biology and Medicine
Nucleic Acids Research
The developmental potential of cells, termed pluripotency, is highly dynamic and progresses throu... more The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved ‘trans-omic’ datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in...
SUMMARYPluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell... more SUMMARYPluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell states. The two states that bookend the pluripotency continuum, naïve and primed, are well characterized, but our understanding of the intermediate states and transitions between them remain incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre-to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of mouse embryonic stem cells transitioning from naïve to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes demarcate discrete phases of pluripotency, characterized by cell-state-specific surface marker expression. Our data...
Cell Systems
Highlights d Multi-omic maps of embryonic stem cells transitioning from naive to primed pluripote... more Highlights d Multi-omic maps of embryonic stem cells transitioning from naive to primed pluripotency d Phosphoproteome dynamics precede changes to epigenome, transcriptome, and proteome d ERK signaling is dispensable beyond the initial phase of exit from naive pluripotency d Comparative analysis of mouse and human naive and primed pluripotent states
ABSTRACTFaithful transcription initiation is critical for accurate gene expression, yet the mecha... more ABSTRACTFaithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription preinitiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene express...
Molecular cell, Jan 5, 2017
Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) re... more Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intra...
Journal of Cellular Biochemistry, Jun 1, 2009
Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have t... more Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27 Kip1 , and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.
Journal of Cancer Research and Clinical Oncology, Apr 1, 2011
Helicase-like transcription factor (HLTF) is a member of the SWI/SNF (mating type switching/sucro... more Helicase-like transcription factor (HLTF) is a member of the SWI/SNF (mating type switching/sucrose non-fermenting) family of ATPases/helicases and also has a RING-finger motif characteristic of ubiquitin ligase proteins. These features have led to suggestions that HLTF functions like yeast Rad5, which promotes replication through DNA lesions via a post-replication repair pathway. However, the function of HLTF in higher eukaryotes is still unknown. Herein, we found the overexpression of HLTF in radiation recurrent human uterine cervical carcinoma tissues when compared to disease free survived patients tissues. In this study, we used RNA interference techniques to investigate the potential function of HLTF in cervical cancer cell line HeLa and found that the cell proliferation was reduced by knockdown (KD) of HLTF. A host-cell reactivation assay showed that the capacity for repair to DNA damage induced by X-ray irradiation was reduced in HLTF KD cells. X-rays also increased apoptosis in HLTF KD cells. These results suggest that HLTF is involved in DNA repair and apoptosis in cancer cells, which might represent a target for gene therapies of human cancer. Keywords HLTF Á Helicase-like transcription factor Á Cervical cancer Á Apoptosis Á Radiation S. Cho and S. Cinghu have contributed equally to the work.
Nature communications, Jan 24, 2015
Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during pube... more Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC mainte...
Acta Biochimica et Biophysica Sinica, 2011
Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation ... more Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (g-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.
Proceedings of the National Academy of Sciences, 2014
Data deposition: The microarray gene expression data generated for this study have been deposited... more Data deposition: The microarray gene expression data generated for this study have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE47872).
Oncogene, 2010
Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molec... more Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3À/À mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3À/À bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3À/À epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3 þ /À mice (B18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3 þ /À mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.
Journal of Cellular Physiology, 2011
The high incidence and fatality rate of uterine cervical cancer warrant effective diagnostic and ... more The high incidence and fatality rate of uterine cervical cancer warrant effective diagnostic and therapeutic target identification for this disease. Here, we have found a novel oncoprotein FTS (Fused Toes Homolog), which is involved in cervical cancer pathogenesis. Immunohistochemical analysis of human cervical biopsy samples revealed that the expression of FTS is absent in normal cervical epithelium but progressively overexpressed in human cervical intraneoplastic lesions (CIN-I to CIN-III), this characteristic phenomenon put this protein, a potential diagnostic marker for the screening of early neoplastic changes of cervix. Using FTS-specific small hairpin RNA (shRNA) in cervical cancer cells, we determined a specific role for FTS protein in, cervical neoplasia. Targeted stable knock down of FTS in HeLa cells led to the growth inhibition, cell-cycle arrest, and apoptosis with concurrent increase in p21 protein. FTS effectively represses the p21 mRNA expression in dual luciferase assay which indicates that p21 is transcriptionally regulated by this oncoprotein which in turn affect the regular cell-cycle process and its components. Consistent with this we found a reciprocal association between these proteins in early cervical neoplastic tissues. These data unraveled the involvement of new oncoprotein FTS in cervical cancer which plays a central role in carcinogenesis. Targeted inhibition of FTS lead to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy for cervical cancer.
Molecular Cell, 2014
Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity ... more Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y's distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways.
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Papers by Senthilkumar Cinghu