Supplementary Materialsba030551-suppl1. GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis. Visual Abstract Open in a separate window Introduction A complete understanding of hematopoietic transcriptional control requires that all functional blocks erythroid cell maturation and causes severe anemia in mice, leading to death by embryonic day 10.5 (e10.5) to e11.5.4,5 Enforced GATA1 expression reprograms alternate hematopoietic lineages into erythroid fates,6,7 and in combination with Tal1, Lmo2, and c-MYC, GATA1 directly converts fibroblasts into erythroid progenitor Rabbit polyclonal to AMPKalpha.AMPKA1 a protein kinase of the CAMKL family that plays a central role in regulating cellular and organismal energy balance in response to the balance between AMP/ATP, and intracellular Ca(2+) levels. cells.8 These findings collectively highlight the dominant role that GATA1 plays in orchestrating erythroid cell fate decision and differentiation. We previously performed a proteomic screen of GATA1-interacting proteins and identified the Krppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as a GATA1 binding partner.9 Tetraploid complementation of gene-trap embryonic stem (ES) cells and purchase OSI-420 chimeric mice showed that Zfp148 deficient cells have reduced contribution to definitive erythroid cells in vivo. Partial depletion of Zfp148 in primary human CD34+ (hCD34+) cells causes a mild impairment of erythroid maturation.10 The locus has been targeted previously in mice by disrupting exon 9, which removes 60% of the protein coding sequence but leaves a portion of the zinc finger DNA binding domain intact.11 This heterozygous allele was reported to cause defects in primordial germ cell development, and the allele was not able to be passed through the germline.11 A conditional knockout (cKO) mouse model targeting exons 8 and 9 deletion was subsequently generated,12,13 and Mx1-CreCmediated deletion in the hematopoietic system showed acute, but transient anemia and thrombocytopenia in adult mice.12 The transcription factor Zfp281 (also called ZBP-99) was originally discovered as a guanine cytosine (GC)-rich DNA sequence binding Krppel-like zinc finger protein that shares amino acid sequence purchase OSI-420 homology with Zfp148 and binds to similar DNA sequences in vitro.14 Zfp281 has been extensively studied in ES cells where it is highly expressed and physically interacts with key stem cell transcription factors, including Nanog, Oct4, and Sox2, to regulate pluripotency genes.15-19 Zfp281 is dispensable for the establishment and maintenance of ES cells, but required for purchase OSI-420 proper ES cell differentiation and embryo survival during the preimplantation blastocyst stage.20-22 Here, purchase OSI-420 we report a new cKO mouse model for Zfp148 that deletes 80% of the protein coding domains, including the entire DNA binding zinc finger region. Hematopoietic selective loss causes mild anemia and delayed recovery from phenylhydrazine-induced hemolysis. We provide evidence that Zfp281 plays an overlapping role with Zfp148 in erythropoiesis, accounting for the mild erythroid phenotype associated with Zfp148 loss alone. Methods Animal studies The locus spans 125 kb in mice and contains 9 exons (GRCm38, Ensembl). A targeting strategy, using purchase OSI-420 pFlexible vector,23 was designed to allow Cre-LoxPCmediated deletion of exons 6 and 7, which introduces a premature translation termination codon TGA in exon 8. Homologous recombination of the targeting construct within locus was achieved in CJ9 (129/Sv) ES cells. A validated ES cell clone was injected into C57BL/6 blastocysts to generate chimeric mice (supplemental Methods). Germline transmission of the loxP allele was confirmed in F1 mice. To obtain hematopoietic-specific deletion of Zfp148, the Zfp148fl/fl mice were crossbred with Vav1-Cre24 transgenic mice. To induce stress erythropoiesis, mice were injected intraperitoneally with 60 mg/kg of phenylhydrazine (Sigma) in sterile phosphate-buffered saline (pH 7.4) on 2 consecutive days, as previously described.25 Zfp148 germline knockout mice were generated by interbreeding with CD-1 mice expressing Cre under the control of the GATA-1 promoter (GATA-1 Cre).26 The resulting Zfp148 heterozygous knockout (Zfp148+/?) F1 hybrids were combined strains of 129/Sv, C57BL/6, and Compact disc-1. All mice had been backcrossed at least 5 to 6 decades with C57BL/6 mice. All experiments involving mice were authorized by the pet Use and Treatment Committee at Boston Childrens Hospital. Antibodies and reagents Era of rabbit polyclonal Zfp148 N14 antibody offers previously been referred to.9 Zfp148 N1-500 antibody was something special from Juanita Vendor (University of Michigan Medical College). Extra antibodies had been purchased from industrial sources (supplemental Strategies), and everything chemicals had been bought from Sigma-Aldrich, unless mentioned otherwise. Blood matters and movement cytometry Mouse peripheral bloodstream was gathered via retroorbital plexus and examined on the Hemavet HV950 multispecies.