Biology subjects

Gertz, J.

Publications and source records attributed to Gertz, J..

28 records · Page 2Linked to original sources

bioRxiv2020

Regulatory Sharing Between Estrogen Receptor α Bound Enhancers

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers, which are approximately 5,000 basepairs apart, exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of the observed enhancer cooperativity. We generated genetic deletions of individual estrogen receptor (ER) bound enhancers and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that a cluster of two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences between enhancers, we found that the genomic location in which a full ERE resides strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing active chromatin environment within an enhancer cluster is also needed in order for estrogen-driven gene regulation to occur.

genetics↗

bioRxiv2019

Reciprocal regulation of pancreatic ductal adenocarcinoma growth and molecular subtype by HNF4α and SIX1/4

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a five-year survival of less than 5%. Transcriptomic analysis has identified two clinically relevant molecular subtypes of PDAC: Classical and Basal-like. The Classical subtype is characterized by a more favorable prognosis and better response to chemotherapy than the Basal-like subtype. The Classical subtype also expresses higher levels of lineage specifiers that regulate endodermal differentiation, including the nuclear receptor HNF4. Using in vitro and in vivo PDAC models, we show that HNF4 restrains tumor growth and drives tumor cells toward an epithelial identity. Gene expression analysis from murine models and human tumors shows that HNF4 activates expression of genes associated with the Classical subtype. Although HNF4 loss is not sufficient for complete conversion to the Basal-like subtype gene expression profile, HNF4 directly represses SIX4 and SIX1, mesodermal lineage specifiers expressed in the Basal-like subtype. Finally, HNF4-negative PDAC cells rely on expression of SIX4 and SIX1 for proliferation in vitro and in vivo. Overall, our data show that HNF4 regulates the growth and molecular subtype of PDAC by multiple mechanisms, including activation of the Classical gene expression program and repression of SIX4 and SIX1, which may represent novel dependencies of the Basal-like subtype.

cancer biology↗

bioRxiv2019

XPRESSyourself: Enhancing and Automating the Ribosome Profiling and RNA-Seq Analysis Toolkit

Ribosome profiling, an application of nucleic acid sequencing for monitoring ribosome activity, has revolutionized our understanding of protein translation dynamics. This technique has been available for a decade, yet the current state and standardization of publicly available computational tools for these data is bleak. We introduce XPRESSyourself, an analytical toolkit that eliminates barriers and bottlenecks associated with this specialized data type by filling gaps in the computational toolset for both experts and non-experts of ribosome profiling. XPRESSyourself automates and standardizes analysis procedures, decreasing time-to-discovery and increasing reproducibility. This toolkit acts as a reference implementation of current best practices in ribosome profiling analysis. We demonstrate this toolkits performance on publicly available ribosome profiling data by rapidly identifying hypothetical mechanisms related to neurodegenerative phenotypes and neuroprotective mechanisms of the small-molecule ISRIB during acute cellular stress. XPRESSyourself brings robust, rapid analysis of ribosome-profiling data to a broad and ever-expanding audience and will lead to more reproducible and accessible measurements of translation regulation. XPRESSyourself software is perpetually open-source under the GPL-3.0 license and is hosted at https://github.com/XPRESSyourself, where users can access additional documentation and report software issues.

bioinformatics↗

bioRxiv2019

ETV4 is necessary for estrogen signaling and growth in endometrial cancer cells

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ERs regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed and in vivo in the context of estrogen dependent growth. Our results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.

molecular biology↗

bioRxiv2018

Sufficiency analysis of estrogen responsive enhancers using synthetic activators

Multiple regulatory regions bound by the same transcription factor have been shown to simultaneously control a single genes expression. However, it remains unclear how these regulatory regions combine to regulate transcription. Here we test the sufficiency of promoter-distal estrogen receptor (ER)-binding sites (ERBS) for activating gene expression by recruiting synthetic activators in the absence of estrogens. Targeting either dCas9-VP16(10x) or dCas9-p300(core) to ERBS induces H3K27ac and activates nearby expression in a manner similar to an estrogen induction, with dCas9-VP16(10x) acting as a stronger activator. The sufficiency of individual ERBS is highly correlated with their necessity, indicating an inherent activation potential that is associated with the binding of RNA polymerase II and several transcription factors. By targeting ERBS combinations, we found that ERBS work independently to control gene expression when bound by synthetic activators. The sufficiency results contrast necessity assays that show synergy between these ERBS, suggesting that synergy occurs between ERBS in terms of activator recruitment, whereas directly recruiting activators leads to independent effects on gene expression.

systems biology↗

bioRxiv2018

Estrogen-independent molecular actions of mutant estrogen receptor alpha in endometrial cancer

Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggests that mutant ESR1 exhibits estrogen independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wildtype ESR1. The D538G mutation impacted expression, including a large set of non-estrogen regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is unique from constitutive ESR1 activity as mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.

cancer biology↗

bioRxiv2017

Clinical and genomic crosstalk between glucocorticoid receptor and estrogen receptor α in endometrial cancer

Steroid hormone receptors are simultaneously active in many tissues and are capable of altering each others function. Estrogen receptor (ER) and glucocorticoid receptor (GR) are expressed in the uterus and their ligands have opposing effects on uterine growth. In endometrial tumors with high ER expression, we surprisingly found that expression of GR is associated with poor prognosis. Dexamethasone reduced normal uterine growth in vivo; however, this growth inhibition was abolished in estrogen-induced endometrial hyperplasia. We observed low genomic binding site overlap when ER and GR are induced with their respective ligands; however, upon simultaneous induction they co-occupy more sites. GR binding is significantly altered by estradiol with GR recruited to ER bound loci that become more accessible upon estradiol induction. Gene expression responses to co-treatment were more similar to estradiol, but with novel regulated genes. Our results suggest phenotypic and molecular interplay between ER and GR in endometrial cancer.

cancer biology↗

bioRxiv2017

Differential Brd4-bound enhancers drive critical sex differences in glioblastoma

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma, we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited opposing responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis, while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET inhibitors than are female cells. Thus, for the first time, Brd4 activity is revealed to drive a sex differences in stem cell and tumorigenic phenotype, resulting in diametrically opposite responses to BET inhibition in male and female GBM cells. This has important implications for the clinical evaluation and use of BET inhibitors. SignificanceConsistent sex differences in incidence and outcome have been reported in numerous cancers including brain tumors. GBM, the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity, including that of GBM. We report that sex-biased Brd4 activity drive sex differences in GBM and render male and female tumor cells differentially sensitive to BET inhibitors. The observed sex differences in BETi treatment strongly indicate that sex differences in disease biology translate into sex differences in therapeutic responses. This has critical implications for clinical use of BET inhibitors further affirming the importance of inclusion of sex as a biological variable.

genomics↗

bioRxiv2017

GIGGLE: a search engine for large-scale integrated genome analysis

GIGGLE is a genomics search engine that identifies and ranks the significance of shared genomic loci between query features and thousands of genome interval files. GIGGLE scales to billions of intervals, is faster (+1,000X) than existing methods, and its speed extends the accessibility and utility of resources such as ENCODE, Roadmap Epigenomics, and GTEX by facilitating data integration and hypothesis generation. GIGGLE is available at https://github.com/ryanlayer/giggle.

bioinformatics↗

bioRxiv2016

Resolving the Functional Significance of BRCA1 RING Domain Missense Substitutions

Part 1Development and calibration of suitably accurate functional assays for BRCA1 RING domain and BRCT domain missense substitutions could dramatically accelerate clinical classification of rare missense substitutions observed in that gene. Leveraging data from 68,000 full sequence tests of BRCA1 and BRCA2, plus data from the limited number of already classified BRCA1 RING domain missense substitutions, we used logistic regression and related techniques to evaluate three BRCA1 RING domain assays. These were recently described high throughput yeast 2-hybrid and E3 ubiquitin ligase assays, plus a newly developed mammalian 2-hybrid assay. While there were concerns about the accuracy of the yeast 2-hybrid assay and the indirect nature of the ubiquitin ligase assay, the mammalian 2-hybrid assay had excellent correlation with existing missense substitution classifications. After calibration, this assay contributed to classification of one newly reported BRCA1 missense substitution. In principal, the mammalian 2-hybrid assay could be converted to a high-throughput format that would likely retain suitable accuracy. Part 2How does one achieve clinically applicable classification of the vast majority of all possible sequence variants in disease susceptibility genes? BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a Mammalian 2-hybrid (M2H) assay. Downstream of the M2H laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that about 20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of about 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 47 with concordant computational and functional assay evidence in favor of pathogenicity; these are particularly likely to be classified as Likely Pathogenic once human observational data become available.

genetics↗