Abstract Epigenetic reprogramming requires extensive remodeling of chromatin landscapes to silence cell-type specific gene expression programs. ATP-dependent chromatin-remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of Bromodomain-containing protein 9 (BRD9) and the associated ncBAF (non-canonical BRG1-associated factors) complex in reprogramming remains unknown. Here, we show that genetic suppression of BRD9 as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase the efficiency by which induced pluripotent stem cells (iPSCs) can be generated from human somatic cells. Chemical inhibition and acute degradation of BRD9 phenocopied this effect. Interestingly, we find that BRD9 is dispensable for establishment and maintenance of human pluripotency but required for mesendodermal lineage commitment during differentiation. Mechanistically, BRD9 inhibition downregulates somatic cell type-specific genes and decreases chromatin accessibility at somatic enhancers. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.
ABSTRACT Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To discover new drug targets for more targeted and personalized therapies, functional interrogation of epigenetic modifiers is essential. We therefore generated an epigenome-wide CRISPR-Cas9 knock-out library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution, depletion of essential genes and steady behaviors of non-targeting sgRNAs. From this, we discovered novel epigenetic modifiers besides previously known ones that regulate triple-negative breast cancer and prostate cancer cell fitness. With further validation assays, we confirmed the growth-regulatory function of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in triple negative breast cancer cells. Overall, we show that EPIKOL, a focused sgRNA library targeting approximately 800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness and serve as a tool to offer novel anti-cancer targets. With its thoroughly generated epigenome-wide gene list, and the relatively high number of sgRNAs per gene, EPIKOL offers a great advantage to study functional roles of epigenetic modifiers in a wide variety of research applications, such as screens on primary cells, patient-derived xenografts as well as in vivo models.
Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.
Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.
Abstract Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.
Many key cellular events determining the thin line between healthy and oncogenic behavior rely on the proper functioning of protein-protein interactions (PPIs). Alterations that affect the affinity of a protein-protein binding site may destabilize a desired healthy interaction, or stabilize an oncogenic interaction. The understanding that there are a few key hot-spot residues that are mainly responsible for the binding energy of an interaction greatly widened the prospects of targeting oncogenic protein-protein interfaces enabling the use of small ligands in addition to biological molecules such as peptides and antibodies. Taming oncogenic signaling requires a deep understanding of protein interactions and their networks. Traditional representation of PPIs in signaling pathways as nodes and edges falls short of expressing interaction specific modulation of signals. Structural networks, deciphering which sites on a protein structure are responsible for each of the many interactions it may carry out, help understanding specific oncogenic mutations on signaling. We describe the key features of PPIs and their targeting, together with the advantages of structural networks, and provide four case studies demonstrating different opportunities for the aim of modulating oncogenic interactions. Keywords: Drug design, Hot-spots, Oncogenic Signaling, Protein interaction networks, Protein-protein interactions, Proteinprotein interface.
Glucocorticoid (GR) and mineralocorticoid receptors (MR) are believed to classically bind DNA as homodimers or MR-GR heterodimers to influence gene regulation in response to pulsatile basal or stress-evoked glucocorticoid secretion. Pulsed corticosterone presentation reveals MR and GR co-occupy DNA only at the peaks of glucocorticoid oscillations, allowing interaction. GR DNA occupancy was pulsatile, while MR DNA occupancy was prolonged through the inter-pulse interval. In mouse mammary 3617 cells MR-GR interacted in the nucleus and at a chromatin-associated DNA binding site. Interactions occurred irrespective of ligand type and receptors formed complexes of higher order than heterodimers. We also detected MR-GR interactions ex-vivo in rat hippocampus. An expanded range of MR-GR interactions predicts structural allostery allowing a variety of transcriptional outcomes and is applicable to the multiple tissue types that co-express both receptors in the same cells whether activated by the same or different hormones.
Abstract Each human chromosome maintains its individuality during the cell cycle, and occupies a spatially limited volume, termed chromosome territory. Each linear chromosomal DNA is folded into multiple loops in the three dimensional space, and further organized into densely packed heterochromatin, less dense euchromatin and nucleosome-free regions that are accessible for transcription factor binding. As the average density of chromatin in the nucleus is very high, size exclusion potentially restricts access of large macromolecules such as RNA polymerase II and Mediator to DNA buried in chromosomal interiors. To examine this idea, we investigated whether increase in chromosome size leads to relative decrease in transcriptional activity of larger chromosomes. We found that the scaling of gene expression relative to chromosome size follows exactly the surface-area-to-volume ratio, suggesting that active genes are located at chromosomal surfaces. To directly test this hypothesis, we developed a scalable probe to assess chromatin accessibility to macromolecules of different sizes. We show that, at the chromosomal level, open chromatin landscapes of small and large molecules are strikingly similar. However, at a finer locus level, regions accessible to small transcription factors were primarily enriched around promoters, whereas regions accessible to large molecules were dispersed along gene bodies. Collectively, our results indicate that DNA accessibility is controlled at two different scales, and suggest that making chromatin accessible to large molecules is a critical step in the control of gene expression.
ABSTRACT Triple-negative breast cancer (TNBC) stands out as a particularly aggressive and frequently recurring form of breast cancer. Due to the absence of hormone receptors, the available treatment avenues are constrained, making chemotherapy the primary approach. Unfortunately, the development of resistance to chemotherapy poses a significant challenge, further restricting the already limited therapeutic alternatives for recurrent cases. Understanding the molecular basis of chemotherapy resistance in TNBC is pivotal for improving treatment outcomes. Here, we generated two different Taxol-resistant TNBC cell lines with a dose-escalation method to mimic chemotherapy resistance in vitro . These cells exhibited hallmark features of resistance, including reduced cell growth, altered morphology, and evasion of apoptosis. Transcriptome analysis uncovered elevated ABCB1 expression and multidrug-resistant phenotype in the resistant cells. To comprehensively investigate the key epigenetic regulators of Taxol resistance, we conducted chromatin-focused genetic and chemical screens and pinpointed Bromodomain and PHD Finger Containing 1 (BRPF1) as a novel regulator of Taxol resistance in TNBC cells. Knockout of BRPF1, the reader protein in the MOZ/MORF histone acetyl-transferase complex, but not the other complex members, sensitized resistant cells to Taxol. Additionally, BRPF1 inhibitors, PFI-4 and OF-1, in combination with Taxol significantly reduced cell viability. Transcriptome analysis upon BRPF1 loss or inhibition revealed a negative impact on ribosome biogenesis-related gene sets, resulting in a global decrease in protein translation in Taxol-resistant cells. Our ChIP-qPCR analysis demonstrated that active BRPF1 directly interacts with the ABCB1 promoter, enhancing its expression towards inducing a multidrug-resistant phenotype. Conversely, knockout or inhibition of BRPF1 leads to decreased ABCB1 expression. This dual mechanism critically sensitizes Taxol-resistant TNBC cells to chemotherapy. Our findings uncover a comprehensive molecular framework, highlighting the pivotal role of epigenetic reader protein BRPF1 in Taxol resistance and providing potential avenues for therapeutic intervention in TNBC.
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