scholarly journals Identification of a novel enhancer of CEBPE essential for granulocytic differentiation

Blood ◽  
2019 ◽  
Vol 133 (23) ◽  
pp. 2507-2517 ◽  
Author(s):  
Pavithra Shyamsunder ◽  
Mahalakshmi Shanmugasundaram ◽  
Anand Mayakonda ◽  
Pushkar Dakle ◽  
Weoi Woon Teoh ◽  
...  
Keyword(s):  
Target Genes ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Open Chromatin ◽  
Sequencing Data ◽  
Granulocytic Differentiation ◽  
Circular Chromosome ◽  
Chromosome Conformation ◽  
Guide Rna

Abstract CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe. In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.

scholarly journals Evidence for a Bigenic Chromatin Subdomain in Regulation of the Fetal-to-Adult Hemoglobin Switch

2008 ◽  
Vol 29 (6) ◽  
pp. 1635-1648 ◽  
Author(s):  
Hugues Beauchemin ◽  
Marie Trudel
Keyword(s):  
Transcriptional Activation ◽  
Gene Activation ◽  
Dna Demethylation ◽  
Open Chromatin ◽  
Chromosome Conformation ◽  
Hemoglobin Switching ◽  
Switching Patterns ◽  
Adult Hemoglobin ◽  
Globin Promoter

ABSTRACT During development, human β-globin locus regulation undergoes two critical switches, the embryonic-to-fetal and fetal-to-adult hemoglobin switches. To define the role of the fetal Aγ-globin promoter in switching, human β-globin-YAC transgenic mice were produced with the Aγ-globin promoter replaced by the erythroid porphobilinogen deaminase (PBGD) promoter (PBGDAγ-YAC). Activation of the stage-independent PBGDAγ-globin strikingly stimulated native Gγ-globin expression at the fetal and adult stages, identifying a fetal gene pair or bigenic cooperative mechanism. This impaired fetal silencing severely suppressed both δ- and β-globin expression in PBGDAγ-YAC mice from fetal to neonatal stages and altered kinetics and delayed switching of adult β-globin. This regulation evokes the two human globin switching patterns in the mouse. Both patterns of DNA demethylation and chromatin immunoprecipitation analysis correlated with gene activation and open chromatin. Locus control region (LCR) interactions detected by chromosome conformation capture revealed distinct spatial fetal and adult LCR bigenic subdomains. Since both intact fetal promoters are critical regulators of fetal silencing at the adult stage, we concluded that fetal genes are controlled as a bigenic subdomain rather than a gene-autonomous mechanism. Our study also provides evidence for LCR complex interaction with spatial fetal or adult bigenic functional subdomains as a niche for transcriptional activation and hemoglobin switching.

scholarly journals Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

2020 ◽  
Author(s):  
Fuhui Han ◽  
Jing Li ◽  
Ranran Zhao ◽  
Lirong Liu ◽  
Lanlan Li ◽  
...  
Keyword(s):  
Target Genes ◽  
Structural Characteristics ◽  
Intramuscular Fat ◽  
Differentially Expressed ◽  
Lipid Deposition ◽  
Sequencing Data ◽  
Dual Purpose ◽  
Intramuscular Lipid ◽  
Non Coding Rnas

Abstract Background: Intramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China's most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12). Results: We identified a total of 26,247 genes and 6,935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.Conclusions: Our study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

scholarly journals Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

2020 ◽  
Author(s):  
Fuhui Han ◽  
Jing Li ◽  
Nan Liu ◽  
Ranran Zhao ◽  
Lirong Liu ◽  
...  
Keyword(s):  
Target Genes ◽  
Structural Characteristics ◽  
Intramuscular Fat ◽  
Differentially Expressed ◽  
Lipid Deposition ◽  
Sequencing Data ◽  
Dual Purpose ◽  
Intramuscular Lipid ◽  
Non Coding Rnas

Abstract Background: Intramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China's most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12). Results: We identified a total of 26,247 genes and 6,935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.Conclusions: Our study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

scholarly journals Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Fuhui Han ◽  
Jing Li ◽  
Ranran Zhao ◽  
Lirong Liu ◽  
Lanlan Li ◽  
...  
Keyword(s):  
Target Genes ◽  
Structural Characteristics ◽  
Intramuscular Fat ◽  
Differentially Expressed ◽  
Lipid Deposition ◽  
Sequencing Data ◽  
Dual Purpose ◽  
Intramuscular Lipid ◽  
Non Coding Rnas

Abstract Background Intramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China’s most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12). Results We identified a total of 26,247 genes and 6935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR. Conclusions Our study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

scholarly journals KMT2B and Neuronal Transdifferentiation: Bridging Basic Chromatin Mechanisms to Disease Actionability

2020 ◽  
Vol 15 ◽  
pp. 263310552092806
Author(s):  
Giulia Barbagiovanni ◽  
Michele Gabriele ◽  
Giuseppe Testa
Keyword(s):  
Target Genes ◽  
Direct Conversion ◽  
Open Chromatin ◽  
Cell Fates ◽  
Embryonic Fibroblasts ◽  
Neural Fate ◽  
Physiological Processes ◽  
Histone H3k4 ◽  
Bona Fide

The role of bona fide epigenetic regulators in the process of neuronal transdifferentiation was until recently largely uncharacterized, despite their key role in the physiological processes of neural fate acquisition and maintenance. In this commentary, we describe the main findings of our recent paper “KMT2B is selectively required for neuronal transdifferentiation, and its loss exposes dystonia candidate genes,” where we investigated the role of this histone H3K4 methyltransferase during mouse embryonic fibroblasts (MEFs) to induced neuronal cells (iNs) direct conversion. Indeed, Kmt2b–/– MEFs, transduced with three neuronal-specific transcription factors (TFs), Brn2, Ascl1, and Myt1l, show lower transdifferentiation efficiency, defective iN maturation, and augmented alternative cell fates acquisition, with respect to controls. Here, we went beyond the data, hypothesizing how KMT2B executes its fundamental role. In particular, we supposed that MYT1L, which has been proven to be fundamental for iN maturation and the switch-off of alternative cell fates, directly or indirectly needs KMT2B. Indeed, KMT2B could be important both to make MYT1L-target genes accessible, because MYT1L is not a pioneer TF and preferentially binds to open chromatin, and to activate MYT1L-downstream genes.

scholarly journals Chromosomal Translocations in NK-Cell Lymphomas Originate from Inter-Chromosomal Contacts of Active rDNA Clusters Possessing Hot Spots of DSBs

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3889
Author(s):  
Nickolai A. Tchurikov ◽  
Leonid A. Uroshlev ◽  
Elena S. Klushevskaya ◽  
Ildar R. Alembekov ◽  
Maria A. Lagarkova ◽  
...  
Keyword(s):  
T Cells ◽  
Hot Spots ◽  
Nk Cell ◽  
Intergenic Spacer ◽  
Whole Genome Sequence ◽  
Open Chromatin ◽  
Dna Double Strand Breaks ◽  
Circular Chromosome ◽  
Chromosome Conformation

Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer. There are nine hot spots of DSBs (denoted Pleiades) in human rDNA units that are located exclusively inside the intergenic spacer (IGS). Profiles of Pleiades coincide with the profiles of γ-H2AX, suggesting a high level of in vivo breakage inside rDNA genes. The data were confirmed by microscopic observation of the largest γ-H2AX foci inside nucleoli in interphase chromosomes. Circular chromosome conformation capture (4C) data indicate that the rDNA units often make contact with a specific set of chromosomal regions containing genes that are involved in differentiation and cancer. Interestingly, these regions also often possess hot spots of DSBs that provide the potential for Robertsonian and oncogenic translocations. In this study, we searched for translocations in which rDNA clusters are involved. The whole genome sequence (WGS) data of normal T cells and NK-cell lymphomas from the same individuals revealed numerous translocations in which Pleiades were involved. The sites of these translocations in normal T cells and in the lymphomas were mostly different, although there were also some common sites. The genes at translocations in normal cells and in lymphomas are associated with predominantly non-overlapping lists of genes that are depleted with silenced genes. Our data indicate that rDNA-mediated translocations occur at about the same frequency in the normal T cells and NK-lymphoma cells but differ at particular sites that correspond to open chromatin. We conclude that oncogenic translocations lead to dysregulation of a specific set of genes controlling development. In normal T cells and in NK cells, there are hot spots of translocations at sites possessing strong H3K27ac marks. The data indicate that Pleiades are involved in rDNA-mediated translocation.

scholarly journals Identification and co-expression analysis of long noncoding RNAs and mRNAs involved in the deposition of intramuscular fat in Aohan fine-wool sheep

2020 ◽  
Author(s):  
Fuhui Han ◽  
Jing Li ◽  
Nan Liu ◽  
Ranran Zhao ◽  
Lirong Liu ◽  
...  
Keyword(s):  
Target Genes ◽  
Structural Characteristics ◽  
Intramuscular Fat ◽  
Differentially Expressed ◽  
Future Research ◽  
Lipid Deposition ◽  
Sequencing Data ◽  
Intramuscular Lipid ◽  
Non Coding Rnas

Abstract Background: Intramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China's most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12). Results: We identified a total of 26,247 genes and 6,935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. Three pathways were significantly enriched, namely tight junction, glycosaminoglycan biosynthesis-heparan sulfate/heparin and lysosome. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed an lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4,MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1,MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR. Conclusions: Our study provided a list of the lncRNAs and mRNAs related to intramuscular lipid deposition in sheep and laid a foundation for future research on regulatory mechanisms.

scholarly journals Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sandra Heller ◽  
Zhijian Li ◽  
Qiong Lin ◽  
Ryan Geusz ◽  
Markus Breunig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Model Systems ◽  
Open Chromatin ◽  
Sequencing Data ◽  
Pancreatic Development ◽  
Pancreatic Differentiation

AbstractCell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

scholarly journals Corticosterone-mediated regulation and functions of miR-218-5p in rat brain

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuta Yoshino ◽  
Bhaskar Roy ◽  
Yogesh Dwivedi
Keyword(s):  
Rat Brain ◽  
Chronic Stress ◽  
Promoter Region ◽  
Noncoding Rna ◽  
Target Genes ◽  
Present Report ◽  
Chromatin Modification ◽  
Sequencing Data ◽  
Regulatory Pathways

AbstractChronic stress is one of the key precipitating factors in major depressive disorder (MDD). Stress associated studies have underscored the mechanistic role of epigenetic master players like microRNAs (miRNAs) in depression pathophysiology at both preclinical and clinical levels. Previously, we had reported changes in miR-218-5p expression in response to corticosterone (CORT) induced chronic stress. MiR-218-5p was one of the most significantly induced miRNAs in the prefrontal cortex (PFC) of rats under chronic stress. In the present report, we have investigated how chronic CORT exposure mechanistically affected miR-218-5p expression in the rat brain and how miR-218 could trigger molecular changes on its downstream regulatory pathways. Elevated expression of miR-218-5p was found in the PFC of CORT-treated rats. A glucocorticoid receptor (GR) targeted Chromatin-Immunoprecipitation (ChIP) assay revealed high GR occupancy on the promoter region of Slit3 gene hosting miR-218-2 in its 3rd intron. RNA-sequencing data based on RNA Induced silencing Complex Immunoprecipitation (RISC-IP) with AGO2 in SH-SY5Y cells detected six consistent target genes of miR-218-5p (APOL4, DTWD1, BNIP1, METTL22, SNAPC1, and HDAC6). The expression of all five genes, except APOL4, was successfully validated with qPCR in CORT-treated rat PFC. Further, Hdac6-based ChIP-seq experiment helped in mapping major genomic loci enriched for intergenic regions in the PFC of CORT-treated rat. A proximity-based gene ontology (GO) analysis revealed a majority of the intergenic sites to be part of key genes implicated in central nervous system functions, notably synapse organization, neuron projection morphogenesis, and axonogenesis. Our results suggest that the upregulation of miR-218-5p in PFC of CORT-treated rats possibly resulted from GR biding in the promoter region of Slit3 gene. Interestingly, Hdac6 was one of the consistent target genes potentially found to regulate CNS related genes by chromatin modification. Collectively, these findings establish the role of miR-218-5p in chronic stress and the epigenetic function it plays to induce chromatin-based transcriptional changes of several CNS genes in triggering stress-induced disorders, including depression. This also opens up the scope to understand the role of miR-218-5p as a potential target for noncoding RNA therapeutics in clinical depression.

scholarly journals Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3834-3834
Author(s):  
Pavithra Shyamsunder ◽  
Mahalakshmi Shanmugasundaram ◽  
Anand Mayakonda ◽  
Weoi Woon Teoh ◽  
Lin Han ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Progenitor Cells ◽  
Core Promoter ◽  
Upstream Region ◽  
Open Chromatin ◽  
Granulocytic Differentiation ◽  
Specific Expression ◽  
Granule Formation ◽  
Granule Proteins ◽  
Secondary Granule

Abstract CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins. Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE. Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp). To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice. In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

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