Sorafenib inhibits epithelial-mesenchymal transition through an epigenetic-based mechanism in human lung epithelial cells
ABSTRACT: The epithelial to mesenchymal transition (EMT) has been well recognized for many decades as an essential early step in the progression of primary tumors towards metastases. Widespread epigenetic reprogramming of DNA and histone modifications tightly regulates gene expression and cellular activity during carcinogenesis, and epigenetic therapy has been developed to design efficient strategies for cancer treatment. As the first oral agent approved for the clinical treatment of cancer, sorafenib has significant inhibitory effects on tumor growth and EMT. However, a detailed understanding of the underlying epigenetic mechanism remains elusive. In this manuscript, we performed a ChIP-Seq assay to evaluate the activity of sorafenib on the genome-wide profiling of histone modifications. We demonstrate that sorafenib largely reverses the changes in histone modifications that occur during EMT in A549 alveolar epithelial cells. Sorafenib also significantly reduces the coordinated epigenetic switching of critical EMT-associated genes in accordance with their expression levels. Furthermore, we show that sorafenib potentiates histone acetylation by regulating the expression levels of histone-modifying enzymes. Collectively, these findings provide the first evidence that sorafenib inhibits the EMT process through an epigenetic mechanism, which holds enormous promise for identifying novel epigenetic candidate diagnostic markers and drug targets for the treatment of human malignancies. To further explore the underlying epigenetic mechanisms of EMT regulation by sorafenib, we chose conventional markers of active euchromatin such as H3K9ac and H3K4me3, and contrasted their architecture with the repressive structures associated with H3K27me3 and H3K9me3. The profiling of these four selected histone modifications was performed using ChIP-seq on control, TGF-β1-treated and sorafenib-treated cells. We further performed pair-wise comparisons among the three treatment conditions to assess the changes in the histone modifications within specific genomic regions during EMT.
Project description:The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined, however the epigenetic landscape that determines their cellular identity and functionality has not been established. We report that LSCs in MLL-associated leukemia are maintained in an epigenetic state defined by relative genome-wide high-level H3K4me3 methylation and low level H3K79me2. LSC differentiation is associated with dynamic reversal of these broad epigenetic profiles and concomitant down-regulation of the LSC maintenance transcriptional program. LSCs also share with embryonic stem cells a large subset of genes with bivalent histone marks related to embryonic development. The histone demethylase KDM5B negatively regulates MLL-induced leukemogenesis demonstrating the crucial role of the H3K4 global methylome for determining leukemia stem cell fate. Investigation of multiple histone modification marks and RNA Pol II in ckit+ and ckit- cells isolated and fractionated from MLL leukemia mice.
Project description:Instructive mechanisms are present for induction of DNA methylation, as shown by methylation of specific CpG islands (CGIs) by specific inducers and in specific cancers. However, instructive factors involved are poorly understood, except for involvement of low transcription and trimethylation of histone H3 lysine 27 (H3K27me3). Here, we used methylated DNA immunoprecipitation (MeDIP) combined with a CGI oligonucleotide microarray analysis, and identified 5510 and 521 genes with promoter CGIs resistant and susceptible, respectively, to DNA methylation in prostate cancer cell lines. Expression analysis revealed that the susceptible genes had low transcription in a normal prostatic epithelial cell line. Chromatin immunoprecipitation with microarray hybridization (CHiP-chip) analysis of RNA polymerase II (Pol II) and histone modifications showed that, even among the genes with low transcription, the presence of Pol II was associated with marked resistance to DNA methylation (OR = 0.22; 95% CI = 0.12-0.38), and H3K27me3 was associated with increased susceptibility (OR = 11.20; 95% CI = 7.14-17.55). The same was true in normal human mammary epithelial cells for 5430 and 733 genes resistant and susceptible, respectively, to DNA methylation in breast cancer cell lines. These results showed that the presence of Pol II, active or stalled, and H3K27me3 can predict the epigenetic fate of promoter CGIs independently of transcription levels. To analyze DNA methylation status in normal and cancer cells, MeDIP-CGI oligonucleotide microarray analysis was performed. To analyze expression and histone modification status in normal cells, GeneChip analysis and ChIP-oligonucleotide microarray analysis were performed.
Project description:Dynamic changes in histone posttranslational modifications (PTMs) are important regulators of chromatin structure and gene transcription in both normal and disease settings. Herein, we describe a novel signaling mechanism of nitric oxide (•NO) by demonstrating its ability to modulate gene expression via alteration of histone PTMs. Having established that •NO exposure induced differential expression of approximately 6500 genes, we set out to determine if there was an epigenetic component to their regulation. •NO exposure led to alterations in the global levels of acetyl and methyl modifications at numerous lysine residues on core histones H3 and H4. Residues H3K9me2/ac were examined further and determined to have differential distribution at various loci throughout the genome in response to •NO. Changes in the enrichment levels of H3K9me2/ac at specific genes correlated with changes in the expression levels of their transcripts. Molecular mechanisms contributing to phenotypic outcomes in •NO-associated cancers remain to be well understood since traditional modes of •NO-signaling do not explain a large proportion of its impact on tumor cell behavior. Our results reveal that •NO drives a significant amount of gene expression changes epigenetically by changing the distribution of numerous histone marks. Cultured cells were treated with 500uM DETA/NO to examine the effects of a physiologically relevant •NO concentration on differential distribution of H3K9ac/H3K9me2. A total of two untreated biological replicates and two •NO-treated biological replicates were harvested. The untreated samples served as control against which comparisons were made to elucidate •NO-mediated changes in the histone landscape.
Project description:Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here, we describe a new class of modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and we demonstrate that Fibrillarin is the equivalent enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using a H2AQ105me specific antibody, show that this modification is exclusively enriched over the 35S rDNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (Facilitator of Transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 exhibits a defect in histone incorporation and shows increased transcription at rDNA genes. This defect is phenocopied by mutations in FACT that decrease its activity. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.
Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we used a mouse model of T-ALL through the overexpression of the intarcellular transcriptionally active part of Notch1 (N1-IC). This model faithfully recapitulates the major characteristics of the human disease. Comparison of the leukemic cells from peripheral tumors(thymoma) of this mouse model to normal thymic cells Double Positive (DP) for the markers CD4 and CD8 that express very low levels of Notch1 showed major expression changes (please see GSE34554) in pathways controlling the transition from physiology to disease. Further correlation of the data to ChIP-Seq data from the same cell populations led us to identify a hitherto unknown antagonism of the Notch1 oncogenic pathway and the polycomb complex (PRC2) in leukemia. Importantly exome sequencing in primary samples from human patients with T-ALL revealed that the PRC2 complex is frequently mutated and inactivated, further supporting the tumor suppressor role of the complex in this disease. Gene expression profiles from CD4+/8+ Double Positive (named DP) derived from normal thymus and Notch1-IC over-expressing tumors (named T-ALL) were analyzed for the genomewide enrichments of two major activating epigenetic marks (Histone 3 Lysine 9 acetylation (H3K9ac) and lysine 4 trimethylation (H3K4me3)), one reprressive mark (Histone 3 lysine 27 trimethylation, H3K27me3) and the major oncogene Notch1.
Project description:Embryonic hematopoiesis is regulated by the coordinated interaction between transcription factors and the epigenetic regulators driving developmental-stage specific gene expression but how this process drives hematopoietic specification and terminal differentiation is poorly understood. Here we generated RNA-Seq, DNase-Seq and ChIP-Seq data for histone marks and transcription factors from ES-cell derived purified cells representing six sequential stages of blood cell specification and differentiation. Our data reveal the binding patterns of specific transcription factors involved in the priming and maintenance of distal elements and inform how binding impacts on promoter activity. Functional studies based on these data uncovered a previously unrecognised role for Hippo signalling in mammalian hematopoietic specification. Finally, we present a dynamic core regulatory network model for hematopoiesis and demonstrate its utility for the design of reprogramming experiments. Our study represents a powerful resource for studying hematopoiesis and demonstrates how such data can advance our understanding of mammalian development. ChIP-seq data of histone modifications and transcription factors, and RNA-seq data obtained from purified cells representing five sequential stages of murine blood cell specification and differentiation
Project description:Normal cell type specific histone H3 acetylation of miRNA genes. HMEC and HMF represent two distinct differentiated cell type present in mammary gland each with a distinct phenotype, a distinct epigenotype as well as distinct miRNA expression pattern. The aim of the study was to determine how epigenetic modifications including histone H3 acetylation affect miRNA expression. Two cell types HMEC vs. HMF. Biological replicates: 3 pairs of HMEC-HMF of 3 distinct genotypes. Immunoprecipitation using anti-acetylated histone H3 antibody (06-599, Millipore).
Project description:Here we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis in primary hematopoietic cells, combining global occupancy of Lsd1, genome-wide analysis of its histone substrates H3K4 mono- and di-methylation and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 function was not restricted to transcription start sites, but is also critical at enhancers. Loss of Lsd1 at these sites was associated with increased H3K4me1 and H3K4me2 methylation levels on HSPC genes and their derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells and mature blood cell lineages. Our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation. To identify direct target genes of Lsd1 in myeloid cells we mapped global occupancy of Lsd1 in 32D granuolocytic progenitor cells and compared H3K4me1/me2/me3 and H3K27ac histone modifications in Lsd1fl/fl (wild type) vs. Lsd1fl/f Mx1Cre (knockout) Gr1dim Mac1 granuolocytic progenitor cells.
Project description:Our group is interested in epithelial-to-mesenchymal transition (EMT), in particular, TGF-beta induced EMT. TGF-beta signalling has been shown to be an important factor in the induction of EMT and it has been demonstrated that adding TGF-beta to epithelial cells in culture is a convenient way to study the process of EMT. In response to TGF-beta, Smad2 and 3 are activated, and form complexes with Smad4, which then regulate transcription of target genes through interactions with other DNA binding transcription factors. In the induction of EMT, the activated Smads mediate transcriptional regulation through three families of transcription factors, resulting in repression of epithelial marker gene expression and activation of mesenchymal gene expression (Xu J, et al. 2009) <br></br> Also investigated in this study is the role of H2A.Z in EMT. H2A.Z is an evolutionary conserved and a metazoan essential histone variant of the H2A class. Mice deficient in H2A.Z die during early development but the reason for this is unknown (Faast et al. 2001). Previously, our laboratory showed that the loss of H2A.Z in Xenpous laevis impaired cell movement required for the formation of the mesoderm and neural crest (Ridgway et al. 2004). Given that mesoderm formation is critically dependent upon EMT, we therefore wondered whether H2A.Z might be a chromatin regulator of EMT. We transfected MDCK cells with a lentiviral vector to express a construct encoding an shRNA targeting canine H2A.Z as we wanted to test the hypothesis that H2A.Z is involved in the maintenance of cellular identity and that its loss might trigger de-differentiation. <br></br> In order to investigate changes in histone variant H2A.Z occupancy associated with TGF-beta induced epithelial-to-mesenchymal transition (EMT) we performed H2A.Z ChIP-Seq in untreated and TGFb-treated MDCK cells. The MDCK cell line has been extensively used as a model system for EMT because they convert fully from the epithelial to the mesenchymal state in response to TGF-beta. <br></br>Please note that RNA-seq data generated in conjunction to this ChIP-seq data set were also deposited at ArrayExpress under accession number E-MTAB-5628 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5628 ).
Project description:The p53-regulated long non-coding RNA, lincRNA-p21, has been proposed to promote apoptosis and to repress in trans the expression of genes in the p53 transcriptional network. Here, we report the generation of a conditional knockout mouse model developed to further examine lincRNA-p21 function. Using this genetic approach, we find that the primary function of lincRNA-p21 is to activate in cis the expression of its neighboring gene, the cyclin-dependent kinase inhibitor p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a co-activator for p53-dependent transcription of p21. Additional phenotypes of lincRNA-p21 deficiency, including deregulated expression and altered chromatin state of a set of Polycomb target genes, defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency could be attributed to diminished p21 levels. This study reveals a novel paradigm, whereby the long non-coding RNA lincRNA-p21 affects global gene expression and influences events in the p53 tumor suppressor pathway by acting in cis as a locus-restricted transcriptional co-activator for p53-mediated expression of p21. Examination of 2 different histone modifications (H3K4me3 and H3K27me3) in 2 cell types (WT and lincRNA-p21 KO) in the presence and absence of Doxorubicin.