Zhang B*, Day DS*, Ho JW, Song L, Cao J, Christodoulou D, Seidman JG, Crawford GE, Park PJ, Pu WT.
A dynamic H3K27ac signature identifies VEGFA-stimulated endothelial enhancers and requires EP300 activity. Genome Res 2013;23(6):917-27.
Abstract
Histone modifications are now well-established mediators of transcriptional programs that distinguish cell states. However, the kinetics of histone modification and their role in mediating rapid, signal-responsive gene expression changes has been little studied on a genome-wide scale. Vascular endothelial growth factor A (VEGFA), a major regulator of angiogenesis, triggers changes in transcriptional activity of human umbilical vein endothelial cells (HUVECs). Here, we used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to measure genome-wide changes in histone H3 acetylation at lysine 27 (H3K27ac), a marker of active enhancers, in unstimulated HUVECs and HUVECs stimulated with VEGFA for 1, 4, and 12 h. We show that sites with the greatest H3K27ac change upon stimulation were associated tightly with EP300, a histone acetyltransferase. Using the variation of H3K27ac as a novel epigenetic signature, we identified transcriptional regulatory elements that are functionally linked to angiogenesis, participate in rapid VEGFA-stimulated changes in chromatin conformation, and mediate VEGFA-induced transcriptional responses. Dynamic H3K27ac deposition and associated changes in chromatin conformation required EP300 activity instead of altered nucleosome occupancy or changes in DNase I hypersensitivity. EP300 activity was also required for a subset of dynamic H3K27ac sites to loop into proximity of promoters. Our study identified thousands of endothelial, VEGFA-responsive enhancers, demonstrating that an epigenetic signature based on the variation of a chromatin feature is a productive approach to define signal-responsive genomic elements. Further, our study implicates global epigenetic modifications in rapid, signal-responsive transcriptional regulation.
pdf Majumdar S, Gong EM, Di Vizio D, Dreyfuss JM, Degraff DJ, Hager MH, Park PJ, Bellmunt J, Matusik RJ, Rosenberg JE, Adam RM.
Loss of Sh3gl2/endophilin A1 is a common event in urothelial carcinoma that promotes malignant behavior. Neoplasia 2013;15(7):749-60.
Abstract
Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis, we identified Sh3gl2 (endophilin A1) as a bladder urothelium-enriched transcript. The gene encoding Sh3gl2 is located on chromosome 9p, a region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to nontumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, which is a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 19 of 20 human UC cell lines but preserved in the low-grade cell line RT4. Stable silencing of Sh3gl2 in RT4 cells by RNA interference 1) enhanced proliferation and colony formation in vitro, 2) inhibited EGF-induced EGFR internalization and increased EGFR activation, 3) stimulated phosphorylation of Src family kinases and STAT3, and 4) promoted growth of RT4 xenografts in subrenal capsule tissue recombination experiments. Conversely, forced re-expression of Sh3gl2 in T24 cells and silenced RT4 clones attenuated oncogenic behaviors, including growth and migration. Together, these findings identify loss of Sh3gl2 as a frequent event in UC development that promotes disease progression.
pdf Kim Y-J, Lee H-J, Kim T-M, Eisinger-Mathason KTS, Zhang AY, Schmidt B, Karl DL, Nakazawa MS, Park PJ, Simon CM, Yoon SS.
Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α. Int J Cancer 2013;132(1):29-41.
Abstract
Increased levels of hypoxia and hypoxia-inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category "Response to hypoxia" was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia-responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF-1α shRNA or Dox at low concentrations blocked HIF-1α induction of VEGF-A by 84-97% and carbonic anhydrase 9 by 83-93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF-1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α target genes that may promote resistance to antiangiogenic and other therapies. HIF-1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.
pdf Gokcumen O, Tischler V, Tica J, Zhu Q, Iskow RC, Lee E, Fritz MH-Y, Langdon A, Stütz AM, Pavlidis P, Benes V, Mills RE, Park PJ, Lee C, Korbel JO.
Primate genome architecture influences structural variation mechanisms and functional consequences. Proc Natl Acad Sci U S A 2013;110(39):15764-9.
Abstract
Although nucleotide resolution maps of genomic structural variants (SVs) have provided insights into the origin and impact of phenotypic diversity in humans, comparable maps in nonhuman primates have thus far been lacking. Using massively parallel DNA sequencing, we constructed fine-resolution genomic structural variation maps in five chimpanzees, five orang-utans, and five rhesus macaques. The SV maps, which are comprised of thousands of deletions, duplications, and mobile element insertions, revealed a high activity of retrotransposition in macaques compared with great apes. By comparison, nonallelic homologous recombination is specifically active in the great apes, which is correlated with architectural differences between the genomes of great apes and macaque. Transcriptome analyses across nonhuman primates and humans revealed effects of species-specific whole-gene duplication on gene expression. We identified 13 gene duplications coinciding with the species-specific gain of tissue-specific gene expression in keeping with a role of gene duplication in the promotion of diversification and the acquisition of unique functions. Differences in the present day activity of SV formation mechanisms that our study revealed may contribute to ongoing diversification and adaptation of great ape and Old World monkey lineages.
pdf Woo CJ, Kharchenko PV, Daheron L, Park PJ, Kingston RE.
Variable requirements for DNA-binding proteins at polycomb-dependent repressive regions in human HOX clusters. Mol Cell Biol 2013;33(16):3274-85.
Abstract
Polycomb group (PcG)-mediated repression is an evolutionarily conserved process critical for cell fate determination and maintenance of gene expression during embryonic development. However, the mechanisms underlying PcG recruitment in mammals remain unclear since few regulatory sites have been identified. We report two novel prospective PcG-dependent regulatory elements within the human HOXB and HOXC clusters and compare their repressive activities to a previously identified element in the HOXD cluster. These regions recruited the PcG proteins BMI1 and SUZ12 to a reporter construct in mesenchymal stem cells and conferred repression that was dependent upon PcG expression. Furthermore, we examined the potential of two DNA-binding proteins, JARID2 and YY1, to regulate PcG activity at these three elements. JARID2 has differential requirements, whereas YY1 appears to be required for repressive activity at all 3 sites. We conclude that distinct elements of the mammalian HOX clusters can recruit components of the PcG complexes and confer repression, similar to what has been seen in Drosophila. These elements, however, have diverse requirements for binding factors, which, combined with previous data on other loci, speaks to the complexity of PcG targeting in mammals.
pdf Davoli T, Xu AW, Mengwasser KE, Sack LM, Yoon JC, Park PJ, Elledge SJ.
Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 2013;155(4):948-62.
Abstract
Aneuploidy has been recognized as a hallmark of cancer for more than 100 years, yet no general theory to explain the recurring patterns of aneuploidy in cancer has emerged. Here, we develop Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method that analyzes the patterns of mutational signatures in tumors and predicts the likelihood that any individual gene functions as a tumor suppressor (TSG) or oncogene (OG). By analyzing >8,200 tumor-normal pairs, we provide statistical evidence suggesting that many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. Integrating our driver predictions with information on somatic copy number alterations, we find that the distribution and potency of TSGs (STOP genes), OGs, and essential genes (GO genes) on chromosomes can predict the complex patterns of aneuploidy and copy number variation characteristic of cancer genomes. We propose that the cancer genome is shaped through a process of cumulative haploinsufficiency and triplosensitivity.
pdf Ferrari F, Plachetka A, Alekseyenko AA, Jung YL, Ozsolak F, Kharchenko PV, Park PJ, Kuroda MI.
"Jump start and gain" model for dosage compensation in Drosophila based on direct sequencing of nascent transcripts. Cell Rep 2013;5(3):629-36.
Abstract
Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately 2-fold. The MSL complex preferentially binds the bodies of active genes on the male X, depositing H4K16ac with a 3' bias. Two models have been proposed for the influence of the MSL complex on transcription: one based on promoter recruitment of RNA polymerase II (Pol II), and a second featuring enhanced transcriptional elongation. Here, we utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. We also compare X and autosomes from published data on paused and elongating polymerase in order to assess the role of Pol II recruitment. Our results support a model for differentially regulated elongation, starting with release from 5' pausing and increasing through X-linked gene bodies. Our results highlight facilitated transcriptional elongation as a key mechanism for the coordinated regulation of a diverse set of genes.
pdf Tzatsos A, Paskaleva P*, Ferrari F*, Deshpande V, Stoykova S, Contino G, Wong K-K, Lan F, Trojer P, Park PJ, Bardeesy N.
KDM2B promotes pancreatic cancer via Polycomb-dependent and -independent transcriptional programs. J Clin Invest 2013;123(2):727-39.
Abstract
Epigenetic mechanisms mediate heritable control of cell identity in normal cells and cancer. We sought to identify epigenetic regulators driving the pathogenesis of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal human cancers. We found that KDM2B (also known as Ndy1, FBXL10, and JHDM1B), an H3K36 histone demethylase implicated in bypass of cellular senescence and somatic cell reprogramming, is markedly overexpressed in human PDAC, with levels increasing with disease grade and stage, and highest expression in metastases. KDM2B silencing abrogated tumorigenicity of PDAC cell lines exhibiting loss of epithelial differentiation, whereas KDM2B overexpression cooperated with KrasG12D to promote PDAC formation in mouse models. Gain- and loss-of-function experiments coupled to genome-wide gene expression and ChIP studies revealed that KDM2B drives tumorigenicity through 2 different transcriptional mechanisms. KDM2B repressed developmental genes through cobinding with Polycomb group (PcG) proteins at transcriptional start sites, whereas it activated a module of metabolic genes, including mediators of protein synthesis and mitochondrial function, cobound by the MYC oncogene and the histone demethylase KDM5A. These results defined epigenetic programs through which KDM2B subverts cellular differentiation and drives the pathogenesis of an aggressive subset of PDAC.
pdf DeGennaro CM, Alver BH, Marguerat S, Stepanova E, Davis CP, Bähler J, Park PJ, Winston F.
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast. Mol Cell Biol 2013;33(24):4779-92.
Abstract
Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the roles of Spt6, we performed genome-wide analyses of transcription, chromatin structure, and histone modifications in a Schizosaccharomyces pombe spt6 mutant. Our results demonstrate dramatic changes to transcription and chromatin structure in the mutant, including elevated antisense transcripts at >70% of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for marks associated with active transcription, including trimethylation of histone H3 on lysine 4, previously observed in humans but not Saccharomyces cerevisiae, and lysine 36. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones.
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