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.

IL-3 Inhibits Terminal Granulocytic Differentiation by Inducing the Binding of Stabilizing Proteins to a 300 Base Pair Region within p21cip1/waf1 Messenger RNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3152-3152
Author(s):  
Louis Ghanem ◽  
Richard A. Steinman
Keyword(s):  
Progenitor Cells ◽  
Adaptor Protein ◽  
Proteinase 3 ◽  
Granulocytic Differentiation ◽  
Induced Differentiation ◽  
Aberrant Expression ◽  
P21 Protein ◽  
Mrna Stabilization ◽  
Granule Proteins ◽  
Primary Granule

Abstract Elevated levels of the molecular adaptor protein p21cip1/waf1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demo−nstrated that p21 undergoes dynamic changes in expression levels and subcellular compartmentalization during cytokine-induced granulocytic differentiation, suggesting that p21 may play an important role in myeloid development. Based on our observation that p21 protein levels decrease during granulocytic differentiation of CD34+ human progenitor cells, we hypothesized that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 is required to maintain the undifferentiated state in murine 32Dcl3 cells and has been shown to slow the kinetics of differentiation of normal human myeloid progenitors (Hevehan DL, 2000). Given that 32Dcl3 myeloblasts express high basal levels of p21, we also hypothesized that IL-3 inhibition of 32Dcl3 differentiation is mediated in part by p21. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3 that are normally not abundant in cells maintained as myeloblasts by IL-3. Rescue of p21 knockdown myeloblasts with human p21 suppressed aberrant expression of granule proteins. The upregulation of myeloperoxidase and proteinase-3 occurred at a posttranscriptional level. These findings indicated that p21 prevented premature expression of primary granule proteins and may contribute to maintenance of the myeloblast phenotype. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF, indicating that p21 antagonized the entire differentiation process rather than only suppressing primary granule proteins. We then determined how IL-3 maintains p21 expression in myeloblast cells. We demonstrated that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3′ untranslated region (UTR) of the p21 transcript. IL-3 also rescued the decrease in p21 mRNA stability noted during G-CSF-induced differentiation. This has been shown to coincide with differentiation blockade. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3′ UTR and are regulated by IL-3 and G-CSF signaling. Association of a ~60–65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a ~40–42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.

scholarly journals Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2725-2725
Author(s):  
Lawrence J Druhan ◽  
Shimena Li ◽  
Sarah A Baxter ◽  
Amanda Lance ◽  
Andrea E Price ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Hematopoietic Progenitor Cells ◽  
Human Neutrophils ◽  
Neutrophil Granulocyte ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Granule Proteins

Abstract Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported Disclosures No relevant conflicts of interest to declare.

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 Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site.

1991 ◽  
Vol 11 (12) ◽  
pp. 6116-6127 ◽  
Author(s):  
K Ravid ◽  
T Doi ◽  
D L Beeler ◽  
D J Kuter ◽  
R D Rosenberg
Keyword(s):  
Tissue Specificity ◽  
Bone Marrow Cells ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Human Growth ◽  
Platelet Factor 4 ◽  
Upstream Region ◽  
Growth Hormone Gene ◽  
Specific Expression ◽  
Platelet Factor

We used various segments of the 5' upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from -97 to the transcriptional start site and an enhancer/silencer domain from -448 to -112. The core promoter contains a GATA site at -31 to -28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from -97 to -83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from -380 to -362, -270 to -257, and -137 to -120 as well as a negatively acting subdomain at -184 to -151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from -380 to -362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocytic lineage. The subdomains from -270 to -257 and -137 to -120 function together with the subdomain from -380 to -362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from -380 to -362 or -137 to -120 reduce transcription in megakaryocytes by 10- to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.

scholarly journals Lhx2 regulates temporal changes in chromatin accessibility and transcription factor binding in retinal progenitor cells

2017 ◽  
Author(s):  
Cristina Zibetti ◽  
Sheng Liu ◽  
Jun Wan ◽  
Jiang Qian ◽  
Seth Blackshaw
Keyword(s):  
Transcription Factors ◽  
Progenitor Cells ◽  
Late Stage ◽  
Chromatin Accessibility ◽  
Developmental Competence ◽  
Open Chromatin ◽  
Retinal Progenitor Cells ◽  
Retinal Progenitor ◽  
Target Sites ◽  
Retinal Neurogenesis

AbstractRetinal progenitor cells (RPCs) pass through multiple stages of developmental competence, where they successively acquire and lose the ability to generate individual cell subtypes. To identify the transcriptional regulatory networks that control these transitions, we conducted epigenomic and transcriptomic profiling of early and late-stage RPCs and observed a developmentally dynamic landscape of chromatin accessibility. Open chromatin regions that showed stage-specificity, as well as those shared by early and late-stage RPCs, were selectively targeted by the homeodomain factor Lhx2, which is expressed throughout retinal neurogenesis but also regulates many stage-specific processes in RPCs. Stage-specific Lhx2 binding sites were frequently associated with target sites for transcription factors that are preferentially expressed in either early or late-stage RPCs, and which were predicted to possess pioneer activity. Lhx2 loss of function in RPCs led to a loss of chromatin accessibility at both direct Lhx2 target sites and more broadly across the genome, as well as a loss of binding by transcription factors associated with stage-specific Lhx2 target sites. These findings demonstrate a central role for Lhx2 in control of chromatin accessibility in RPCs, and identify transcription factors that may guide stage-specific target site selection by Lhx2.SummaryLhx2 is a central regulator of chromatin accessibility in retinal progenitor cells, and interacts with stage-specific transcription factors to regulate genes that are dynamically expressed during retinal neurogenesis.

Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site

1991 ◽  
Vol 11 (12) ◽  
pp. 6116-6127
Author(s):  
K Ravid ◽  
T Doi ◽  
D L Beeler ◽  
D J Kuter ◽  
R D Rosenberg
Keyword(s):  
Tissue Specificity ◽  
Bone Marrow Cells ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Human Growth ◽  
Platelet Factor 4 ◽  
Upstream Region ◽  
Growth Hormone Gene ◽  
Specific Expression ◽  
Platelet Factor

We used various segments of the 5' upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from -97 to the transcriptional start site and an enhancer/silencer domain from -448 to -112. The core promoter contains a GATA site at -31 to -28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from -97 to -83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from -380 to -362, -270 to -257, and -137 to -120 as well as a negatively acting subdomain at -184 to -151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from -380 to -362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocytic lineage. The subdomains from -270 to -257 and -137 to -120 function together with the subdomain from -380 to -362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from -380 to -362 or -137 to -120 reduce transcription in megakaryocytes by 10- to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.

scholarly journals Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Lago ◽  
Matteo Nadai ◽  
Filippo M. Cernilogar ◽  
Maryam Kazerani ◽  
Helena Domíniguez Moreno ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Specific Gene ◽  
Open Chromatin ◽  
Rna Seq ◽  
Transcript Levels ◽  
Promoter Sequences ◽  
G Quadruplex ◽  
Cell Type Specific ◽  
Folding State

AbstractCell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.

scholarly journals The core promoter region of the tumor necrosis factor alpha gene confers phorbol ester responsiveness to upstream transcriptional activators.

1992 ◽  
Vol 12 (3) ◽  
pp. 1352-1356 ◽  
Author(s):  
D C Leitman ◽  
E R Mackow ◽  
T Williams ◽  
J D Baxter ◽  
B L West
Keyword(s):  
Transcription Factors ◽  
Tumor Necrosis Factor ◽  
Promoter Region ◽  
Tumor Necrosis ◽  
Core Promoter ◽  
Core Promoter Region ◽  
Necrosis Factor Alpha ◽  
The Core ◽  
Factor Alpha ◽  
Necrosis Factor

Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways.

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