FOXA1 Binding Sites Research Articles

FOXA1 regulates alternative splicing in prostate cancer.

SummaryDysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an “exon definition” mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer.

A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.

Down-regulation of HaABCC3, potentially mediated by a cis-regulatory mechanism, is involved in resistance to Cry1Ac in the cotton bollworm, Helicoverpa armigera.

Evolution of resistance to Cry proteins in multiple pest insects has been threatening the sustainable use of Bacillus thuringiensis (Bt)-transgenic crops. Better understanding about the mechanism of resistance to Cry proteins in insects is needed. Our preliminary study reported that the transcription of HaABCC3 was significantly decreased in a near-isogenic line (LFC2) of a Cry1Ac-resistant strain (LF60) of the global pest Helicoverpa armigera. However, the causality between HaABCC3 downregulation and resistance to Cry1Ac remains to be verified, and the regulatory mechanism underlying the HaABCC3 downregulation is still unclear. In this study, our data showed that both HaABCC3 and HaABCC3 downregulation were genetically linked to resistance to Cry1Ac in LF60. However, no InDels were observed in the coding sequence of HaABCC3 from LF60. Furthermore, F1 offspring from the cross of LF60 and a HaABCC2/3-knockout mutant exhibited moderate resistance to Cry1Ac toxin; this indicated that the high resistance to Cry1Ac toxin in LF60 may have resulted from multiple genetic factors, including HaABCC2 mis-splicing and HaABCC3 downregulation. Results from luciferase reporter assays showed that promoter activity of HaABCC3 in LF60 was significantly lower than that in the susceptible strain, which indicated that HaABCC3 downregulation was likely mediated by promoter variation. Consistently, multiple variations of the GATA- or FoxA-binding sites in the promoter region of HaABCC3 were identified. Collectively, all results in this study suggested that the downregulation of HaABCC3 observed in the H.armigera LF60 strain, which is resistant to Cry1Ac, may be mediated by a cis-regulatory mechanism.

Abstract 1458: Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues

Abstract Single cell mRNA atlases of multiple organs have been reported using scRNA-seq. We had previously reported single cell mRNA atlas of the breast. In this study, we performed scATAC-seq and scRNA-seq of breast tissues from 22 women of Ashkenazi (A) ancestry (19,935 nuclei), 20 women of European (non-A) ancestry (19,090 nuclei), six BRCA1 (6,261 nuclei) and five BRCA2 (3,400 nuclei) mutation carriers to correlate cell type-specific gene expression with chromatin accessibility. An integrated analysis revealed 17 cell clusters including basal, luminal progenitor, and mature luminal epithelial cells, two endothelial subtypes and two fibroblast/mesenchymal stem-like cell types. Basal cells could be further subdivided into two subclusters that differentially expressed genes such as TSHZ2, POSTN and ETV6. Similarly, there were four luminal progenitor subclusters that differentially expressed genes such as MALAT1, SOX13, and MDM4. Although ESR1 expression was observed predominantly in mature luminal cells, binding sites for ESR1 were accessible in multiple epithelial and non-epithelial cells with the exception of basal cells. FOXA1 expression and binding site enrichment were observed mostly in mature luminal cells. GATA3 expression was observed in all epithelial cell types with highest expression in mature luminal cells but binding sites for GATA3 were accessible in multiple cell types. Expression and binding site enrichment of alveolar differentiation-associated EHF were predominant in luminal progenitor cells. TP63 expression and binding site enrichment were restricted to basal cells, which is consistent with the proposed role of TP63 in maintaining stem cells among the basal cell populations. EMT-associated ZEB1 showed an intriguing pattern. Similar to our published immunohistochemistry-based analysis of breast tissues, ZEB1 expression was detected in fibroblasts/mesenchymal cells, endothelial cells and adipocytes but not in epithelial cells. However, binding sites for this transcription factor were enriched in epithelial cell types and in T-cells. ZEB1 mRNA stability is tightly controlled by the epithelial cell-enriched miR-200 family of microRNAs, and it is possible that ZEB1 transcribed in epithelial cells is immediately targeted for degradation through miR-200. To gain insight into this possibility, we examined the chromatin accessibility status of ZEB1 regulatory regions in fibroblasts/mesenchymal cells and in epithelial cells. ZEB1 regulatory regions displayed open chromatin status in both epithelial cells and fibroblasts. With the exception of a few genes such as SIGLEC1 in macrophages and IL7R in T cells, cell type-specific expression of many genes is not controlled at the level of chromatin accessibility of their regulatory regions. Collectively, these results reveal complexities in gene expression in different cell types of the breast. Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1458.

Abstract PD1-05: Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer

Abstract Background: Aberrant activation of the pioneer transcription factor (TF) FOXA1 contributes to endocrine resistance and metastasis in ER+ breast cancer (BC) by promoting genome-wide enhancer and transcriptional reprogramming that engages the AP-1 TF complex. Identification of central transcriptional nexuses in this deregulated network is key for developing new therapeutic interventions focusing on transcriptional programs. We previously identified FRA1, among the AP-1 components, as the top super-enhancer target forming a FOXA1/FRA1-centered transcriptional axis to activate genes enriched in luminal B-subtype BC and ER+ metastases. To further dissect the FOXA1/FRA1-centered transcriptional axis and its potential therapeutic role, we employed integrative multi-omics data analysis and functional studies targeting FRA1 using our MCF7-parental (P) and FOXA1-amplifed tamoxifen-resistant (TamR) preclinical models. Methods: RNA-seq data were obtained in MCF7-P and TamR cells with FOXA1 and/or AP-1 (FRA1 and c-Jun) perturbation via overexpression and/or si/shRNA knockdown (KD). Differential gene expression was analyzed using DESeq2 or the limma-voom R package. ChIP-seq-based genome-wide FOXA1 binding sites were further refined by intersection with promoter-tethered regions (PTRs) denoting Hi-C-mapped chromatin looping. Genomic binding of FOXA1/c-Jun and H3K27ac modification at target gene loci were aligned and visualized by IGV. Significance of gene set enrichment was determined using a chi-square test adjusted for multiple comparisons. Clinical relevance was examined using an RNA-seq dataset of ER+ metastatic BC (SABCS19-GS2-02). Clonogenicity, soft agar, and wound-healing assays were performed using MCF7-TamR cell derivatives engineered for doxycycline (Dox)-inducible KD of FRA1 or NS shRNA. Significance of the KD effects in functional assays was determined using a linear mixed-effects model. Results: We found that FRA1 and the two embryonic TFs SOX9 and KLF4 that harbor FOXA1-bound PTRs were commonly down-regulated in TamR cells upon KD of FOXA1, FRA1, or c-Jun. We observed increased c-Jun binding at the FOXA1-bound super-enhancer and the PTR looping to the FRA1 gene locus in TamR vs. P cells, suggesting a feed-forward mechanism by which FRA1 transcription is strengthened by the AP-1-engaged super-enhancer in TamR cells. Among the FOXA1-activated genes in TamR cells, a subset of secretory protein-encoding genes was more enriched in the genes commonly dependent on both FRA1 and c-Jun, vs. the genes depending on either FRA1 or c-Jun alone. This FOXA1/FRA1/c-Jun-activated secretome is enriched for multiple biological processes engaged in tumor metastasis and associated microenvironmental niches, and was upregulated in clinical ER+ metastases vs. primary tumors. A larger proportion of this secretome, including CXCL8 and S100P, also relies on ER preferentially in TamR vs. P cells (40% vs. 16%, respectively). FRA1 KD using two different shRNA sequences in TamR cells reduced SOX9 and KLF4 expression levels, and significantly diminished clonogenicity, soft agar colony formation, and wound healing, compared to the control NS KD. Conclusions: Our integrative bioinformatics analyses reveal a feed-forward mechanism on FRA1 activation in amplifying high-FOXA1-induced transcriptional reprogramming in endocrine resistance. A prometastatic secretome activated by FRA1/c-Jun may represent a main transcriptional output of the FOXA1/FRA1-dependent nexus in promoting ER+ disease progression. These identified key transcriptional nexuses may present network hotspots susceptible to therapeutic interventions in which FRA1 inhibition could be used as a new transcriptional program-oriented therapy to treat advanced ER+ BC. Citation Format: Chia Chia Liu, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Resel Pereira, Martin J. Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Zhijie Liu, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Xiaoyong Fu. Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-05.

Subtype-Independent ANP32E Reduction During Breast Cancer Progression in Accordance with Chromatin Relaxation

BackgroundChromatin state provides a clear decipherable blueprint for maintenance of transcriptional patterns, exemplifying a mitotically stable form of cellular programming in dividing cells. In this regard, genomic studies of chromatin states within cancerous tissues have the potential to uncover novel aspects of tumor biology and unique mechanisms associated with disease phenotypes and outcomes. The degree to which chromatin state differences occur in accordance with breast cancer features has not been established.MethodsWe applied a series of unsupervised computational methods to identify chromatin and molecular differences associated with discrete physiologies across human breast cancer tumors.ResultsChromatin patterns alone are capable of stratifying tumors in association with cancer subtype and disease progression. Major differences occur at DNA motifs for the transcription factor FOXA1, in hormone receptor-positive tumors, and motifs for SOX9 in Basal-like tumors. We find that one potential driver of this effect, the histone chaperone ANP32E, is inversely correlated with tumor progression and relaxation of chromatin at FOXA1 binding sites. Tumors with high levels of ANP32E exhibit an immune response and proliferative gene expression signature, whereas tumors with low ANP32E levels appear programmed for differentiation.ConclusionsOur results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity. This study sets a precedent for future computational studies of chromatin changes in carcinogenesis.

FoxA-dependent demethylation of DNA initiates epigenetic memory of cellular identity.

Tissue-specific DNA methylation patterns are created by transcription factors that recruit methylation and demethylation enzymes to cis-regulatory elements. To date, it is not known whether transcription factors are needed to continuously maintain methylation profiles in development and mature tissues or whether they only establish these marks during organ development. We queried the role of the pioneer factor FoxA in generating hypomethylated DNA at liver enhancers. We discovered a set of FoxA-binding sites that undergo regional, FoxA-dependent demethylation during organ development. Conditional ablation of FoxA genes in the adult liver demonstrated that continued FoxA presence was not required to maintain the hypomethylated state, even when massive cell proliferation was induced. This study provides strong evidence for the stable, epigenetic nature of tissue-specific DNA methylation patterns directed by lineage-determining transcription factors during organ development.

Application of alternative de novo motif recognition models for analysis of structural heterogeneity of transcription factor binding sites: a case study of FOXA2 binding sites.

The most popular model for the search of ChIP-seq data for transcription factor binding sites (TFBS) is the positional weight matrix (PWM). However, this model does not take into account dependencies between nucleotide occurrences in different site positions. Currently, two recently proposed models, BaMM and InMoDe, can do as much. However, application of these models was usually limited only to comparing their recognition accuracies with that of PWMs, while none of the analyses of the co-prediction and relative positioning of hits of different models in peaks has yet been performed. To close this gap, we propose the pipeline called MultiDeNA. This pipeline includes stages of model training, assessing their recognition accuracy, scanning ChIP-seq peaks and their classification based on scan results. We applied our pipeline to 22 ChIP-seq datasets of TF FOXA2 and considered PWM, dinucleotide PWM (diPWM), BaMM and InMoDe models. The combination of these four models allowed a significant increase in the fraction of recognized peaks compared to that for the sole PWM model: the increase was 26.3 %. The BaMM model provided the main contribution to the recognition of sites. Although the major fraction of predicted peaks contained TFBS of different models with coincided positions, the medians of the fraction of peaks containing the predictions of sole models were 1.08, 0.49, 4.15 and 1.73 % for PWM, diPWM, BaMM and InMoDe, respectively. Thus, FOXA2 BSs were not fully described by only a sole model, which indicates theirs heterogeneity. We assume that the BaMM model is the most successful in describing the structure of the FOXA2 BS in ChIP-seq datasets under study.

Epigenetic Memory: Lessons From iPS Cells Derived From Human β Cells.

Incomplete reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) may be responsible for the heterogeneity in differentiation capacity observed among iPSC lines. It remains unclear whether it results from stochastic reprogramming events, or reflects consistent genetic or cell-of-origin differences. Some evidence suggests that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type. We investigated iPSCs reprogrammed from human pancreatic islet β cells (BiPSCs), as a step in development of a robust differentiation protocol for generation of β-like cells. BiPSCs derived from multiple human donors manifested enhanced and reproducible spontaneous and induced differentiation towards insulin-producing cells, compared with iPSCs derived from isogenic non-β-cell types and fibroblast-derived iPSCs (FiPSCs). Genome-wide analyses of open chromatin in BiPSCs and FiPSCs identified thousands of differential open chromatin sites (DOCs) between the two iPSC types. DOCs more open in BiPSCs (Bi-DOCs) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites. Bi-DOCs were associated with genes related to pancreas development and β-cell function. These studies provide evidence for reproducible epigenetic memory in BiPSCs. Bi-DOCs may provide clues to genes and pathways involved in the differentiation process, which could be manipulated to increase the efficiency and reproducibility of differentiation of pluripotent stem cells from non-β-cell sources.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

Abstract DL-1: Essential Genes and Cistromes in Breast Cancer

Abstract Steroid hormones mediate critical lineage-specific developmental and physiologic responses. While estrogen is required for normal breast development, the genes regulated by estrogen and the genomic targets of the estrogen receptor (ER) are altered in ER+ breast cancers. The requirement of most breast cancers for estrogen has led to the development of endocrine therapies that block ER action. While initial endocrine interventions are successful, in the advanced disease setting resistance to ER-targeted therapy almost invariably arises. As with other targeted therapies, gain of function mutations play a significant role in the development of therapeutic resistance to ER directed treatments in breast cancer. In addition, pathways downstream of ER are activated in cases in which ER itself is not genetically altered suggesting that these pathways remain essential. Activation of these pathways may depend on mutations in other transcription factors involved in steroid receptor networks or chromatin modifying enzymes involved in transcriptional regulation mediated by ER. These mutations may be present in the protein coding regions of the genes or in the cis-regulatory elements regulating expression of key genes in the pathways. Renewed success in targeting ER and promising advances in inhibiting the activity of the chromatin modifying enzymes provides new opportunities for the treatment of patients with breast cancer. Using genome-wide CRISPR-Cas9 knockout screens we have identified the genes essential for hormone-dependent breast cancer growth including key genes in the receptor-centered transcriptional networks. Although large sets transcription factor binding sites or cistromes have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR-Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of more than 10,000 FOXA1 and CTCF binding sites in breast cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed two classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at TAD boundaries and display distinct characteristics. Using regression methods trained on the screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts non-coding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given transcription factor and can inform the development of predictive models of cistrome function. In addition, defining the essential cistrome may shed light on the noncoding regions of the genome most likely to harbor oncogenic driver mutations. Citation Format: M Brown. Essential Genes and Cistromes in Breast Cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr DL-1.

Deciphering essential cistromes using genome-wide CRISPR screens

Although millions of transcription factor binding sites, or cistromes, have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR/Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of over 10,000 FOXA1 and CTCF binding sites in breast and prostate cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed 2 classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at topologically associated domain (TAD) boundaries and display distinct characteristics. Using regression methods trained on our screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts noncoding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given factor and can inform the development of predictive models of cistrome function.

Comprehensive Genomic Analysis Reveals that the Pioneering Function of FOXA1 Is Independent of Hormonal Signaling

SummaryConsiderable work has linked hormone receptors, such as estrogen receptor-alpha (ER), with the pioneer factor FOXA1. Altered FOXA1 levels contribute to endocrine-resistant breast cancer, where it maintains ER-chromatin interactions, even in contexts in which cells are refractory to ER-targeted drugs. A recent study controversially suggests that FOXA1 binding can be induced by hormonal pathways, including the estrogen-ER complex. We now show that the vast majority (>99%) of FOXA1 binding events are unaffected by steroid activation. A small number (<1%) of FOXA1 binding sites appear to be induced by estrogen, but these are created from chromatin interactions between ER binding sites and adjacent FOXA1 binding sites and do not represent genuine new FOXA1-pioneering elements. FOXA1 is therefore not regulated by estrogen and remains a bone fide pioneer factor that is entirely upstream of the ER complex.

Age-specific changes in genome-wide methylation enrich for Foxa2 and estrogen receptor alpha binding sites.

The role of DNA methylation patterns in complex phenotypes remains unclear. To explore this question, we adapted our methods for rare variant analysis to characterize genome-wide murine DNA hybridization array to investigate methylation at CpG islands, shores, and regulatory elements. We have applied this platform to compare age and tissue- specific methylation differences in the brain and spleen of young and aged mice. As expected from prior studies, there are clear global differences in organ-specific, but not age-specific, methylation due mostly to changes at repetitive elements. Surprisingly, out of 200,000 loci there were only 946 differentially methylated cytosines (DMCs) between young and old samples (529 hypermethylated, 417 hypomethylated in aged mice) compared to thousands of tissue-specific DMCs. Hypermethylated loci were clustered around the promoter region of Sfi1, exon 2 of Slc11a2, Drg1, Esr1 and Foxa2 transcription factor binding sites. In particular, there were 75 hypermethylated Foxa2 binding sites across a 2.7 Mb region of chromosome 11. Hypomethylated loci were clustered around Mid1, Isoc2b and genome-wide loci with binding sites for Foxa2 and Esr1, which are known to play important roles in development and aging. These data suggest discreet tissue-independent methylation changes associated with aging processes such as cell division (Sfi1, Mid1), energy production (Drg1, Isoc2b) and cell death (Foxa2, Esr1).

Integrative functional genomics identifies regulatory genetic variant modulating RAB31 expression and altering susceptibility to breast cancer.

Despite the successes of genome-wide association study (GWAS) in identifying breast cancer (BC) risk-associated variants, only a small fraction of the heritability can be explained. The greatest challenge in the post-GWAS is to identify causal variants and underlying mechanisms responsible for BC susceptibility. In this study, we integrated functional genomic data from ENCODE ChIP-seq, ANNOVAR, and the TRANSFAC matrix to identify potentially regulatory variants with modulating FOXA1-binding affinity across the whole genome, and then conducted a two-stage case-control study including 2164 cases and 2382 controls to investigate the associations between candidate SNPs and BC susceptibility. We identified a BC susceptibility SNP, rs6506689 G>T, with an odds ratio (OR) of 1.23 (95% confidence interval = 1.07-1.40, P = 0.003) under a dominant model in the combined study. Biological assays indicated that the germline G>T variation at rs6506689 creates a FOXA1-binding site and up-regulates the expression of RAB31, thus playing an important role in the development of BC. Our results highlight the importance of regulatory genetic variants in the development of BC by influencing TF-DNA interaction and provide critical insights to pinpoint causal genetic variants.

BRG1 governs glucocorticoid receptor interactions with chromatin and pioneer factors across the genome.

The Glucocorticoid Receptor (GR) alters transcriptional activity in response to hormones by interacting with chromatin at GR binding sites (GBSs) throughout the genome. Our work in human breast cancer cells identifies three classes of GBSs with distinct epigenetic characteristics and reveals that BRG1 interacts with GBSs prior to hormone exposure. The GBSs pre-occupied by BRG1 are more accessible and transcriptionally active than other GBSs. BRG1 is required for a proper and robust transcriptional hormone response and knockdown of BRG1 blocks recruitment of the pioneer factors FOXA1 and GATA3 to GBSs. Finally, GR interaction with FOXA1 and GATA3 binding sites was restricted to sites pre-bound by BRG1. These findings demonstrate that BRG1 establishes specialized chromatin environments that define multiple classes of GBS. This in turn predicts that GR and other transcriptional activators function via multiple distinct chromatin-based mechanisms to modulate the transcriptional response.

Role of Semaphorin Signaling During Cardiovascular Development.

Semaphorins are a family of secreted and membrane‐bound molecules that play critical functions in diverse biological processes.[1][1], [2][2], [3][3], [4][4], [5][5], [6][6], [7][7], [8][8], [9][9] SEMA1A/Fasciclin IV is the founding member of the semaphorin family and was first reported in 1992

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.

Genes Associated with Pancreas Development and Function Maintain Open Chromatin in iPSCs Generated from Human Pancreatic Beta Cells.

SummaryCurrent in vitro islet differentiation protocols suffer from heterogeneity and low efficiency. Induced pluripotent stem cells (iPSCs) derived from pancreatic beta cells (BiPSCs) preferentially differentiate toward endocrine pancreas-like cells versus those from fibroblasts (FiPSCs). We interrogated genome-wide open chromatin in BiPSCs and FiPSCs via ATAC-seq and identified ∼8.3k significant, differential open chromatin sites (DOCS) between the two iPSC subtypes (false discovery rate [FDR] < 0.05). DOCS where chromatin was more accessible in BiPSCs (Bi-DOCS) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites (FDR < 0.05). Bi-DOCS were associated with genes related to pancreas development and beta-cell function, including transcription factors mutated in monogenic diabetes (PDX1, NKX2-2, HNF1A; FDR < 0.05). Moreover, Bi-DOCS correlated with enhanced gene expression in BiPSC-derived definitive endoderm and pancreatic progenitor cells. Bi-DOCS therefore highlight genes and pathways governing islet-lineage commitment, which can be exploited for differentiation protocol optimization, diabetes disease modeling, and therapeutic purposes.

Abstract 2847: Loss of heterozygosity (LOH) within the hypoxia inducible factor 3A (HIF3A) locus in liver metastasis (LM) from primary colorectal cancer (CRC)

Abstract Background and Aims: HIF3α is a hypoxia-regulated transcription factor that represses the effects of HIF1α and HIF2α under hypoxia at transcriptional and posttranslational levels. We previously found that LOH at the HIF3A locus is frequent in LM (59%) and the expression of HIF3A protein is reduced in LM compared to primary CRC. At present, the smallest overlapping LOH (SOL-LOH) region has been mapped to the intron 8 of the HIF3A, spanning from 46,312,911 to 46,315,651 on chromosome 19 in LMs. This genomic region contains multiple FOXA1 binding sites embedded in the two DNase I hypersensitive sites (UCSC Genome Browser, https://genome.UCSC.edu). About 30% of the human population carries a deletion within our SOL-LOH region (1000 Genomes Consortium: esv3644527, Chr19: 46,314,109-46,315,478). In this study, we determined whether esv3644527 deletion affects on the expressions of HIF3A, HIF2A, HIF1A and their target genes under normoxic and hypoxic conditions. Methods: For LOH mapping, 75 resected LMs derived from primary CRC were analyzed for LOH using 8 polymorphic markers. We obtained three EBV-transformed B cell lines with heterozygous esv3644527 deletion and the one with the homozygous deletion from the Coriell Institute. The expression levels of 23 genes related to HIF pathways were determined by qRT-PCR. Results and Discussion: The expression levels of HIF3A mRNA were too low to compare among these B cell lines by qRT-PCR. However, the expression levels of HIF2A and its target gene, angiopoietin 2 (ANGPT2), are significantly elevated in B cells with a homozygous deletion compared to B cells with a heterozygous deletion under both normoxic and hypoxic conditions. These observations are compatible with the results obtained from a previous study where pulmonary endothelial cells from HIF3A-knockout mice expressed elevated levels of HIF2A and ANGPT2 (Kobayashi-S et al., Genes Cells, 2015, 20:224-241), suggesting that deletion of the FOXA1 sites present in this region may regulate the HIF3A expression. Conclusions: Our results show that about 60% of LMs exhibit LOH in the 2.7 Kb region surrounding intron 8 of the HIF3A locus. Deletion within this region may lead to upregulation of HIF2A and ANGPT2 by negatively regulating HIF3A expression. To prove our hypothesis, colon cancer cell lines with deleted FOXA1 sites will be assessed. Citation Format: Takahito Kitajima, Minoru Koi, Alexander Worix, John M. Carethers. Loss of heterozygosity (LOH) within the hypoxia inducible factor 3A (HIF3A) locus in liver metastasis (LM) from primary colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2017-2847