Person:

Park, Peter

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only (\sim)2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to “chromatin entry sites,” which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal) complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

Background: Recent advances in sequencing technologies have enabled generation of large-scale genome sequencing data. These data can be used to characterize a variety of genomic features, including the DNA copy number profile of a cancer genome. A robust and reliable method for screening chromosomal alterations would allow a detailed characterization of the cancer genome with unprecedented accuracy. Results: We develop a method for identification of copy number alterations in a tumor genome compared to its matched control, based on application of Smith-Waterman algorithm to single-end sequencing data. In a performance test with simulated data, our algorithm shows >90% sensitivity and >90% precision in detecting a single copy number change that contains approximately 500 reads for the normal sample. With 100-bp reads, this corresponds to a ~50 kb region for 1X genome coverage of the human genome. We further refine the algorithm to develop rSW-seq, (recursive Smith-Waterman-seq) to identify alterations in a complex configuration, which are commonly observed in the human cancer genome. To validate our approach, we compare our algorithm with an existing algorithm using simulated and publicly available datasets. We also compare the sequencing-based profiles to microarray-based results. Conclusion: We propose rSW-seq as an efficient method for detecting copy number changes in the tumor genome.

Background: Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) allows genome-wide discovery of protein-DNA interactions such as transcription factor bindings and histone modifications. Previous reports only compared a small number of profiles, and little has been done to compare histone modification profiles generated by the two technologies or to assess the impact of input DNA libraries in ChIP-seq analysis. Here, we performed a systematic analysis of a modENCODE dataset consisting of 31 pairs of ChIP-chip/ChIP-seq profiles of the coactivator CBP, RNA polymerase II (RNA PolII), and six histone modifications across four developmental stages of Drosophila melanogaster. Results: Both technologies produce highly reproducible profiles within each platform, ChIP-seq generally produces profiles with a better signal-to-noise ratio, and allows detection of more peaks and narrower peaks. The set of peaks identified by the two technologies can be significantly different, but the extent to which they differ varies depending on the factor and the analysis algorithm. Importantly, we found that there is a significant variation among multiple sequencing profiles of input DNA libraries and that this variation most likely arises from both differences in experimental condition and sequencing depth. We further show that using an inappropriate input DNA profile can impact the average signal profiles around genomic features and peak calling results, highlighting the importance of having high quality input DNA data for normalization in ChIP-seq analysis. Conclusions: Our findings highlight the biases present in each of the platforms, show the variability that can arise from both technology and analysis methods, and emphasize the importance of obtaining high quality and deeply sequenced input DNA libraries for ChIP-seq analysis.

Type 2 diabetes mellitus is a complex disorder associated with multiple genetic, epigenetic, developmental, and environmental factors. Animal models of type 2 diabetes differ based on diet, drug treatment, and gene knockouts, and yet all display the clinical hallmarks of hyperglycemia and insulin resistance in peripheral tissue. The recent advances in gene-expression microarray technologies present an unprecedented opportunity to study type 2 diabetes mellitus at a genome-wide scale and across different models. To date, a key challenge has been to identify the biological processes or signaling pathways that play significant roles in the disorder. Here, using a network-based analysis methodology, we identified two sets of genes, associated with insulin signaling and a network of nuclear receptors, which are recurrent in a statistically significant number of diabetes and insulin resistance models and transcriptionally altered across diverse tissue types. We additionally identified a network of protein–protein interactions between members from the two gene sets that may facilitate signaling between them. Taken together, the results illustrate the benefits of integrating high-throughput microarray studies, together with protein–protein interaction networks, in elucidating the underlying biological processes associated with a complex disorder.

Bmi1 is required for the self-renewal of stem cells in many tissues including the lung epithelial stem cells, Bronchioalveolar Stem Cells (BASCs). Imprinted genes, which exhibit expression from only the maternally- or paternally-inherited allele, are known to regulate developmental processes but their role in adult cells remains a fundamental question. Many imprinted genes were de-repressed in Bmi1 knockout mice, and knockdown of Cdkn1c (p57) and other imprinted genes partially rescued the self-renewal defect of Bmi1 mutant lung cells. Expression of p57 and other imprinted genes was required for lung cell self-renewal in culture and correlated with repair of lung epithelial cell injury in vivo. Our data suggest that Bmi1-dependent regulation of expressed alleles at imprinted loci, distinct from imprinting per se, is required for control of lung stem cells. We anticipate that the regulation and function of imprinted genes is crucial for self-renewal in diverse adult tissue-specific stem cells.

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. What’s new? Despite their initial promise, anti-angiogenic therapies have been a disappointment in the clinic. One reason is that solid tumors often become resistant to these drugs. Tumors that respond poorly to this type of therapy have increased activation of the hypoxia-induced transcription factor HIF-1α which can enhance tumor survival and progression. In this study, the authors report that this evasive resistance can be overcome by adding low-dose doxorubicin or shRNA to inhibit HIF-1α activity. They are thus developing a clinical trial combining the angiogenesis inhibitor bevacizumab with metronomic doxorubicin in sarcoma patients.

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.