Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines.

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

LSD1 promotes prostate cancer reprogramming by repressing TP53 signaling independently of its demethylase function.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cell survival in cancers such as prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since the widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC versus adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors - but not catalytic inhibitors - reduced NEPC cell survival. RNA-Seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1's catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1's noncatalytic function may be a promising treatment strategy for NEPC.

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3' end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3' processing are deployed across human tissues, with nearly half of multi-transcript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

KDM6A Loss Recruits Tumor-Associated Neutrophils and Promotes Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, the impact of KDM6A loss on the PDAC tumor immune microenvironment is not known. This study used a genetically engineered, pancreas-specific Kdm6a knockout (KO) PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased tumor-associated neutrophils and neutrophil extracellular traps (NET) formation, which are known to contribute to PDAC progression. Genome-wide bromouridine sequencing analysis to evaluate nascent RNA synthesis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), was upregulated in KDM6A KO PDAC cells. KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruited neutrophils. Furthermore, in a syngeneic orthotopic mouse model, treatment with a CXCL1 neutralizing antibody blocked the chemotactic and NET-promoting properties of KDM6A-deficient PDAC cells and suppressed tumor growth, confirming CXCL1 as a key mediator of chemotaxis and PDAC growth driven by KDM6A loss. These findings shed light on how KDM6A regulates the tumor immune microenvironment and PDAC progression and suggests that the CXCL1-CXCR2 axis may be a candidate target in PDAC with KDM6A loss. SIGNIFICANCE: KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1-CXCR2 signaling axis in tumors with low KDM6A.

CDK12 regulates co-transcriptional splicing and RNA turnover in human cells.

The cyclin-dependent kinase CDK12 has garnered interest as a cancer therapeutic target as DNA damage response genes are particularly suppressed by loss of CDK12 activity. In this study, we assessed the acute effects of CDK12 inhibition on transcription and RNA processing using nascent RNA Bru-seq and BruChase-seq. Acute transcriptional changes were overall small after CDK12 inhibition but over 600 genes showed intragenic premature termination, including DNA repair and cell cycle genes. Furthermore, many genes showed reduced transcriptional readthrough past the end of genes in the absence of CDK12 activity. RNA turnover was dramatically affected by CDK12 inhibition and importantly, caused increased degradation of many transcripts from DNA damage response genes. We also show that co-transcriptional splicing was suppressed by CDK12 inhibition. Taken together, these studies reveal the roles of CDK12 in regulating transcription elongation, transcription termination, co-transcriptional splicing, and RNA turnover.

Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons.

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs), and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not the unrelated phosphatase MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Our findings thus establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell-type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development. METHODS: We performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis. RESULTS: Loss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial-mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata. CONCLUSIONS: Loss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

Co-transcriptional splicing efficiencies differ within genes and between cell types.

Pre-mRNA splicing is carried out by the spliceosome and involves splice site recognition, removal of introns, and ligation of exons. Components of the spliceosome have been shown to interact with the elongating RNA polymerase II (RNAPII) which is thought to allow splicing to occur concurrently with transcription. However, little is known about the regulation and efficiency of co-transcriptional splicing in human cells. In this study, we used Bru-seq and BruChase-seq to determine the co-transcriptional splicing efficiencies of 17,000 introns expressed across 6 human cell lines. We found that less than half of all introns across these 6 cell lines were co-transcriptionally spliced. Splicing efficiencies for individual introns showed variations across cell lines, suggesting that splicing may be regulated in a cell-type specific manner. Moreover, the splicing efficiency of introns varied within genes. The efficiency of co-transcriptional splicing did not correlate with gene length, intron position, splice site strengths, or the intron/neighboring exons GC content. However, we identified binding signals from multiple RNA binding proteins (RBPs) that correlated with splicing efficiency, including core spliceosomal machinery components-such as SF3B4, U2AF1 and U2AF2 showing higher binding signals in poorly spliced introns. In addition, multiple RBPs, such as BUD13, PUM1 and SND1, showed preferential binding in exons that flank introns with high splicing efficiencies. The nascent RNA splicing patterns presented here across multiple cell types add to our understanding of the complexity in RNA splicing, wherein RNA-binding proteins may play important roles in determining splicing outcomes in a cell type- and intron-specific manner.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.

Characterization of novel primary miRNA transcription units in human cells using Bru-seq nascent RNA sequencing.

MicroRNAs (miRNAs) are key contributors to gene regulatory networks. Because miRNAs are processed from RNA polymerase II transcripts, insight into miRNA regulation requires a comprehensive understanding of the regulation of primary miRNA transcripts. We used Bru-seq nascent RNA sequencing and hidden Markov model segmentation to map primary miRNA transcription units (TUs) across 32 human cell lines, allowing us to describe TUs encompassing 1443 miRNAs from miRBase and 438 from MirGeneDB. We identified TUs for 61 miRNAs with an unknown CAGE TSS signal for MirGeneDB miRNAs. Many primary transcripts containing miRNA sequences failed to generate mature miRNAs, suggesting that miRNA biosynthesis is under both transcriptional and post-transcriptional control. In addition to constitutive and cell-type specific TU expression regulated by differential promoter usage, miRNA synthesis can be regulated by transcription past polyadenylation sites (transcriptional read through) and promoter divergent transcription (PROMPTs). We identified 197 miRNA TUs with novel promoters, 97 with transcriptional read-throughs and 3 miRNA TUs that resemble PROMPTs in at least one cell line. The miRNA TU annotation data resource described here reveals a greater complexity in miRNA regulation than previously known and provides a framework for identifying cell-type specific differences in miRNA transcription in cancer and cell transition states.

The role of H3K79 methylation in transcription and the DNA damage response.

Chromatin plays a critical role in organizing and protecting DNA. However, chromatin acts as an impediment for transcription and DNA repair. Histone modifications, such as H3K79 methylation, promote transcription and genomic stability by enhancing transcription elongation and by serving as landing sites for proteins involved in the DNA damage response. This review summarizes the current understanding of the role of H3K79 methylation in transcription, how it affects genome stability and opportunities to develop impactful therapeutic interventions for cancer.

A Bifunctional MAPK/PI3K Antagonist for Inhibition of Tumor Growth and Metastasis.

Responses to targeted therapies frequently are brief, with patients relapsing with drug-resistant tumors. For oncogenic MEK and BRAF inhibition, drug resistance commonly occurs through activation of PI3K/AKT/mTOR signaling and immune checkpoint modulation, providing a robust molecular target for concomitant therapy. Here, we evaluated the efficacy of a bifunctional kinase inhibitor (ST-162) that concurrently targets MAPK and PI3K signaling pathways. Treatment with ST-162 produced regression of mutant KRAS- or BRAF-addicted xenograft models of colorectal cancer and melanoma and stasis of BRAF/PTEN-mutant melanomas. Combining ST-162 with immune checkpoint blockers further increased efficacy in a syngeneic KRAS-mutant colorectal cancer model. Nascent transcriptome analysis revealed a unique gene set regulated by ST-162 related to melanoma metastasis. Subsequent mouse studies revealed ST-162 was a potent inhibitor of melanoma metastasis to the liver. These findings highlight the significant potential of a single molecule with multikinase activity to achieve tumor control, overcome resistance, and prevent metastases through modulation of interconnected cell signaling pathways. Mol Cancer Ther; 16(11); 2340-50. ©2017 AACR.

Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53.

In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells.

Gene length as a biological timer to establish temporal transcriptional regulation.

Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarily conserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs.

Genome stability versus transcript diversity.

Our genome is protected from the introduction of mutations by high fidelity replication and an extensive network of DNA damage response and repair mechanisms. However, the expression of our genome, via RNA and protein synthesis, allows for more diversity in translating genetic information. In addition, the splicing process has become less stringent over evolutionary time allowing for a substantial increase in the diversity of transcripts generated. The result is a diverse transcriptome and proteome that harbor selective advantages over a more tightly regulated system. Here, we describe mechanisms in place that both safeguard the genome and promote translational diversity, with emphasis on post-transcriptional RNA processing.

Capturing the dynamic nascent transcriptome during acute cellular responses: The serum response.

Dynamic regulation of gene expression via signal transduction pathways is of fundamental importance during many biological processes such as cell state transitioning, cell cycle progression and stress responses. In this study we used serum stimulation as a cell response paradigm to apply the nascent RNA Bru-seq technique in order to capture early dynamic changes in the nascent transcriptome. Our data provides an unprecedented view of the dynamics of genome-wide transcription during the first two hours of serum stimulation in human fibroblasts. While some genes showed sustained induction or repression, other genes showed transient or delayed responses. Surprisingly, the dynamic patterns of induction and suppression of response genes showed a high degree of similarity, suggesting that these opposite outcomes are triggered by a common set of signals. As expected, early response genes such as those encoding components of the AP-1 transcription factor and those involved in the circadian clock were immediately but transiently induced. Surprisingly, transcription of important DNA damage response genes and histone genes were rapidly repressed. We also show that RNA polymerase II accelerates as it transcribes large genes and this was independent of whether the gene was induced or not. These results provide a unique genome-wide depiction of dynamic patterns of transcription of serum response genes and demonstrate the utility of Bru-seq to comprehensively capture rapid and dynamic changes of the nascent transcriptome.

Identifying transcription start sites and active enhancer elements using BruUV-seq.

BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5'-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3'-5' degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.

Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.