Chromatin Sites Research Articles

A Mediator Switch Tunes Transcription Pausing to Drive Erythropoiesis

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development. To address whether composition of the Mediator complex undergoes dynamic changes during erythropoiesis, we detected its expression in primary human erythroblasts derived from CD34 + hematopoietic stem and progenitor cells. We found that while the protein levels of most Mediator subunits decreased substantially in the terminal erythroid differentiation, MED26 remained relatively abundant. A gain-of-function assay in the primary human erythroid culture system showed that MED26 overexpression promoted erythroid differentiation. Furthermore, Med26 conditional knockout (cKO) mice exhibited severe erythroid and hematological defects. Erythroid precursors (especially the Cd71+Ter119+ stage) were absent in the bone marrow of Med26-cKO mice, which phenocopies the cKO mice of Gata1. Prussian blue staining of the spleen section showed more iron deposition in the cKO mice. Moreover, the cKO mice were unable to recover after phenylhydrazine-induced hemolytic anemia. To dissect the molecular basis underlying MED26 function, we analyzed the chromatin occupancy signals of MED26 by CUT&Tag assay and compared them to those of MED1, which is often regarded as a representative subunit of Mediator, in human CD34 + erythroid culture cells on Day 4. Intriguingly, we found that ~60% of chromatin sites with MED1 or MED26 occupancy did not colocalize, suggesting these subunits exert context-dependent gene regulation. Notably, MED26 colocalizes with GATA1 and GATA2 better than MED1. PRO-seq analysis further revealed that MED26-enriched loci were associated with RNA polymerase II (Pol II) pausing. To examine how MED26 might mediate transcription pausing, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify interacting proteins of MED1 and MED26. While MED26 and MED1 similarly coimmunoprecipitated with the majority of other Mediator subunits and the elongation complex, MED26 exhibited a substantially higher interaction with transcription pausing related factors (NELF, DSIF, and PAF complexes) compared to MED1. Knocking out MED26 significantly impairs the recruitment of PAF1 to MED26 occupancy sites, indicating the presence of MED26 facilitates the recruitment of PAF1. Our results also revealed that MED26 enrichment is associated with increased transcription pausing during terminal erythroid stages, which can promote erythroid development. Moreover, we uncovered that MED26 exhibited pronounced biomolecular condensate-forming capability. The intrinsically disordered region (IDR) of MED26 was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors to the MED26-enriched condensates. Taken together, our findings demonstrate that the relative enrichment of MED26 in terminal erythropoiesis leads to a shift of transcription condensates towards a MED26-enriched form. This shift, in turn, facilitates the recruitment of pausing related factors, thereby achieving genome-wide transcription repression during erythropoiesis. This study not only illustrates the indispensable function of MED26 in erythropoiesis but also elucidates the functional coordination of MED1 and MED26 during different erythroid stages. The dynamic switch of Mediator stoichiometry might serve as an important example for understanding the mechanism of the versatile Mediator function during development.

Chromatin accessibility and pioneer factor FOXA1 restrict glucocorticoid receptor action in prostate cancer.

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.

Epigenomic mapping reveals distinct B cell acute lymphoblastic leukemia chromatin architectures and regulators

B cell lineage acute lymphoblastic leukemia (B-ALL) is composed of diverse molecular subtypes, and while transcriptional and DNA methylation profiling has been extensively examined, the chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq in primary B-ALL cells from 156 patients spanning ten molecular subtypes and present this dataset as a resource. Differential chromatin accessibility and transcription factor (TF) footprint profiling were employed and identified B-ALL cell of origin, TF-target gene interactions enriched in B-ALL, and key TFs associated with accessible chromatin sites preferentially active in B-ALL. We further identified over 20% of accessible chromatin sites exhibiting strong subtype enrichment and candidate TFs that maintain subtype-specific chromatin architectures. Over 9,000 genetic variants were uncovered, contributing to variability in chromatin accessibility among patient samples. Our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants that promote unique gene-regulatory networks.

The N-Terminal Part of Drosophila CP190 Is a Platform for Interaction with Multiple Architectural Proteins.

CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.

STAG2 loss Induces HSC Programs By Modulating Accessibility of AP-1 Bound Enhancers

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood disorders characterized by clonal expansion of mutant hematopoietic stem and progenitor cells (HSPCs). Mutations in genes encoding the cohesin complex, including STAG2, RAD21, SMC3 and SMC1A, are frequent genetic drivers in MDS and AML. However, the mechanisms by which cohesin mutations lead to clonal expansion of HSPCs are not well understood. Cohesin is essential for sister chromatid cohesion, DNA damage repair, maintenance of chromatin architecture, and gene regulation. To investigate the impact of cohesin mutations on chromatin accessibility, we performed ATAC-Seq in a panel of six isogenic STAG2 wild type (WT) and knockout (KO) U937 leukemia cell lines. Among the total of 97,798 ATAC-Seq peaks, we identified significantly more STAG2 KO (8,859) versus WT (1,622) uniquely accessible chromatin sites, which were strongly enriched for intergenic regions. Integration of ChIP-Seq profiling of cohesin binding and histone modifications demonstrated cohesin binding and an increase in the active enhancer and promoter histone mark H3K27Ac in the STAG2 KO newly accessible chromatin, suggesting a direct role for cohesin in regulating the activity of such enhancers. Together, our data demonstrate that STAG2 KO-induced open chromatin may be enriched for novel STAG2 KO-specific enhancers. To further infer the regulatory function of these putative enhancers, we predicted binding of transcription factors (TFs) in ATAC-Seq-defined regions by performing TF footprinting using the Transcription factor Occupancy Prediction By Investigation of ATAC-seq Signal (TOBIAS) algorithm. We observed that binding of members of the AP-1 transcription family, including JUN FOS andATF, previously implicated in oncogenic transformation, was significantly enriched in the STAG2 KO cells (Figure 1A). Conversely, STAG2 KO cells lost CEBP TF binding in regions that lost chromatin accessibility. To identify potential regulatory targets of these TFs, we mapped TF footprints to annotated promoter regions and to enhancer-gene pairs determined by the Activity by Contact (ABC) model. We observed a strong correlation between predicted AP-1 binding and gene expression of putative regulated genes in STAG2 KO cells, further supporting the role of these newly accessible AP-1 bound sites as regulatory elements. To validate our findings using in vivo models of cohesin-mutant disease, we performed RNA-Seq and ATAC-Seq in HSPC isolated from a Tet2/Stag2 mouse model of MDS previously developed in our lab. We confirmed an increase in accessible chromatin and demonstrated a strong enrichment of predicted Ap-1 binding in intergenic regions, in agreement with our observations in the U937 model. Gene set enrichment analysis in the Tet2/Stag2-mutant versus WT HSPC revealed that putative targets of Ap-1 binding were enriched for hematopoietic stem cell gene signatures (Figure 1B) . This is consistent with our observation of clonal expansion of HSPCs in this model, and suggests a mechanism by which novel Ap-1 bound enhancers may facilitate expression of key genes involved in HSPC expansion and development of MDS/AML. To explore whether our findings were applicable to other published models of Stag2 mutant myeloid neoplasms, we performed TF footprinting using publicly available ATAC-Seq datasets generated in HSPC isolated from Stag2 and Runx1/Stag2 conditional knockout mice. We recapitulated increased chromatin accessibility and significant enrichment of cohesin-bound Ap-1 footprints in Stag2 mutant over WT conditions. Together, these data further validate conservation of STAG2 KO-induced AP-1 bound regulatory elements in human and mouse models of MDS and AML. In summary, we show that STAG2 loss leads to the establishment of a novel set of accessible intergenic regions with features of enhancer activity in vitro and in vivo. We identify multiple members of the AP-1 complex with enriched predicted binding at these putative enhancers, and demonstrate that AP-1 may drive expression of HSPC signatures in models of Stag2 mutant MDS in vivo. We illustrate here a potential role for the AP-1 complex in cohesin mutant myeloid malignancies, which we are currently functionally interrogating.

Molecular genetic abnormalities in ACTH-secreting pituitary tumors (corticotropinomas): fundamental research and prospects for use in clinical practice

In recent years, a large number of studies have been carried out to research molecular genetic abnormalities in ACTH--secreting pituitary tumors. This review presents a comprehensive analysis of exome studies results (germline and somatic mutations, chromosomal abnormalities in corticotropinomas which developed as part of hereditary syndromesMEN 1, 2, 4, DICER1, Carney complex etc., and isolated tumors, respectively) and transcriptome (specific genes expression profiles in hormonally active and inactive corticotropinomas, regulation of cell cycles and signal pathways). Modern technologies (next-generation sequencing - NGS) allow us to study the state of the microRNAome, DNA methylome andinactive chromatin sites, in particular using RNA sequencing. Thus, a wide range of fundamental studies is shown, theresults of which allow us to identify and comprehend the key previously known and new pathogenesis mechanisms andbiomarkers of corticotropinomas. The characteristics of the most promising molecular genetic factors that can be used in clinical practice for screening and earlier diagnosis of hereditary syndromes and isolated corticotropinomas, differential diagnosis of various forms of endogenous hypercorticism, sensitivity to existing and potential therapies andpersonalized outcome determination of Cushing`s disease.

UBE2A/B is the trans-acting factor mediating mechanotransduction and contact inhibition.

Mechanotransduction and contact inhibition (CI) control gene expression to regulate proliferation, differentiation, and even tumorigenesis of cells. However, their downstream trans-acting factors (TAFs) are not well known due to a lack of a high-throughput method to quantitatively detect them. Here, we developed a method to identify TAFs on the cis-acting sequences that reside inopen chromatin or DNase I-hypersensitive sites (DHSs) and to detect nucleocytoplasmic shuttling TAFs using computational and experimental screening. The DHS-proteomics revealed over 1,000 potential mechanosensing TAFs and UBE2A/B (Ubiquitin-conjugating enzyme E2 A) was experimentally identified as a force- and CI-dependent nucleocytoplasmic shuttling TAF. We found that translocation of YAP/TAZ and UBE2A/B are distinctively regulated by inhibition of myosin contraction, actin-polymerization, and CI depending on cell types. Next generation sequence analysis revealed many downstream genes including YAP are transcriptionally regulated by ubiquitination of histone by UBE2A/B. Our results suggested a YAP-independent mechanotransduction and CI pathway mediated by UBE2A/B.

The MADF-BESS Protein CP60 Is Recruited to Insulators via CP190 and Has Redundant Functions in Drosophila.

Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. In this study, we show that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein-protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, our results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein.

OR22-04 The Oncogenic Action Of Glucocorticoid Receptor In Prostate Cancer

Abstract Disclosure: V. Paakinaho: None. Steroid receptors, such as androgen (AR), and glucocorticoid receptor (GR), are hormone-controlled transcription factors (TFs) that regulate transcription in multiple central cellular systems. The action of steroid receptors has a major role in the development and progression diseases, especially endocrine-related cancers, such as prostate cancer. Since AR is the key oncogenic TF driving the development and progression of prostate cancer, the receptor is the major precision drug target in the disease. Therefore, antiandrogen treatment, i.e., the inhibition of AR activity, is a common form of cancer therapy. Despite the initial effectiveness of the therapy, antiandrogen resistance often occurs and there is no curable treatment in many cases. In contrast to AR, GR has been thought to possess tumor suppressive properties in therapy-naïve prostate cancer. Nonetheless, GR can hijack the oncogenic role of AR in prostate cancer by replacing the antiandrogen inactivated AR thus mediating antiandrogen-resistance and becoming the driver of the disease. Yet, the genome-wide effects of GR in prostate cancer have largely remained elusive. The tumor suppressive versus the oncogenic consequences of GR activation are important to acknowledge since prostate cancer patients regularly receive glucocorticoids to reduce inflammation and alleviate therapy-related side effects. To unveil these issues, we integrated ChIP-seq, RNA-seq, ATAC-seq data and uncover several pivotal features of GR action in prostate cancer cells. We detected that the replacement of AR by GR in antiandrogen-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying the occupancy of FOXA1. Counterintuitively to the classical pioneer factor model, the silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Additionally, we discovered that the chromatin binding of GR as well as transcriptional regulation by the receptor differs between different prostate cancer subtypes. Moreover, GR activity in normal prostate cells diverges drastically from that of the cancer cells. In sum, our data highlight the impact FOXA1 harbors in GR-mediated antiandrogen-resistant prostate cancer. Furthermore, the distinct GR transcriptional activity in prostate cancer subtypes could potentially be used to determine if glucocorticoid exposure possess potential harm for the prostate cancer patient. Presentation: Saturday, June 17, 2023

Mechanism of BLIMP1/TRIM66/COX2 in human decidua participates in parturition†.

The mechanism underlying the initiation of parturition remains unclear. Cyclooxygenase 2 and prostaglandins in decidual membrane tissue play an important role in the "parturition cascade." With the advancement of gestation, the expression of the transcriptional suppressor B lymphocyte-induced maturation protein 1 in the decidual membrane gradually decreases. Through chromatin immunoprecipitation sequencing, we found that B lymphocyte-induced maturation protein 1 has a binding site in the distal intergenic of PTGS2(COX2). Tripartite motif-containing protein 66 is a chromatin-binding protein that usually performs transcriptional regulatory functions by "reading" histone modification sites in chromatin. In this study, tripartite motif-containing protein 66 exhibits the same trend of expression as B lymphocyte-induced maturation protein 1 in the decidua during gestation. Moreover, the co-immunoprecipitation assay revealed that tripartite motif-containing protein 66 combined with B lymphocyte-induced maturation protein 1. This finding indicated that tripartite motif-containing protein 66 formed a transcription complex with B lymphocyte-induced maturation protein 1, which coregulated the expression of COX2. In animal experiments, we injected si-Blimp1 adenoviruses (si-Blimp1), Blimp1 overexpression plasmid (Blimp1-OE), and Trim66 overexpression plasmid (Trim66-OE) through the tail vein of mice. The results showed that B lymphocyte-induced maturation protein 1 and tripartite motif-containing protein 66 affected the initiation of parturition in mice. Therefore, the present evidence suggests that B lymphocyte-induced maturation protein 1 and tripartite motif-containing protein 66 partially participate in the initiation of labor, which may provide a new perspective for exploring the mechanism of term labor.

Mapping disease regulatory circuits at cell-type resolution from single-cell multiomics data

Resolving chromatin-remodeling-linked gene expression changes at cell-type resolution is important for understanding disease states. Here we describe MAGICAL (Multiome Accessibility Gene Integration Calling and Looping), a hierarchical Bayesian approach that leverages paired single-cell RNA sequencing and single-cell transposase-accessible chromatin sequencing from different conditions to map disease-associated transcription factors, chromatin sites, and genes as regulatory circuits. By simultaneously modeling signal variation across cells and conditions in both omics data types, MAGICAL achieved high accuracy on circuit inference. We applied MAGICAL to study Staphylococcus aureus sepsis from peripheral blood mononuclear single-cell data that we generated from subjects with bloodstream infection and uninfected controls. MAGICAL identified sepsis-associated regulatory circuits predominantly in CD14 monocytes, known to be activated by bacterial sepsis. We addressed the challenging problem of distinguishing host regulatory circuit responses to methicillin-resistant and methicillin-susceptible S. aureus infections. Although differential expression analysis failed to show predictive value, MAGICAL identified epigenetic circuit biomarkers that distinguished methicillin-resistant from methicillin-susceptible S. aureus infections.

Hypomethylation and Overexpression of Th17-Associated Genes is a Hallmark of Intestinal CD4+ Lymphocytes in Crohn's Disease.

The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated. Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells. CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites. The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells.

Abstract A011: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A011.

Abstract 1445: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1445.

Abstract NG10: Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets

Abstract Untreated prostate cancers rely on androgen receptor (AR) signaling for growth and survival, forming the basis for the initial efficacy of androgen deprivation therapy (ADT). Yet the disease can relapse and progress to a lethal stage termed castration-resistant prostate cancer (CRPC). Reactivation of AR signaling represents the most common driver of CRPC growth, and next-generation AR signaling inhibitors (ARSIs) are now used in combination with ADT as first-line therapy. However, ARSIs can result in selective pressure, thereby generating AR-independent tumors. The transition from AR dependence frequently accompanies a change in a phenotype resembling developmental transdifferentiation or “lineage plasticity”. Neuroendocrine prostate cancer, which lacks a defined pathologic classification, is the most studied type of lineage plasticity. However, most AR-null tumors do not exhibit neuroendocrine features and are classified as “double-negative prostate cancer”, the drivers of which are poorly defined. Lineage plasticity studies in CRPC are limited by the lack of genetically defined patient-derived models that recapitulate the disease spectrum. To address this, we developed a biobank of organoids generated from patient biopsies to study the landscape of metastatic CRPC and allow for functional validation assays. Proteins called transcription factors (TFs) are drivers of tumor lineage plasticity. To identify the key TFs that drive the growth of AR-independent tumors, we integrated epigenetic and transcriptomic data generated from CRPC models. We generated ATAC-seq (assay for transposase-accessible chromatin sequencing) and RNA-seq data from 22 metastatic human prostate cancer organoids, six patient-derived xenografts (PDXs), and 12 derived or traditional cell lines. We classified the 40 models into four subtypes and predicted key TFs of each subtype. Besides the well-characterized AR-dependent (CRPC-AR) and neuroendocrine subtypes (CRPC-NE), we identified two novel AR-negative/low groups, including a Wnt-dependent subtype (CRPC-WNT), driven by TCF/LEF TFs, and a stem cell-like (SCL) subtype (CRPC-SCL), driven by the AP-1 family of TFs. To apply the subtype classification to patient samples, we derived RNA-seq signatures from the organoids and applied them to 366 patient samples from two independent CRPC cohorts. The generated signatures recapitulated the four-subtype classification and revealed that CRPC-SCL is the second most prevalent group. Patients from CRPC-SCL are also associated with shorter time under ARSI treatment compared to CRPC-AR, indicating that the ARSI treatments were less effective for CRPC-SCL patients. Additional chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicated that AP-1 (FOSL1) collaboratively binds with TEAD and transcription coactivators, YAP and TAZ. Knocking down of AP-1 (FOSL1), YAP/TAZ decreased cell growth of CRPC-SCL and showed a decrease of chromatin accessibility at CRPC-SCL-specific open chromatin sites and down-regulation of YAP/TAZ target gene expression. In addition, the expression of AP-1 (FOSL1) decreased upon YAP/TAZ knockdown suggesting a positive feedback loop as well as YAP/TAZ as actional targets in CRPC-SCL. We used two small-molecule inhibitors, verteporfin and T-5224, that act on the YAP/TAZ/AP-1 pathway for their potential use as therapeutics for CRPC-SCL tumors, both inhibited the growth of samples from CRPC-SCL but not CRPC-AR. By overexpressing an AP-1 family gene (FOSL1) in AR-high cells, we observed an increase in chromatin accessibility at CRPC-SCL-specific open chromatin sites as well as significant up-regulation of CRPC-SCL signature genes, suggesting that AP-1 functions as a pioneering factor and master regulator for CRPC-SCL. All this work was recently published in Science (Tang, Xu et al. Science, 2022) where I am the co-first author. In summary, by using a diverse biobank of organoids, PDXs, and cell lines that recapitulate the heterogeneity of metastatic prostate cancer, we created a map of the chromatin accessibility and transcriptomic landscape of CRPC. We validated the CRPC-AR and CRPC-NE subtypes and report two novel subtypes of AR-negative/low samples, CRPC-SCL and CRPC-WNT, as well as their respective key TFs. Additional analysis revealed a model in which YAP, TAZ, TEAD, and AP-1 function together and drive oncogenic growth in CRPC-SCL samples. In addition, we proposed small inhibitors of YAP and TAZ that can potentially be used to treat CRPC-SCL patients. Overall, our results show how the stratification of CRPC patients into four subtypes using their transcriptomes can potentially inform appropriate clinical decisions. Citation Format: Fanying Tang, Duo Xu, Shangqian Wang, Chen Khuan Wong, Alexander Martinez-Fundichely, Cindy J. Lee, Sandra Cohen, Jane Park, Corinne E. Hill, Kenneth Eng, Rohan Bareja, Teng Han, Eric Minwei Liu, Ann Palladino, Wei Di, Dong Gao, Wassim Abida, Shaham Beg, Loredana Puca, Maximiliano Meneses, Elisa de Stanchina, Michael F. Berger, Anuradha Gopalan, Lukas E. Dow, Juan Miguel Mosquera, Himisha Beltran, Cora N. Sternberg, Ping Chi, Howard I. Scher, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG10.

Mechanism of Action p53

P53 is a 393 residue protein in humans made up of five proposed domains, with which the central DNA binding domain with 100-300 sequences very important for the direct binding of p53 in the promoters of its target genes to specific response elements. P53 is a tumor suppressor gene with cellular stress like oxygen deficiency, oxidative stress, radiation and carcinogens substances, is stimulated has major roles in translational regulation and feedback processes. A wide variety of damage signals that relate to the stability, post-translational alteration and recruitment of p53 to binding sites in chromatin which activate the p53 pathway. As a transcriptional activation, p53 mediates transcriptional changes which facilitate cell death, senescence or reversing and protective arrest of the cell cycle. P53 is a protein under intense investigation because it is necessary to prevent tumor, in human tumors have been found to deregulation of p53 activity. On this article study focuses the mechanism of suppressive p53 effects in the response to any stress and correlation of the mutation p53 with different tumor.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

Genome-wide analysis of the chromatin sites targeted by HEX 70a storage protein in the honeybee brain and fat body.

Hexamerins, the proteins massively stored in the larval haemolymph of insects, are gradually used throughout metamorphosis as a source of raw material and energy for the development of adult tissues. Such behaviour defined hexamerins as storage proteins. Immunofluorescence experiments coupled with confocal microscopy show a hexamerin, HEX 70a, in the nucleus of the brain and fat body cells from honeybee workers, an unexpected localization for a storage protein. HEX 70a colocalizes with fibrillarin, a nucleolar-specific protein and H3 histone, thus suggesting a potential role as a chromatin-binding protein. This was investigated through chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq). The significant HEX 70a-DNA binding sites were mainly localized at the intergenic, promoter and intronic regions. HEX 70a targeted DNA stretches mapped to the genomic regions encompassing genes with relevant functional attributes. Several HEX 70a targeted genes were associated with H3K27ac or/and H3K27me3, known as active and repressive histone marks. Brain and fat body tissues shared a fraction of the HEX 70 targeted genes, and tissue-specific targets were also detected. The presence of overrepresented DNA motifs in the binding sites is consistent with specific HEX 70a-chromatin association. In addition, a search for HEX 70a targets in RNA-seq public libraries of fat bodies from nurses and foragers revealed differentially expressed targets displaying hex 70a-correlated developmental expression, thus supporting a regulatory activity for HEX 70a. Our results support the premise that HEX 70a is a moonlighting protein that binds chromatin and has roles in the brain and fat body cell nuclei, apart from its canonical role as a storage protein.

Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.