Transcriptional Pause Release Research Articles

Unraveling the CDK9/PP2A/ERK Network in Transcriptional Pause Release and Complement Activation in KRAS-mutant Cancers.

Selective inhibition of the transcription elongation factor (P-TEFb) complex represents a promising approach in cancer therapy, yet CDK9 inhibitors (CDK9i) are currently limited primarily to certain hematological malignancies. Herein, while initial responses to CDK9-targeted therapies are observed in vitro across various KRAS-mutant cancer types, their efficacy is far from satisfactory in nude mouse xenograft models. Mechanistically, CDK9 inhibition leads to compensatory activation of ERK-MYC signaling, accompanied by the recovery of proto-oncogenes, upregulation of immediate early genes (IEGs), stimulation of the complement C1r-C3-C3a cascade, and induction of tumor immunosuppression. The "paradoxical" regulation of PP2Ac activity involving the CDK9/Src interplay contributes to ERK phosphorylation and pause-release of RNA polymerase II (Pol II). Co-targeting of CDK9 and KRAS/MAPK signaling pathways eliminates ERK-MYC activation and prevents feedback activation mediated by receptor tyrosine kinases, leading to more effective control of KRAS-mutant cancers and overcoming KRASi resistance. Moreover, modulating the tumor microenvironment (TME) by complement system intervention enhances the response to CDK9i and potently suppresses tumor growth. Overall, the preclinical investigations establish a robust framework for conducting clinical trials employing KRASi/SOS1i/MEKi or immunomodifiers in combination with CDK9i to simultaneously target cancer cells and their crosstalk with the TME, thereby yielding improved responses in KRAS-mutant patients.

YTHDC1 m6A-dependent and m6A-independent functions converge to preserve the DNA damage response

Cells have evolved a robust and highly regulated DNA damage response to preserve their genomic integrity. Although increasing evidence highlights the relevance of RNA regulation, our understanding of its impact on a fully efficient DNA damage response remains limited. Here, through a targeted CRISPR-knockout screen, we identify RNA-binding proteins and modifiers that participate in the p53 response. Among the top hits, we find the m6A reader YTHDC1 as a master regulator of p53 expression. YTHDC1 binds to the transcription start sites of TP53 and other genes involved in the DNA damage response, promoting their transcriptional elongation. YTHDC1 deficiency also causes the retention of introns and therefore aberrant protein production of key DNA damage factors. While YTHDC1-mediated intron retention requires m6A, TP53 transcriptional pause-release is promoted by YTHDC1 independently of m6A. Depletion of YTHDC1 causes genomic instability and aberrant cancer cell proliferation mediated by genes regulated by YTHDC1. Our results uncover YTHDC1 as an orchestrator of the DNA damage response through distinct mechanisms of co-transcriptional mRNA regulation.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase(Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and isthen released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

BOD1L Facilitates Chromatin Binding of SETD1A and Promotes Leukemia Cell Growth

The H3K4 methyltransferase SETD1A plays an essential role in leukemia through a non-catalytic activity which regulates transcriptional pause release of RNA polymerase II. SETD1A localizes to the transcriptional start site of numerous actively transcribed genes, but primarily regulates the expression of a restricted set of genes within the DNA damage response as well as heme biosynthesis pathway. However, the roles of upstream regulators or essential components of SETD1A chromatin binding remain unclear. Using the DepMap database and a CRISPR-tiling screen method, we found that BOD1L is the most correlated co-dependency with SETD1A in human cancer cell lines and that the Shg1 domain of BOD1L plays an indispensable role in human MOLM-13 leukemia cell line. BOD1L knockout increases apoptosis and reduces cell cycle. The strong BOD1L dependency in AML cells was also confirmed using a mouse MLL-r leukemia model in vitro and in vivo. SETD1A catalytic domain mutant AML cells still responded to the BOD1L sgRNAs that suggests the role of BOD1L is independent from the SETD1A catalytic function. Importantly, BOD1L knockout mimics the transcriptional profiles of SETD1A knockout cells and reduces the expression of genes in DNA damage response and heme biosynthesis. BOD1L loss immediately reduces SETD1A distribution on chromatin and increased the NELFE and RNA polymerase II signals at transcriptional start sites. These results show that BOD1L is an essential component of the non-catalytic SETD1A function in transcriptional pause release. We also performed an immunoprecipitation assay using tagged constructs to examine the interaction between BOD1L and SETD1A. Our data indicate that SETD1A associates with BOD1L through the FLOS domain, especially in the C-terminal region. In addition, we found that the loop structure in the Shg1 domain of BOD1L is associated with the nonenzymatic domain of SETD1A. A split luciferase complementation assay reveals that the BOD1L Shg1 domain and SETD1A FLOS domain interact. To demonstrate the role of BOD1L chromatin interaction, we enforced localization of the Shg1 domain fragment at a silent gene locus and it successfully induced ectopic SETD1A recruitment to chromatin and formation of the COMPASS complex. However, we observed neither induced H3K4me3 modification nor RNA polymerase II recruitment. Taken together, while the BOD1L-SETD1A complex is not sufficient to act as a pioneer complex for active transcription, our study identified that BOD1L facilitates chromatin binding of SETD1A through the Shg1-FLOS domain interaction and then regulates transcriptional pause release, and that this interaction is a potential therapeutic opportunity for SETD1A-targeting therapy.

Myc beyond Cancer: Regulation of Mammalian Tissue Regeneration.

Myc is one of the most well-known oncogenes driving tumorigenesis in a wide variety of tissues. From the brain to blood, its deregulation derails physiological pathways that grant the correct functioning of the cell. Its action is carried out at the gene expression level, where Myc governs basically every aspect of transcription. Indeed, in addition to its role as a canonical, chromatin-bound transcription factor, Myc rules RNA polymerase II (RNAPII) transcriptional pause-release, elongation and termination and mRNA capping. For this reason, it is evident that minimal perturbations of Myc function mirror malignant cell behavior and, consistently, a large body of literature mainly focuses on Myc malfunctioning. In healthy cells, Myc controls molecular mechanisms involved in pivotal functions, such as cell cycle (and proliferation thereof), apoptosis, metabolism and cell size, angiogenesis, differentiation and stem cell self-renewal. In this latter regard, Myc has been found to also regulate tissue regeneration, a hot topic in the research fields of aging and regenerative medicine. Indeed, Myc appears to have a role in wound healing, in peripheral nerves and in liver, pancreas and even heart recovery. Herein, we discuss the state of the art of Myc's role in tissue regeneration, giving an overview of its potent action beyond cancer.

H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation1,2. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation3,4. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.

A New Insight into MYC Action: Control of RNA Polymerase II Methylation and Transcription Termination

MYC oncoprotein deregulation is a common catastrophic event in human cancer and limiting its activity restrains tumor development and maintenance, as clearly shown via Omomyc, an MYC-interfering 90 amino acid mini-protein. MYC is a multifunctional transcription factor that regulates many aspects of transcription by RNA polymerase II (RNAPII), such as transcription activation, pause release, and elongation. MYC directly associates with Protein Arginine Methyltransferase 5 (PRMT5), a protein that methylates a variety of targets, including RNAPII at the arginine residue R1810 (R1810me2s), crucial for proper transcription termination and splicing of transcripts. Therefore, we asked whether MYC controls termination as well, by affecting R1810me2S. We show that MYC overexpression strongly increases R1810me2s, while Omomyc, an MYC shRNA, or a PRMT5 inhibitor and siRNA counteract this phenomenon. Omomyc also impairs Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcription elongation. ChIP-seq experiments show that Omomyc replaces MYC and reshapes RNAPII distribution, increasing occupancy at promoter and termination sites. It is unclear how this may affect gene expression. Transcriptomic analysis shows that transcripts pivotal to key signaling pathways are both up- or down-regulated by Omomyc, whereas genes directly controlled by MYC and belonging to a specific signature are strongly down-regulated. Overall, our data point to an MYC/PRMT5/RNAPII axis that controls termination via RNAPII symmetrical dimethylation and contributes to rewiring the expression of genes altered by MYC overexpression in cancer cells. It remains to be clarified which role this may have in tumor development.

The super elongation complex (SEC) mediates phase transition of SPT5 during transcriptional pause release.

Release of promoter-proximally paused RNA Pol II into elongation is a tightly regulated and rate-limiting step in metazoan gene transcription. However, the biophysical mechanism underlying pause release remains unclear. Here, we demonstrate that the pausing and elongation regulator SPT5 undergoes phase transition during transcriptional pause release. SPT5 per se is prone to form clusters. The disordered domain in SPT5 is required for pause release and gene activation. During early elongation, the super elongation complex (SEC) induces SPT5 transition into elongation droplets. Depletion of SEC increases SPT5 pausing clusters. Furthermore, disease-associated SEC mutations impair phase properties of elongation droplets and transcription. Our study suggests that SEC-mediated SPT5 phase transition might be essential for pause release and early elongation and that aberrant phase properties could contribute to transcription abnormality in diseases.

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.

DNA Replication Stress Due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

Introduction: Myelodysplastic syndromes (MDS) with SF3B1 mutation represent a subtype of hematopoietic stem cell disorders with bone marrow (BM) erythroid dysplasia and ring sideroblasts. This mutation in a splicing factor gene causes multiple alterations of mRNA maturation. Other mutated splicing factors SRSF2 or U2AF1, produce splicing alterations and formation of unscheduled R-loops, which trigger DNA damage response and genetic instability. Mechanistically, mutant SRSF2 impairs the transcription pause release downstream of TSS allowing invasion of relaxed DNA double helix by nascent RNA. Although previous studies suggested that R-loops inappropriately form in SF3B1 mutant cells, investigations are required to elucidate why MDS with SF3B1 mutation are less prone to genetic instability. Methods: A total of 96 patients including 53 MDS with SF3B1 mutation and 43 other low risk MDS was enrolled in this study. RNA-seq of BM mononuclear cells (MNC) was performed in 21 MDS with and 6 MDS w/o SF3B1 mutation. BM CD34+-derived erythroblasts were expanded from 69 MDS and 22 healthy controls to analyze transcriptome, proteome, R-loop landscape, and DNA replication stress. For global proteome analysis, after protein digestion, peptides were fractionated on SCX column and analyzed by mass spectrometry. R-loops were detected by DNA-RNA immunoprecipitation (DRIP-seq) using S9.6 antibody. Immunofluorescence labeling of pRPA32, 53BP1 and γH2AX, DNA combing, BrdU assay and metabolomics were used to investigate DNA replication stress and DNA biosynthesis in human primary erythroblasts or in murine proerythroblastic (proE) G1E-ER4 Crispr-cas9 Sf3b1K700E/+ cell line. expressing GATA1 under the control of estrogen receptor. Results: In SF3B1-mutated BM MNC samples, DeSeq2 analysis of RNA-seq data revealed 1,058 upregulated genes (log2(FC)>0.6, BH-adj P-value<0.05) involved in DNA replication, cell cycle, nucleotide biosynthesis, DNA repair, and erythroid differentiation. Analysis of splicing identified 3,937 differential splicing events (ΔPSI>0.10, BH-adj P-value<0.05) including 1,256 events of intron retention (IR). Among IR events, 384 loss of IR events correlated with altered expression of 293 genes. In SF3B1-mutated erythroblasts, loss of IR events were more frequent in polychromatophilic erythroblasts (polyE: 383) than in proE/early basophilic erythroblasts (eBasoE: 171) suggesting that gene extinction mediated by IR during normal differentiation was altered. Quantitative proteomics revealed 924 significantly deregulated proteins with little overlap with IR transcripts, consistently involved in DNA damage, DNA repair, and nucleotide biosynthesis pathways. DRIP-seq was performed in proE/eBasoE of 8 MDS w/o or with SF3B1 mutation to investigate R-loop formation. Stringent peak calling identified 2,208 shared peaks in wild-type samples compared to 159 shared peaks in SF3B1-mutated samples, suggesting a loss of R-loops. Integrative analysis and DRIP-qPCR demonstrated that R-loop losses overlapped IR loss events, consistent with the notion that intron excision prevents R-loop formation. Compared to SRSF2 or U2AF1-mutated erythroblasts, SF3B1-mutated cells exhibited a significant increase of DNA synthesis by BrdU labeling and of replication fork speed by DNA combing. Immunofluorescence analysis detected pRPA32 foci marking single strand DNA but no evidence of double strand breaks in contrast to SRSF2 mutant cells in which 53BP1 or γH2AX foci were detected. Compared to isogenic cell lines, Sf3b1K700E/+ proE G1E-ER4 cells demonstrated higher proliferation rate and fork speed in proliferation and in estradiol-induced differentiation conditions, and significantly lower amounts of deoxynucleotides (dATP, dCTP, TTP) suggesting persistence of DNA synthesis during differentiation. Targeting of DNA polymerase by aphidicolin or ribonucleotide reductase by low doses of hydroxyurea partially rescued erythroid differentiation. Conclusion: Our data demonstrated a highly specific signature of DNA replication stress at transcriptomic and proteomic level in SF3B1-mutant cells. High replication fork speed reflects the loss of R-loops in these cells, which may reduce the risk of genetic instability at the expense of normal differentiation. Pharmacological targeting of the DNA replication stress could improve erythropoiesis.

SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia.

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.

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.

Abstract 2362: Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets

Abstract Background: Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocations such as the the t(2;13)(q35;q14), which creates the oncogenic transcription factor, PAX3-FOXO1, in alveolar rhabdomyosarcoma (aRMS). Identification of the direct transcriptional targets and mechanisms of control by translocated transcription factors is critical to our understanding of disease etiology and the development of targeted therapeutic approaches. However, defining the genes directly controlled by DNA binding proteins and the mechanisms of regulation at those direct target genes is challenging, because traditional genetic approaches to inactivate transcription factors cannot distinguish direct from indirect changes in gene expression. Methods: A CRISPR-based approach was used to engineer the endogenous PAX3-FOXO1 locus to incorporate the FKBP12F36V degron tag. This allowed degradation of PAX3-FOXO1 within 2 hr of treatment with the proteolysis targeting chimera, dTAG-47. We performed short time courses to analyze nascent transcription (precision nuclear run-on sequencing; PRO-seq), chromatin accessibility (ATAC-seq), and genome-wide mapping of transcriptional complexes (CUT&amp;RUN). This identified a small cohort of high-confidence PAX3-FOXO1 gene targets and associated PAX3-FOXO1-regulated enhancer elements. In addition, we used CRISPR editing to add APEX2 and 3XFLAG tags to PAX3-FOXO1 to enable deep proteomics to identify PAX3-FOXO1-associated transcriptional complexes in aRMS cell lines. Results: Degradation of PAX3-FOXO1-FKBP12F36V led to growth inhibition, hallmarks of myogenic differentiation, and reduced growth in soft agar. PAX3-FOXO1 binding was detected at over 44,000 sites throughout the genome using CUT&amp;RUN, and loss of chromatin bound PAX3-FOXO1 was observed within 2-4hr of PAX3-FOXO1 degradation. PRO-seq analysis defined a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Unexpectedly, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. Deep proteomic analysis showed that PAX3-FOXO1 was in close proximity to many transcriptional regulatory complexes, but appeared to coordinate the activity of these complexes at only a few hundred sites. Moreover, the continued presence of PAX3-FOXO1 at these elements was required to maintain chromatin accessibility. Conclusion: This work demonstrates the utility of using rapid degradation of endogenous transcription factors to define their direct transcription targets and the mechanisms by which these factors control gene expression. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network, and emphasizes the utility of PAX3-FOXO1 as a therapeutic target in aRMS. Citation Format: Susu Zhang, Jing Wang, Qi Liu, Hayes McDonald, Monica Bomber, Hillary Layden, Jacob Ellis, Scott Borinstein, Scott Hiebert, Kristy Stengel. Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets [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 2362.

Abstract 6050: JMJD6 regulates super-enhancer mediated transcriptional dependencies in group 3 medulloblastoma

Abstract Medulloblastoma (MB) is a heterogenous cerebellar tumor that comprises four molecular and clinically significant subgroups. MB is the most common brain malignancy in children with a five-year survival of 70%, however, Group 3 MB patients have the poorest survival due to the amplification of MYC and overexpression of key oncogenes that underly the Group 3 phenotype. Recently, epigenetic modifications bearing super-enhancer, H3K27ac, marks have been suggested to contribute to transcriptional dependencies that define Group 3 MB and represents an emerging target for therapeutic intervention. Currently, the mechanism that establishes and maintains these modifications to the DNA architecture remains unclear. In our study, we hypothesize that an epigenetic modifying protein, JMJD6, regulates the promoter-pause release associated with super-enhancer mediated oncogene addiction in Group 3 MB. This hypothesis is based on evidence in the literature showing that JMJD6 regulates the transcriptional pause release at enhancer regions. Moreover, the promoter-pause release has been shown to be critical for super-enhancer mediated transcription. In our preliminary studies, we demonstrate elevated levels of JMJD6 in Group 3 MB and survival analysis indicating JMJD6 as a predictor for poor prognosis in MB patients. Immunoprecipitation revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers. Inducible knockdown of JMJD6 decreased MYC expression, cellular proliferation and stemness in vitro while inhibiting tumor formation and promoting survival in mice. Together, these studies identify JMJD6 as a promising therapeutic target for treating aggressive epigenetically driven Groups 3 MB tumors. Citation Format: Matthew Kling, Sutapa Ray, Don W. Coulter, Nagendra Chaturvedi. JMJD6 regulates super-enhancer mediated transcriptional dependencies in group 3 medulloblastoma [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 6050.

MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets

c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Analyses of clinical specimens reveal that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq uncover an increase in RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.

Transcription Pause and Escape in Neurodevelopmental Disorders.

Transcription pause-release is an important, highly regulated step in the control of gene expression. Modulated by various factors, it enables signal integration and fine-tuning of transcriptional responses. Mutations in regulators of pause-release have been identified in a range of neurodevelopmental disorders that have several common features affecting multiple organ systems. This review summarizes current knowledge on this novel subclass of disorders, including an overview of clinical features, mechanistic details, and insight into the relevant neurodevelopmental processes.

Abstract LB193: Lysine demethylase 5A is required for MYC driven transcription in multiple myeloma

Abstract Multiple myeloma (MM) is a malignant plasma cell disorder accounting for 10% of hematologic malignancies. Although high dose chemotherapy and targeted agents have improved patient outcomes, MM remains an incurable disorder. Thus, there is an urgent need to develop novel therapeutic strategies. We discovered that the distinct lysine demethylase 5A (KDM5A) plays a critical role in MM cell growth, through a unique link to MYC-driven transcriptional programs. KDM5A is a dominant regulator of histone H3K4 trimethylation, a histone mark associated with activated gene transcription. We identify that KDM5A interacts with P-TEFb complex and cooperates with MYC to control MYC targeted genes in MM cells. We develop a novel, cell-permeable and selective KDM5 inhibitor, JQKD82 that increases histone H3K4me3, and inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Utilizing genetic ablation together with our inhibitor, we established a detail mechanism that KDM5A is required for transcriptional pause release by P-TEFb at MYC target genes. These data identify KDM5A as a unique vulnerability in MM through regulation of the driving MYC oncogene in MM, and defined JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM. Citation Format: Jun Qi, hiroto Ohguchi, Paul MC Park, tingjian wang, Kenneth C. Anderson, Teru Hideshima, Catrine Johansson, Udo Oppermann, Adam D. Durbin, Berkley Gryder, Javed Khan, xiaofeng zhang. Lysine demethylase 5A is required for MYC driven transcription in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB193.

Shape of promoter antisense RNAs regulates ligand-induced transcription activation.

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required therecruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17β-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to Tcell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.