Abstract
We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.
Highlights
GATA-1 is the founding member of the GATA family of transcription factors that bind to (T/A)GATA(A/G) motifs in DNA via C4-type zinc fingers
We designed degenerate primers corresponding to several zinc fingers of the CCHC-type, murine transcription factor Friend-of-GATA-1 (mFOG-1) fingers 5, 6, 7, and 9, as we reasoned that these regions might be conserved in other FOG-like genes
We have identified a novel factor, hFOG-2, and have shown that it is related to mFOG-1, not just at the amino acid level but in its ability to associate with certain protein partners
Summary
Cell Lines and Growth Conditions—The human cell lines Meg-01, CHRF-288, and Dami/HEL were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), streptomycin, and penicillin. A fragment extending between the two RACE primers was generated by PCR, creating fog2race3–5 This fragment was 32P-labeled and used as a hFOG-2-specific probe. The cDNA fragments corresponding to the largest RACE products that hybridized to the fog2race probe were excised from the agarose gel and cloned into the plasmid pGEM-Teasy (Promega). A 32P-labeled hFOG-2-specific PCR fragment, fog2race, was used as a probe and hybridization was performed under high stringency conditions using ExpressHyb (CLONTECH). This fragment was used to probe two commercial filters, a multiple tissue Northern (CLONTECH) containing poly(A)ϩ RNA from 12 human tissues and a dot-blot array of 50 RNA samples isolated from human adult and embryonic tissues (CLONTECH). Allegro GH assay kits according to the manufacturer’s instructions
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