Unknown,Transcriptomics,Genomics,Proteomics

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KLF1, KLF2 and c-myc control a regulatory network essential for embryonic erythropoiesis

ABSTRACT: The Krüppel-like factors, KLF1 and KLF2, positively regulate embryonic β-globin expression, and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1-/-KLF2-/- double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1-/- and KLF1-/-KLF2-/-. Among these, c-myc emerged as a central node in the most significant gene network. c-myc expression is synergistically regulated by KLF1 and KLF2, and both factors bind the c-myc promoters. To characterize the role of c-myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia analogous to KLF1-/-KLF2-/-. In the absence of c-myc, circulating erythroid cells do not show the normal increase in α- and β-like globin expression, but interestingly, have accelerated erythroid maturation, between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate c-myc, to control the primitive erythropoietic program. Timed-pregnant KLF1+/-, KLF1+/- KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 8 different wild-type, 3 KLF1-/-, 3 KLF1-/- KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Christopher Pang

PROVIDER: E-GEOD-36427 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression. Timed-pregnant KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron KLF2-/- frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 4 different KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays

Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO. The cells were further cultured in the presence of EPO, and formaldehyde crosslinked chromatin was isolated after cells differentiated into R3/R4 nucleated erythroid cells. Chromatin Immunoprecipitation followed by sequencing (chIP-seq) was performed using antibodies against GATA1, KLF1, NFE2, TAL1, p300, H3K4me2 and H3K4me3, along with a total input control. Raw data (fastq, SRA) is missing for the TAL1 chIP-seq dataset

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KLF1, KLF2 and c-myc control a regulatory network essential for embryonic erythropoiesis

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