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Project description:The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.
| S-EPMC8347936 | biostudies-literature
Project description:A Positive Regulatory Feedback Loop Between EKLF/ KLF1 and TAL1/SCL Sustaining the Erythropoiesis
| PRJNA719026 | ENA
Project description:Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.
| S-EPMC7932694 | biostudies-literature
Project description:The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the cardiac valves and septa. Specification and differentiation of these two cell layers are among the earliest events in heart development, but the embryonic origins and genetic regulation of early endocardial development remain largely undefined. We have analyzed early endocardial development in the zebrafish using time-lapse confocal microscopy and show that the endocardium seems to originate from a region in the lateral plate mesoderm that will give rise to hematopoietic cells of the primitive myeloid lineage. Endocardial precursors appear to rapidly migrate to the site of heart tube formation, where they arrive prior to the bilateral myocardial primordia. Analysis of a newly discovered zebrafish Scl/Tal1 mutant showed an additional and previously undescribed role of this transcription factor during the development of the endocardium. In Scl/Tal1 mutant embryos, endocardial precursors are specified, but migration is severely defective and endocardial cells aggregate at the ventricular pole of the heart. We further show that the initial fusion of the bilateral myocardial precursor populations occurs independently of the endocardium and tal1 function. Our results suggest early separation of the two components of the primitive heart tube and imply Scl/Tal1 as an indispensable component of the molecular hierarchy that controls endocardium morphogenesis.
| S-EPMC1950955 | biostudies-literature
Project description:Purpose: We characterized the role of EKLF in erythroblast island macrophages in E13.5 mouse fetal liver by analysing the transcriptomes of F4/80+ macrophages from WT and EKLF-/- by RNA-Seq. In addition we analyzed the transcriptomes of F4/80+ fetal liver macrophages from an EKLF/GFP mouse, where GFP acts as a surrogate for EKLF expression, to determine the genes enriched in EKLF/GFP+ macrophages compared to EKLF/GFP- macrophages where EKLF is not expressed. Finally, we used single cell RNA sequencing to resolve the heterogeneous population of F4/80+ macrophages from WT. Methods: For RNA-Seq from WT and EKLF-/- F4/80+ macrophages were FACS sorted from primary E13.5 mouse fetal livers and RNA was isolated using the Trizol method. F4/80+ macrophages were also FACS sorted from the EKLF/GFP mouse and the GFP+ and GFP- populations were collected. The GFP+ population had low cell numbers and therefore RNA was isolated using an Agilent RNA Nanoprep kit. For single cell sequencing, E13.5 fetal livers were stained with F4/80-PE antibody and the F4/80-PE+ cells were purified using an EZ-Sep PE selection kit (mouse). 25,000 cells were submitted for single cell sequencing for the Chromium V3 platform. Library preparation was done using the standard Illumina Truseq workflow, and libraries were sequenced in a Hiseq 2500 for WT and EKLF-/-, or Novaseq for EKLF/GFP and single cell sequencing. Results: We found that 1954 genes are differentially expressed in EKLF-/- F4/80+ macrophages vs WT and 2330 genes are differentially enriched in EKLF/GFP+ F4/80+ macrophages using DESeq2. Of these, 504 are common and constitute the EKLF-dependent gene expression program in F4/80+ fetal liver macrophages. We resolved the F4/80+ WT macrophages into 13 clusters based on gene expression and find that 23% of the total F4/80+ cells express EKLF. Conclusions: We find that the F4/80+ fetal liver macrophage are a unique cell type. We identify the EKLF-dependent gene expression program in these macrophages and determine an important transcription circuit governed by EKLF that constitute cell cycle and other Klf and Sp family transcription factors. Single cell sequencing showed a highly heterogeneous population of macrophages with activated macrophage as well as erythro-myeloid characteristics. Based on the expression of EKLF, we identified specific cell surface markers for EKLF+ F4/80+ macrophages.
2021-03-16 | GSE156153 | GEO
Project description:Erythroid Krüppel-like factor (EKLF; KLF1) is an erythroid-specific transcription factor required for the transcription of genes that regulate erythropoiesis. In this paper, we describe the identification of a novel EKLF interactor, Ppm1b, a serine-threonine protein phosphatase that has been implicated in the attenuation of NFκB signaling and the regulation of Cdk9 phosphorylation status. We show that Ppm1b interacts with EKLF via its PEST1 sequence. However, its genetic regulatory role is complex. Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the β-globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity. Conversely, depletion of Ppm1b in CD34(+) cells leads to a higher level of endogenous β-globin gene activation after differentiation. We also observe that Ppm1b likely has an indirect role in regulating EKLF turnover via its zinc finger domain. Together, these studies show that Ppm1b plays a multilayered role in regulating the availability and optimal activity of the EKLF protein in erythroid cells.
| S-EPMC3346094 | biostudies-literature
Project description:Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.
| S-EPMC2742862 | biostudies-other
Project description:Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.
| S-EPMC3392972 | biostudies-literature
Project description:The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.
| S-EPMC4034424 | biostudies-literature
Project description:BackgroundHepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms. The newly defined epigenetics, epitranscriptomics regulation, has been reported to involve in multiple cancers including Hepatocellular Carcinoma (HCC). Our previous study found that HBx, HBV encodes X protein, mediated H3K4me3 modification in WDR5-dependent manner to involve in HBV infection and contribute to oncogene expression. AlkB Homolog 5 (ALKBH5), one of epitranscriptomics enzymes, has been identified to be associated with various cancers. However, whether and how ALKBH5 is dysregulated in HBV-related HCC remains unclear yet. This study aims to investigate ALKBH5 function, clinical significance and mechanism in HBV related HCC (HBV-HCC) patients derived from Chinese people.MethodsThe expression pattern of ALKBH5 was evaluated by RT-qPCR, Western blot, data mining and immunohistochemistry in total of 373 HBV-HCC tissues and four HCC cell lines. Cell Counting Kit 8 (CCK8) assay, Transwell and nude mouse model were performed to assess ALKBH5 function by both small interference RNAs and lentiviral particles. The regulation mechanism of ALKBH5 was determined in HBx and WDR5 knockdown cells by CHIP-qPCR. The role of ALKBH5 in HBx mRNA N6-methyladenosine (m6A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.ResultALKBH5 increased in tumor tissues and predicts a poor prognosis of HBV-HCC. Mechanically, the highly expressed ALKBH5 is induced by HBx-mediated H3K4me3 modification of ALKBH5 gene promoter in a WDR5-dependent manner after HBV infection. The increased ALKBH5 protein catalyzes the m6A demethylation of HBx mRNA, thus stabilizing and favoring a higher HBx expression level. Furthermore, there are positive correlations between HBx and ALKBH5 in HBV-HCC tissues, and depletion of ALKBH5 significantly inhibits HBV-driven tumor cells' growth and migration in vitro and in vivo.ConclusionsHBx-ALKBH5 may form a positive-feedback loop to involve in the HBV-induced liver carcinogenesis, and targeting the loop at ALKBH5 may provide a potential way for HBV-HCC treatment.
| S-EPMC8194239 | biostudies-literature