Abstract
Zfp206 (recently renamed Zscan10) encodes a zinc finger transcription factor specifically expressed in human and mouse embryonic stem cells (ESC). It has been shown that Zfp206 is required to maintain ESC in an undifferentiated, pluripotent state. Presented here are data showing that Zfp206 works together with two other transcription factors, Oct4 and Sox2, which are also essential regulators of ESC pluripotency. We show that Zfp206 binds to the Oct4 promoter and directly regulates Oct4 expression. Genome-wide mapping of Zfp206-binding sites in ESC identifies more than 3000 target genes, many of which encode transcription factors that are also targeted for regulation by Oct4 and Sox2. In addition, we show that Zfp206 physically interacts with both Oct4 and Sox2. These data demonstrate that Zfp206 is a key component of the core transcriptional regulatory network and together with Oct4 and Sox2 regulates differentiation of ESC.
Highlights
Pluripotency, the potential to give rise to all lineages of the developing embryo, is a unique and defining characteristic of mammalian embryonic stem cells (ESC).2 Pluripotent ESC, like the inner cell mass of the embryo from which they were derived, exist in a developmental state that is poised to respond to extracellular signals that specify unique patterns of cellular differentiation
Thousands of direct target genes regulated by Oct4 and Sox2 have been identified through comprehensive, genome-wide chromatin immunoprecipitation studies [5, 6]
Zfp206 was implicated as a pluripotency factor because it was found highly expressed in undifferentiated ESC and the inner cell mass of the preimplantation embryo, but not in differentiated ESC or trophectoderm
Summary
ESC Culture—E14 cells, a mouse ESC cell line (American Type Culture Collection), were cultured under feeder-free conditions in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum (ESC-qualified; Invitrogen), 0.055 mM -mercaptoethanol (Invitrogen), 2 mM L-glutamine, 0.1 mM nonessential amino acid, 5000 units/ml penicillin/streptomycin, and 1000 units/ml of leukemia inhibitory factor (Chemicon) and maintained at 37 °C with 5% CO2. Commercial ChIP-on-chip promoter microarrays (Agilent Technologies, Palo Alto, CA), which tiled the genomic regions from 5.5 kb upstream to 2.5 kb downstream of the transcription start site of 17,000 annotated mouse genes, were used to interrogate genome-wide occupancy of Zfp206 from two independent biological replicates. DNA was fluorophore-labeled using an Invitrogen CGH labeling kit (ChIP samples with Cy5; whole cell extract with Cy3). Min at room temperature to cross-link transcription factors to Luciferase Reporter Assays—The promoter regions, includchromatin for each ChIP experiment. Sonicated chromatin Meis, Meis, mir124a1, mir124a2, Jarid1c-truncated, and extracts containing DNA fragments with an average size of 500 mir-124-a2-truncated were cloned into pGL4-basic vector bp were immunoprecipitated using 10 g of Zfp206 antibody (Promega), and the Klf intron region was cloned into the [13], rabbit IgG (Santa Cruz Biotechnology), or V5 control pGL4.23-minimal promoter vector (Promega). Enhanced expression of Oct4 [15]
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