Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation

Riboswitches are a class of nonprotein-coding RNAs that directly sense cellular metabolites to regulate gene expression. They are model systems for analyzing RNA-ligand interactions and are established targets for antibacterial agents. Many studies have analyzed the ligand-binding properties of riboswitches, but this work has outpaced our understanding of the underlying chemical pathways that govern riboswitch-controlled gene expression. To address this knowledge gap, we prepared 15 mutants of the preQ1-II riboswitch—a structurally and biochemically well-characterized HLout pseudoknot that recognizes the metabolite prequeuosine1 (preQ1). The mutants span the preQ1-binding pocket through the adjoining Shine–Dalgarno sequence (SDS) and include A-minor motifs, pseudoknot-insertion helix P4, U·A-U base triples, and canonical G-C pairs in the anti-SDS. As predicted—and confirmed by in vitro isothermal titration calorimetry measurements—specific mutations ablated preQ1 binding, but most aberrant binding effects were corrected by compensatory mutations. In contrast, functional analysis in live bacteria using a riboswitch-controlled GFPuv-reporter assay revealed that each mutant had a deleterious effect on gene regulation, even when compensatory changes were included. Our results indicate that effector binding can be uncoupled from gene regulation. We attribute loss of function to defects in a chemical interaction network that links effector binding to distal regions of the fold that support the gene-off RNA conformation. Our findings differentiate effector binding from biological function, which has ramifications for riboswitch characterization. Our results are considered in the context of synthetic ligands and drugs that bind tightly to riboswitches without eliciting a biological response.

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CHD8 Regulates Cellular Homeostasis and Neuronal Function Genes Across Multiple Models of CHD8 Haploinsufficiency

10.1101/375238 ◽

2018 ◽

Author(s):

A. Ayanna Wade ◽

Kenneth Lim ◽

Rinaldo Catta-Preta ◽

Alex S. Nord

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Chromatin Remodeling ◽

De Novo ◽

Autism Spectrum ◽

Loss Of Function ◽

Cell Functions ◽

High Affinity

ABSTRACTThe packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Recent sequencing of patient mutations has linked de novo loss-of-function mutations to chromatin remodeling factors with specific, causal roles in neurodevelopmental disorders. Characterizing cellular and molecular phenotypes arising from haploinsufficiency of chromatin remodeling factors could reveal convergent mechanisms of pathology. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor gene and has among the highest de novo loss-of-function mutations rates in patients with autism spectrum disorder (ASD). Mutations to CHD8 are expected to drive neurodevelopmental pathology through global disruptions to gene expression and chromatin state, however, mechanisms associated with CHD8 function have yet to be fully elucidated. We analyzed published transcriptomic and epigenomic data across CHD8 in vitro and in vivo knockdown and knockout models to identify convergent mechanisms of gene regulation by CHD8. We found reproducible high-affinity interactions of CHD8 near promoters of genes necessary for basic cell functions and gene regulation, especially chromatin organization and RNA processing genes. Overlap between CHD8 interaction and differential expression suggests that reduced dosage of CHD8 directly relates to decreased expression of these genes. In addition, genes important for neuronal development and function showed consistent dysregulation, though there was a reduced rate and decreased affinity for CHD8 interactions near these genes. This meta-analysis verifies CHD8 as a critical regulator of gene expression and reveals a consistent set of high affinity CHD8 interaction targets observed across human and mouse in vivo and in vitro studies. Our findings highlight novel core functions of CHD8 and indicate direct and downstream gene regulatory impacts that are likely to be associated with neuropathology underlying CHD8-associated neurodevelopmental disorder.

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Macrophage Modification Strategies for Efficient Cell Therapy

Cells ◽

10.3390/cells9061535 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1535 ◽

Cited By ~ 5

Author(s):

Anastasiya S. Poltavets ◽

Polina A. Vishnyakova ◽

Andrey V. Elchaninov ◽

Gennady T. Sukhikh ◽

Timur Kh. Fatkhudinov

Keyword(s):

Gene Expression ◽

Gene Editing ◽

Target Selection ◽

Model Systems ◽

Attractive Therapeutic Target ◽

Specific Alteration ◽

Inflammatory Phenotypes ◽

Vector Delivery

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

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Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

Journal of Bacteriology ◽

10.1128/jb.00225-16 ◽

2016 ◽

Vol 198 (12) ◽

pp. 1735-1742 ◽

Cited By ~ 29

Author(s):

Nastaran Hadizadeh ◽

Reid C. Johnson ◽

John F. Marko

Keyword(s):

Gene Regulation ◽

Dna Binding ◽

Protein Concentration ◽

Model Systems ◽

Bacterial Chromosome ◽

Regulatory Function ◽

Content Type ◽

E Coli ◽

Dna Double Helix

ABSTRACTOff-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the majorEscherichia colinucleoid protein Fis on isolated bacterial chromosomes. We isolatedE. colinucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant (kexch) of ≈104M−1s−1, comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observedin vitroon chromosomes assembledin vivo.IMPORTANCEBacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolatedE. colichromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed inin vitroexperiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

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Variation in the Group B Streptococcus CsrRS Regulon and Effects on Pathogenicity

Journal of Bacteriology ◽

10.1128/jb.01677-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 1956-1965 ◽

Cited By ~ 44

Author(s):

Sheng-Mei Jiang ◽

Nadeeza Ishmael ◽

Julie Dunning Hotopp ◽

Manuela Puliti ◽

Luciana Tissi ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Virulence Factors ◽

Group B Streptococcus ◽

Regulatory System ◽

Systemic Infection ◽

Global Gene Expression ◽

Pathogenic Potential ◽

Group B

ABSTRACT CsrRS (or CovRS) is a two-component regulatory system that controls expression of multiple virulence factors in the important human pathogen group B Streptococcus (GBS). We now report global gene expression studies in GBS strains 2603V/R and 515 and their isogenic csrR and csrS mutants. Together with data reported previously for strain NEM316, the results reveal a conserved 39-gene CsrRS regulon. In vitro phosphorylation-dependent binding of recombinant CsrR to promoter regions of both positively and negatively regulated genes suggests that direct binding of CsrR can mediate activation as well as repression of target gene expression. Distinct patterns of gene regulation in csrR versus csrS mutants in strain 2603V/R compared to 515 were associated with different hierarchies of relative virulence of wild-type, csrR, and csrS mutants in murine models of systemic infection and septic arthritis. We conclude that CsrRS regulates a core group of genes including important virulence factors in diverse strains of GBS but also displays marked variability in the repertoire of regulated genes and in the relative effects of CsrS signaling on CsrR-mediated gene regulation. Such variation is likely to play an important role in strain-specific adaptation of GBS to particular host environments and pathogenic potential in susceptible hosts.

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Synthesis and in vitro assessment of chemically modified siRNAs targeting BCL2 that contain 2′-ribose and triazole-linked backbone modifications

MedChemComm ◽

10.1039/c5md00147a ◽

2015 ◽

Vol 6 (6) ◽

pp. 1210-1215 ◽

Cited By ~ 2

Author(s):

Gordon Hagen ◽

Brandon J. Peel ◽

John Samis ◽

Jean-Paul Desaulniers

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Chemically Modified ◽

Naturally Occurring ◽

In Vitro Assessment ◽

Transcriptional Gene Regulation ◽

Deleterious Genes

Short-interfering RNAs (siRNAs) are naturally occurring biomolecules used for post-transcriptional gene regulation, and therefore hold promise as a future therapeutic by silencing gene expression of overexpressed deleterious genes.

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Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

10.1101/2021.09.30.462680 ◽

2021 ◽

Author(s):

Genevieve Housman ◽

Emilie Briscoe ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Bone Cells ◽

Cell Types ◽

Differentially Expressed ◽

Functional Genomic ◽

Cell Culture Model ◽

Culture Model ◽

Study Gene Regulation

AbstractThe evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.Author SummaryPrimates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear. Studies of gene expression variation in primate skeletal tissues are extremely restricted due to the ethical and practical challenges associated with collecting samples. Nevertheless, the ability to study gene regulation in primate skeletal tissues is crucial for understanding how the primate skeleton has evolved. We therefore developed a comparative primate skeletal cell culture model that allows us to access a spectrum of human and chimpanzee cell types as they differentiate from stem cells into bone cells. While most gene expression patterns are conserved across species, we also identified hundreds of differentially expressed genes between humans and chimpanzees within and across stages of differentiation. We also classified cells by osteogenic stage and identified additional interspecific differentially expressed genes which may contribute to skeletal trait differences. We anticipate that this model will be extremely useful for exploring questions related to gene regulation variation in primate bone biology and development.

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Mir-30d Regulates Cardiac Remodeling by Intracellular and Paracrine Signaling

Circulation Research ◽

10.1161/circresaha.120.317244 ◽

2021 ◽

Vol 128 (1) ◽

Author(s):

Jin Li ◽

Ane M. Salvador ◽

Guoping Li ◽

Nedyalka Valkov ◽

Olivia Ziegler ◽

...

Keyword(s):

Extracellular Vesicles ◽

Ventricular Remodeling ◽

Left Ventricular Remodeling ◽

Chronic Phase ◽

Left Ventricular ◽

Model Systems ◽

Locked Nucleic Acid ◽

Loss Of Function ◽

Paracrine Signaling

Rationale: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. Objective: To investigate the mechanism of miR-30d–mediated cardioprotection in HF. Methods and Results: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid–based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. Conclusions: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle–contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.

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ARID1a as a marker of prognosis and increased sensitivity to CDK4/6, mTOR 1/2 and Src homology region 2 phosphatase (SHP 1/2) inhibitors in breast cancer (BC).

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.1082 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 1082-1082 ◽

Cited By ~ 1

Author(s):

Veronica Mariotti ◽

Howard L. McLeod ◽

Hatem Hussein Soliman

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Sensitivity Analyses ◽

Tissue Loss ◽

Loss Of Function ◽

Her2 Receptor ◽

Luminal A ◽

Genomic Databases ◽

Metastatic Tissue

1082 Background: ARID1a (AT Rich Interactive Domain 1A) is part of the SWI/SNF complex, which regulates gene transcription, and is believed to be a tumor suppressor gene. Low ARID1a expression has been associated with poor prognosis in BC. The aim of this study was to explore the clinical significance of ARID1a mutation and expression loss, and its potential as a therapeutic target in BC. Methods: We analyzed publicly available genomic databases to study the clinical implication of ARID1a mutations and gene expression in BC. Results: ARID1a was mutated in ~5-7 % of BCs within TCGA/METABRIC/MSK (5511 samples), but did not show differences in frequency between histology, grade, or estrogen receptor (ER)/HER2 receptor status. MSK metastatic tissue samples had higher incidence of ARID1a mutation compared to primary tumor samples (7.6% vs 4.4%, χ2 P = 0.0073). Analysis of ARID1a in KMPLOT showed that lower gene expression was associated with worse relapse-free survival and overall survival across all BCs, but the difference was primarily in molecularly classified luminal A tumors. Mutations in ARID1a did not show an association with outcomes in TCGA/METABRIC/MSK datasets. Pathway analysis of ARID1a showed it is involved in regulating ER ligand driven signaling and interacts with targets regulated by CDK4 and mTOR activity. CancerRxgene drug sensitivity analyses on BC cell lines revealed that ARID1a mutated BC cell lines were significantly more sensitive to palbociclib, SHP1/2, and mTOR1/2 inhibitors compared to ARID1a wildtype cell lines. Conclusions: Reduced activity of ARID1a in luminal BC cells may negatively affect prognosis by altering ER signaling leading to activation of druggable resistance mechanisms, particularly in metastatic tissue. Loss of function ARID1a mutations may sensitize cancer cells to CDK4/6, mTOR1/2, and SHP1/2 inhibitors in vitro. Further research in ARID1a mutated ER+ BCs using combinations of these inhibitors is warranted.

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Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.

Blood ◽

10.1182/blood.v106.11.4255.4255 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4255-4255

Author(s):

Ewa Carrier ◽

Shermila Kausal ◽

Anand S. Srivastava

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Growth Factors ◽

Es Cells ◽

Embryonic Stem ◽

Globin Genes ◽

Rt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.

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ASXL1 Mutations Promote Myeloid Transformation Through Inhibition of PRC2-Mediated Gene Repression

Blood ◽

10.1182/blood.v118.21.405.405 ◽

2011 ◽

Vol 118 (21) ◽

pp. 405-405 ◽

Cited By ~ 2

Author(s):

Omar Abdel-Wahab ◽

Mazhar Adli ◽

Lindsay Saunders ◽

Jie Gao ◽

Alan H. Shih ◽

...

Keyword(s):

Gene Expression ◽

Research Funding ◽

Hematopoietic Cells ◽

Loss Of Function ◽

Genome Wide ◽

Hoxa Gene Expression ◽

Primary Leukemia ◽

Hoxa Gene

Abstract Abstract 405 Somatic mutations in ASXL1 have been identified in patients with myeloid malignancies and are associated with worsened overall survival in AML and MDS patients. However the mechanisms of myeloid transformation of ASXL1 mutations had not been delineated. We therefore performed extensive in vitro and in vivo studies to assess the functional implications of ASXL1 mutations in the hematopoietic compartment. Transcriptional and Western blot analysis demonstrated loss of ASXL1 protein in primary leukemia samples with endogenous ASXL1 mutations indicating that these mutations are loss-of-function disease alleles. Further, ASXL1 depletion by shRNA in normal and malignant hematopoietic cells leads to robust upregulation of a set of genes including the posterior HOXA cluster (HoxA5-HoxA13). Increased HoxA gene expression was confirmed in human hematopoietic stem progenitor cells targeted with ASXL1 siRNA and in mice with conditional deletion of Asxl1 in the hematopoietic compartment. Previous studies in Drosophila had revealed that Asxl forms the polycomb-repressive deubiquitinase (PR-DUB) complex with BAP1, which normally opposes the function of polycomb repressive complex 1 (PRC1) by removing H2AK119 ubiquitination. We verified that wild-type, but not mutant ASXL1 associates with BAP1 in co-immunoprecipitation studies. However, BAP1 depletion in hematopoietic cells did not result in significant changes in HoxA gene expression, suggesting that ASXL1 regulates gene expression in hematopoietic cells independent of its role in the PR-DUB complex. We therefore performed CHIP sequencing for known activating and repressive chromatin marks and histone mass spectrometry to elucidate the genome-wide effects of ASXL1 loss on chromatin state in hematopoietic cells. This allowed us to show that ASXL1 loss resulted in genome-wide loss of the transcriptionally repressive mark H3K27me3 in hematopoietic cells and primary patient samples with ASXL1 mutations. These data were supported by western blot analysis and histone mass spectrometry demonstrating a significant loss of H3K27 trimethylation in ASXL1-mutant cells. Moreover, ASXL1 mutations in primary leukemia samples are characterized by loss of H3K27 trimethylation at the HoxA locus. These data led us to hypothesize that ASXL1 interacts with the PRC2 complex; co-immunoprecipitation studies revealed that ASXL1 associates with members of the PRC2 complex including EZH2 and SUZ12 but not with the PRC1 repressive complex. Importantly, ASXL1 downregulation resulted in loss of EZH2 recruitment to the HOXA locus indicating a role of ASXL1 in recruiting the PRC2 complex to known leukemogenic loci. We next assessed the effects of ASXL1 loss in vivo by generating a conditional knock-out model of ASXL1 and also by employing shRNA to deplete ASXL1 in hematopoietic cells expressing the NRASG12D oncogene. Consonant with the in vitro data, we observed HOXA9 overexpression with ASXL1 loss/depletion in vivo. Preliminary analysis reveals that conditional, hematopoietic specific ASXL1-knockout (ASXL1fl/fl Vav-Cre) mice are characterized by progressive expansion of LSK and myeloid progenitor cells in mice less than 6 months of age. After 6 months of age a significant proportion of ASXL1fl/fl Vav-Cre mice developed leukocytosis, anemia, thrombocytopenia, and splenomegaly; pathologic analysis of tissues revealed a phenotype consistent with myelodysplasia with myeloproliferative features. Moreover, loss of ASXL1 in cooperation with expression of NRasG12D resulted in impaired survival, increased myeloproliferation, and progressive anemia consistent with MPN/MDS in vivo. Taken together, these results reveal that ASXL1 mutations result in a loss-of-function and suggest a specific role for ASXL1 in epigenetic regulation of gene expression by facilitating PRC2-mediated transcriptional repression of known leukemic oncogenes. Moreover, our in vivo data validate the importance of ASXL1 mutations in the pathogenesis of myeloid malignancies and provide insight into how mutations that inhibit PRC2 function contribute to myeloid transformation through epigenetic dysregulation of specific target genes. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

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