Publications by authors named "Venkat S Malladi"

33 Publications

Perivascular mesenchymal cells control adipose-tissue macrophage accrual in obesity.

Nat Metab 2020 11 2;2(11):1332-1349. Epub 2020 Nov 2.

Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Chronic low-grade white adipose tissue (WAT) inflammation is a hallmark of metabolic syndrome in obesity. Here, we demonstrate that a subpopulation of mouse WAT perivascular (PDGFRβ) cells, termed fibro-inflammatory progenitors (FIPs), activate proinflammatory signalling cascades shortly after the onset of high-fat diet feeding and regulate proinflammatory macrophage accumulation in WAT in a TLR4-dependent manner. FIPs activation in obesity is mediated by the downregulation of zinc-finger protein 423 (ZFP423), identified here as a transcriptional corepressor of NF-κB. ZFP423 suppresses the DNA-binding capacity of the p65 subunit of NF-κB by inducing a p300-to-NuRD coregulator switch. Doxycycline-inducible expression of Zfp423 in PDGFRβ cells suppresses inflammatory signalling in FIPs and attenuates metabolic inflammation of visceral WAT in obesity. Inducible inactivation of Zfp423 in PDGFRβ cells increases FIP activity, exacerbates adipose macrophage accrual and promotes WAT dysfunction. These studies implicate perivascular mesenchymal cells as important regulators of chronic adipose-tissue inflammation in obesity and identify ZFP423 as a transcriptional break on NF-κB signalling.
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http://dx.doi.org/10.1038/s42255-020-00301-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669663PMC
November 2020

Total Functional Score of Enhancer Elements Identifies Lineage-Specific Enhancers That Drive Differentiation of Pancreatic Cells.

Bioinform Biol Insights 2020 1;14:1177932220938063. Epub 2020 Jul 1.

Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

The differentiation of embryonic stem cells into various lineages is highly dependent on the chromatin state of the genome and patterns of gene expression. To identify lineage-specific enhancers driving the differentiation of progenitors into pancreatic cells, we used a previously described computational framework called Total Functional Score of Enhancer Elements (TFSEE), which integrates multiple genomic assays that probe both transcriptional and epigenomic states. First, we evaluated and compared TFSEE as an enhancer-calling algorithm with enhancers called using GRO-seq-defined enhancer transcripts (method 1) versus enhancers called using histone modification ChIP-seq data (method 2). Second, we used TFSEE to define the enhancer landscape and identify transcription factors (TFs) that maintain the multipotency of a subpopulation of endodermal stem cells during differentiation into pancreatic lineages. Collectively, our results demonstrate that TFSEE is a robust enhancer-calling algorithm that can be used to perform multilayer genomic data integration to uncover cell type-specific TFs that control lineage-specific enhancers.
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http://dx.doi.org/10.1177/1177932220938063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331761PMC
July 2020

Urethral luminal epithelia are castration-insensitive cells of the proximal prostate.

Prostate 2020 08 4;80(11):872-884. Epub 2020 Jun 4.

Department of Urology, UT Southwestern Medical Center, Dallas, Texas.

Background: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors.

Methods: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry.

Results: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor.

Conclusions: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.
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http://dx.doi.org/10.1002/pros.24020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339731PMC
August 2020

Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation.

Cancer Cell 2020 04 26;37(4):584-598.e11. Epub 2020 Mar 26.

Bioinformatics Core Facility of the Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX 75390, USA.

Metastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.
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http://dx.doi.org/10.1016/j.ccell.2020.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292228PMC
April 2020

Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21.

Mol Cell 2019 09 24;75(6):1270-1285.e14. Epub 2019 Jul 24.

Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduces DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi.
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http://dx.doi.org/10.1016/j.molcel.2019.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754283PMC
September 2019

Open collaborative writing with Manubot.

PLoS Comput Biol 2019 06 24;15(6):e1007128. Epub 2019 Jun 24.

Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Open, collaborative research is a powerful paradigm that can immensely strengthen the scientific process by integrating broad and diverse expertise. However, traditional research and multi-author writing processes break down at scale. We present new software named Manubot, available at https://manubot.org, to address the challenges of open scholarly writing. Manubot adopts the contribution workflow used by many large-scale open source software projects to enable collaborative authoring of scholarly manuscripts. With Manubot, manuscripts are written in Markdown and stored in a Git repository to precisely track changes over time. By hosting manuscript repositories publicly, such as on GitHub, multiple authors can simultaneously propose and review changes. A cloud service automatically evaluates proposed changes to catch errors. Publication with Manubot is continuous: When a manuscript's source changes, the rendered outputs are rebuilt and republished to a web page. Manubot automates bibliographic tasks by implementing citation by identifier, where users cite persistent identifiers (e.g. DOIs, PubMed IDs, ISBNs, URLs), whose metadata is then retrieved and converted to a user-specified style. Manubot modernizes publishing to align with the ideals of open science by making it transparent, reproducible, immediate, versioned, collaborative, and free of charge.
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http://dx.doi.org/10.1371/journal.pcbi.1007128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611653PMC
June 2019

Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation.

Elife 2019 05 28;8. Epub 2019 May 28.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States.

Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation.

Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter).
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http://dx.doi.org/10.7554/eLife.40226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538373PMC
May 2019

Cardiac Reprogramming Factors Synergistically Activate Genome-wide Cardiogenic Stage-Specific Enhancers.

Cell Stem Cell 2019 07 9;25(1):69-86.e5. Epub 2019 May 9.

Department of Molecular Biology, the Hamon Center for Regenerative Science and Medicine, and Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Electronic address:

The cardiogenic transcription factors (TFs) Mef2c, Gata4, and Tbx5 can directly reprogram fibroblasts to induced cardiac-like myocytes (iCLMs), presenting a potential source of cells for cardiac repair. While activity of these TFs is enhanced by Hand2 and Akt1, their genomic targets and interactions during reprogramming are not well studied. We performed genome-wide analyses of cardiogenic TF binding and enhancer profiling during cardiac reprogramming. We found that these TFs synergistically activate enhancers highlighted by Mef2c binding sites and that Hand2 and Akt1 coordinately recruit other TFs to enhancer elements. Intriguingly, these enhancer landscapes collectively resemble patterns of enhancer activation during embryonic cardiogenesis. We further constructed a cardiac reprogramming gene regulatory network and found repression of EGFR signaling pathway genes. Consistently, chemical inhibition of EGFR signaling augmented reprogramming. Thus, by defining epigenetic landscapes these findings reveal synergistic transcriptional activation across a broad landscape of cardiac enhancers and key signaling pathways that govern iCLM reprogramming.
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http://dx.doi.org/10.1016/j.stem.2019.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754266PMC
July 2019

Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Genes Dev 2019 06 11;33(11-12):626-640. Epub 2019 Apr 11.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of in fusion-negative RMS. Knockdown of in RMS cells results in up-regulation of and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.
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http://dx.doi.org/10.1101/gad.324467.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546057PMC
June 2019

Suppression of Myeloid Cell Arginase Activity leads to Therapeutic Response in a NSCLC Mouse Model by Activating Anti-Tumor Immunity.

J Immunother Cancer 2019 02 6;7(1):32. Epub 2019 Feb 6.

Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Background: Tumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models.

Methods: RNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent Kras GEMM of lung adenocarcinoma model.

Results: Analysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of Kras GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro.

Conclusions: We show that arginase expression is elevated in mouse and patient lung tumors. In a KRAS GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.
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http://dx.doi.org/10.1186/s40425-019-0504-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366094PMC
February 2019

High-Phosphate Diet Induces Exercise Intolerance and Impairs Fatty Acid Metabolism in Mice.

Circulation 2019 03;139(11):1422-1434

Department of Internal Medicine, Hypertension Section (P.P.-O., H.K.K., W.V.), University of Texas Southwestern Medical Center, Dallas.

Background: Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in the Western diet. Physical inactivity, a common feature of Western societies, is associated with increased cardiovascular morbidity and mortality. It is unknown whether dietary Pi excess contributes to exercise intolerance and physical inactivity.

Methods: To determine an association between Pi excess and physical activity in humans, we assessed the relationship between serum Pi and actigraphy-determined physical activity level, as well as left ventricular function by cardiac magnetic resonance imaging, in DHS-2 (Dallas Heart Study phase 2) participants after adjusting for relevant variables. To determine direct effects of dietary Pi on exercise capacity, oxygen uptake, serum nonesterified fatty acid, and glucose were measured during exercise treadmill test in C57/BL6 mice fed either a high-Pi (2%) or normal-Pi (0.6%) diet for 12 weeks. To determine the direct effect of Pi on muscle metabolism and expression of genes involved in fatty acid metabolism, additional studies in differentiated C2C12 myotubes were conducted after subjecting to media containing 1 to 3 mmol/L Pi (pH 7.0) to simulate in vivo phosphate conditions.

Results: In participants of the DHS-2 (n=1603), higher serum Pi was independently associated with reduced time spent in moderate to vigorous physical activity ( P=0.01) and increased sedentary time ( P=0.004). There was no association between serum Pi and left ventricular ejection fraction or volumes. In animal studies, compared with the control diet, consumption of high-Pi diet for 12 weeks did not alter body weight or left ventricular function but reduced maximal oxygen uptake, treadmill duration, spontaneous locomotor activity, fat oxidation, and fatty acid levels and led to downregulation of genes involved in fatty acid synthesis, release, and oxidation, including Fabp4, Hsl, Fasn, and Pparγ, in muscle. Similar results were recapitulated in vitro by incubating C2C12 myotubes with high-Pi media.

Conclusions: Our data demonstrate a detrimental effect of dietary Pi excess on skeletal muscle fatty acid metabolism and exercise capacity that is independent of obesity and cardiac contractile function. Dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.037550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411426PMC
March 2019

A Cellular Anatomy of the Normal Adult Human Prostate and Prostatic Urethra.

Cell Rep 2018 12;25(12):3530-3542.e5

Department of Urology, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

A comprehensive cellular anatomy of normal human prostate is essential for solving the cellular origins of benign prostatic hyperplasia and prostate cancer. The tools used to analyze the contribution of individual cell types are not robust. We provide a cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single-cell RNA sequencing (scRNA-seq) and flow cytometry on ∼98,000 cells taken from different anatomical regions. Immunohistochemistry with newly derived cell type-specific markers revealed the distribution of each epithelial and stromal cell type on whole mounts, revising our understanding of zonal anatomy. Based on discovered cell surface markers, flow cytometry antibody panels were designed to improve the purification of each cell type, with each gate confirmed by scRNA-seq. The molecular classification, anatomical distribution, and purification tools for each cell type in the human prostate create a powerful resource for experimental design in human prostate disease.
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http://dx.doi.org/10.1016/j.celrep.2018.11.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411034PMC
December 2018

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Cancer Immunol Immunother 2019 Mar 18;68(3):407-420. Epub 2018 Dec 18.

Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390, USA.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.
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http://dx.doi.org/10.1007/s00262-018-2284-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428600PMC
March 2019

Identification of a multipotent Twist2-expressing cell population in the adult heart.

Proc Natl Acad Sci U S A 2018 09 20;115(36):E8430-E8439. Epub 2018 Aug 20.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Twist transcription factors function as ancestral regulators of mesodermal cell fates in organisms ranging from to mammals. Through lineage tracing of Twist2 (Tw2)-expressing cells with tamoxifen-inducible Tw2-CreERT2 and tdTomato (tdTO) reporter mice, we discovered a unique cell population that progressively contributes to cardiomyocytes (CMs), endothelial cells, and fibroblasts in the adult heart. Clonal analysis confirmed the ability of Tw2-derived tdTO (Tw2-tdTO) cells to form CMs in vitro. Within the adult heart, Tw2-tdTO CMs accounted for ∼13% of total CMs, the majority of which resulted from fusion of Tw2-tdTO cells with existing CMs. Tw2-tdTO cells also contribute to cardiac remodeling after injury. We conclude that Tw2-tdTO cells participate in lifelong maintenance of cardiac function, at least in part through de novo formation of CMs and fusion with preexisting CMs, as well as in the genesis of other cellular components of the adult heart.
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http://dx.doi.org/10.1073/pnas.1800526115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130356PMC
September 2018

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes.

BMC Genomics 2018 02 20;19(1):150. Epub 2018 Feb 20.

The Department of Epigenetics and Molecular Carcinogenesis, University of Texas Graduate School of Biomedical Sciences at Houston and The Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, Houston, Texas, 77030, USA.

Background: Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy.

Results: To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells.

Discussion: Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway.

Conclusions: Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.
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http://dx.doi.org/10.1186/s12864-018-4533-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819162PMC
February 2018

Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis.

Genome Res 2018 02 22;28(2):159-170. Epub 2017 Dec 22.

Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences and Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized ("normal") human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif -values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.
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http://dx.doi.org/10.1101/gr.226019.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793780PMC
February 2018

Transcription pausing regulates mouse embryonic stem cell differentiation.

Stem Cell Res 2017 12 16;25:250-255. Epub 2017 Nov 16.

Cecil H. and Ida Green Center for Reproductive Biology Sciences and Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address:

The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation.
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http://dx.doi.org/10.1016/j.scr.2017.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732849PMC
December 2017

Dynamic Change of Transcription Pausing through Modulating NELF Protein Stability Regulates Granulocytic Differentiation.

Blood Adv 2017 Aug;1(18):1358-1367

Laboratory of Molecular Genetics of Blood Development, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The NELF complex is a metazoan-specific factor essential for establishing transcription pausing. Although NELF has been implicated in cell fate regulation, the cellular regulation of NELF and its intrinsic role in specific lineage differentiation remains largely unknown. Using mammalian hematopoietic differentiation as a model system, here we identified a dynamic change of NELF-mediated transcription pausing as a novel mechanism regulating hematopoietic differentiation. We found a sharp decrease of NELF protein abundance upon granulocytic differentiation and a subsequent genome-wide reduction of transcription pausing. This loss of pausing coincides with activation of granulocyte-affiliated genes and diminished expression of progenitor markers. Functional studies revealed that sustained expression of NELF inhibits granulocytic differentiation, whereas NELF depletion in progenitor cells leads to premature differentiation towards the granulocytic lineage. Our results thus uncover a previously unrecognized regulation of transcription pausing by modulating NELF protein abundance to control cellular differentiation.
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http://dx.doi.org/10.1182/bloodadvances.2017008383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578628PMC
August 2017

SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

PLoS One 2017 12;12(4):e0175310. Epub 2017 Apr 12.

Stanford University School of Medicine, Department of Genetics, Stanford, California, United States of America.

The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175310PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389787PMC
April 2017

Computational Approaches for Mining GRO-Seq Data to Identify and Characterize Active Enhancers.

Methods Mol Biol 2017 ;1468:121-38

The Laboratory of Signaling and Gene Expression, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-8511, USA.

Transcriptional enhancers are DNA regulatory elements that are bound by transcription factors and act to positively regulate the expression of nearby or distally located target genes. Enhancers have many features that have been discovered using genomic analyses. Recent studies have shown that active enhancers recruit RNA polymerase II (Pol II) and are transcribed, producing enhancer RNAs (eRNAs). GRO-seq, a method for identifying the location and orientation of all actively transcribing RNA polymerases across the genome, is a powerful approach for monitoring nascent enhancer transcription. Furthermore, the unique pattern of enhancer transcription can be used to identify enhancers in the absence of any information about the underlying transcription factors. Here, we describe the computational approaches required to identify and analyze active enhancers using GRO-seq data, including data pre-processing, alignment, and transcript calling. In addition, we describe protocols and computational pipelines for mining GRO-seq data to identify active enhancers, as well as known transcription factor binding sites that are transcribed. Furthermore, we discuss approaches for integrating GRO-seq-based enhancer data with other genomic data, including target gene expression and function. Finally, we describe molecular biology assays that can be used to confirm and explore further the function of enhancers that have been identified using genomic assays. Together, these approaches should allow the user to identify and explore the features and biological functions of new cell type-specific enhancers.
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http://dx.doi.org/10.1007/978-1-4939-4035-6_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522910PMC
January 2018

Principles of metadata organization at the ENCODE data coordination center.

Database (Oxford) 2016 15;2016. Epub 2016 Mar 15.

Department of Genetics, Stanford University School of Medicine Department of Genetics, Stanford, CA, USA

The Encyclopedia of DNA Elements (ENCODE) Data Coordinating Center (DCC) is responsible for organizing, describing and providing access to the diverse data generated by the ENCODE project. The description of these data, known as metadata, includes the biological sample used as input, the protocols and assays performed on these samples, the data files generated from the results and the computational methods used to analyze the data. Here, we outline the principles and philosophy used to define the ENCODE metadata in order to create a metadata standard that can be applied to diverse assays and multiple genomic projects. In addition, we present how the data are validated and used by the ENCODE DCC in creating the ENCODE Portal (https://www.encodeproject.org/). Database URL: www.encodeproject.org.
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http://dx.doi.org/10.1093/database/baw001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792520PMC
October 2016

ENCODE data at the ENCODE portal.

Nucleic Acids Res 2016 Jan 2;44(D1):D726-32. Epub 2015 Nov 2.

Stanford University School of Medicine, Department of Genetics, Stanford, CA, 94305, USA

The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments.
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http://dx.doi.org/10.1093/nar/gkv1160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702836PMC
January 2016

Ontology application and use at the ENCODE DCC.

Database (Oxford) 2015 16;2015. Epub 2015 Mar 16.

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA and Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA

The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a catalog of genomic annotations. To date, the project has generated over 4000 experiments across more than 350 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory network and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All ENCODE experimental data, metadata and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage and distribution to community resources and the scientific community. As the volume of data increases, the organization of experimental details becomes increasingly complicated and demands careful curation to identify related experiments. Here, we describe the ENCODE DCC's use of ontologies to standardize experimental metadata. We discuss how ontologies, when used to annotate metadata, provide improved searching capabilities and facilitate the ability to find connections within a set of experiments. Additionally, we provide examples of how ontologies are used to annotate ENCODE metadata and how the annotations can be identified via ontology-driven searches at the ENCODE portal. As genomic datasets grow larger and more interconnected, standardization of metadata becomes increasingly vital to allow for exploration and comparison of data between different scientific projects.
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http://dx.doi.org/10.1093/database/bav010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4360730PMC
September 2015

A comparative encyclopedia of DNA elements in the mouse genome.

Nature 2014 Nov;515(7527):355-64

Bioinformatics and Genomics, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003 Barcelona, Catalonia, Spain.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.
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http://dx.doi.org/10.1038/nature13992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266106PMC
November 2014

ENCODE data in the UCSC Genome Browser: year 5 update.

Nucleic Acids Res 2013 Jan 27;41(Database issue):D56-63. Epub 2012 Nov 27.

Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz (UCSC), Santa Cruz, CA 95064, USA.

The Encyclopedia of DNA Elements (ENCODE), http://encodeproject.org, has completed its fifth year of scientific collaboration to create a comprehensive catalog of functional elements in the human genome, and its third year of investigations in the mouse genome. Since the last report in this journal, the ENCODE human data repertoire has grown by 898 new experiments (totaling 2886), accompanied by a major integrative analysis. In the mouse genome, results from 404 new experiments became available this year, increasing the total to 583, collected during the course of the project. The University of California, Santa Cruz, makes this data available on the public Genome Browser http://genome.ucsc.edu for visual browsing and data mining. Download of raw and processed data files are all supported. The ENCODE portal provides specialized tools and information about the ENCODE data sets.
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http://dx.doi.org/10.1093/nar/gks1172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531152PMC
January 2013

The UCSC Genome Browser database: extensions and updates 2013.

Nucleic Acids Res 2013 Jan 15;41(Database issue):D64-9. Epub 2012 Nov 15.

Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz (UCSC), Santa Cruz, CA 95064, USA.

The University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation 'tracks' are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.
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http://dx.doi.org/10.1093/nar/gks1048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531082PMC
January 2013

An encyclopedia of mouse DNA elements (Mouse ENCODE).

Genome Biol 2012 Aug 13;13(8):418. Epub 2012 Aug 13.

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.
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http://dx.doi.org/10.1186/gb-2012-13-8-418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491367PMC
August 2012

The UCSC Genome Browser database: extensions and updates 2011.

Nucleic Acids Res 2012 Jan 15;40(Database issue):D918-23. Epub 2011 Nov 15.

Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

The University of California Santa Cruz Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analyzing and sharing both publicly available and user-generated genomic data sets. In the past year, the local database has been updated with four new species assemblies, and we anticipate another four will be released by the end of 2011. Further, a large number of annotation tracks have been either added, updated by contributors, or remapped to the latest human reference genome. Among these are new phenotype and disease annotations, UCSC genes, and a major dbSNP update, which required new visualization methods. Growing beyond the local database, this year we have introduced 'track data hubs', which allow the Genome Browser to provide access to remotely located sets of annotations. This feature is designed to significantly extend the number and variety of annotation tracks that are publicly available for visualization and analysis from within our site. We have also introduced several usability features including track search and a context-sensitive menu of options available with a right-click anywhere on the Browser's image.
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http://dx.doi.org/10.1093/nar/gkr1055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245018PMC
January 2012

ENCODE whole-genome data in the UCSC Genome Browser: update 2012.

Nucleic Acids Res 2012 Jan 9;40(Database issue):D912-7. Epub 2011 Nov 9.

Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

The Encyclopedia of DNA Elements (ENCODE) Consortium is entering its 5th year of production-level effort generating high-quality whole-genome functional annotations of the human genome. The past year has brought the ENCODE compendium of functional elements to critical mass, with a diverse set of 27 biochemical assays now covering 200 distinct human cell types. Within the mouse genome, which has been under study by ENCODE groups for the past 2 years, 37 cell types have been assayed. Over 2000 individual experiments have been completed and submitted to the Data Coordination Center for public use. UCSC makes this data available on the quality-reviewed public Genome Browser (http://genome.ucsc.edu) and on an early-access Preview Browser (http://genome-preview.ucsc.edu). Visual browsing, data mining and download of raw and processed data files are all supported. An ENCODE portal (http://encodeproject.org) provides specialized tools and information about the ENCODE data sets.
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http://dx.doi.org/10.1093/nar/gkr1012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245183PMC
January 2012

The effect of SNP discovery method and sample size on estimation of population genetic data for Chinese and Indian rhesus macaques (Macaca mulatta).

Primates 2011 Apr 5;52(2):129-38. Epub 2011 Jan 5.

Department of Anthropology, University of California, Davis, 330 Young Hall, One Shields Avenue, Davis, CA 95616, USA.

This study was designed to address issues regarding sample size and marker location that have arisen from the discovery of SNPs in the genomes of poorly characterized primate species and the application of these markers to the study of primate population genetics. We predict the effect of discovery sample size on the probability of discovering both rare and common SNPs and then compare this prediction with the proportion of common and rare SNPs discovered when different numbers of individuals are sequenced. Second, we examine the effect of genomic region on estimates of common population genetic data, comparing markers from both coding and non-coding regions of the rhesus macaque genome and the population genetic data calculated from these markers, to measure the degree and direction of bias introduced by SNPs located in coding versus non-coding regions of the genome. We found that both discovery sample size and genomic region surveyed affect SNP marker attributes and population genetic estimates, even when these are calculated from an expanded data set containing more individuals than the original discovery data set. Although none of the SNP detection methods or genomic regions tested in this study was completely uninformative, these results show that each has a different kind of genetic variation that is suitable for different purposes, and each introduces specific types of bias. Given that each SNP marker has an individual evolutionary history, we calculated that the most complete and unbiased representation of the genetic diversity present in the individual can be obtained by incorporating at least 10 individuals into the discovery sample set, to ensure the discovery of both common and rare polymorphisms.
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http://dx.doi.org/10.1007/s10329-010-0232-4DOI Listing
April 2011