Translational Isoforms of FOG1 Regulate GATA1-interacting Complexes

Jonathan W Snow,Stuart H Orkin

doi:10.1074/jbc.m109.043497

Abstract

Erythropoietic and megakaryocytic programs are directed by the transcription factor GATA1. Friend of GATA1 (FOG1), a protein interaction partner of GATA1, is critical for GATA1 function in multiple contexts. Previous work has shown that FOG1 recruits two multi-protein complexes, the nucleosome remodeling domain (NuRD) complex and a C-terminal binding protein (CTBP)-containing complex, into association with GATA1 to mediate activation and repression of target genes. To elucidate mechanisms that might differentially regulate the association of FOG1, as well as GATA1, with these two complexes, we characterized a previously unrecognized translational isoform of FOG1. We found that an N-terminally truncated version of FOG1 is produced from an internal ATG and that this isoform, designated FOG1S, lacks the nucleosome remodeling domain-binding domain, altering the complexes with which it interacts. Both isoforms interact with the C-terminal binding protein complex, which we show also contains lysine-specific demethylase 1 (LSD1). FOG1S is preferentially excluded from the nucleus by unknown mechanisms. These data reveal two novel mechanisms for the regulation of GATA1 interaction with FOG1-dependent protein complexes through the production of two translational isoforms with differential interaction profiles and independent nuclear localization controls.

Highlights

Leads to expression of a truncated form of GATA1 that is associated with acute megakaroblastic leukemia [9]
The interaction of Friend of GATA1 (FOG1) with these complexes may account in part for GATA-mediated gene repression, no clear mechanism has been put forward for how FOG1 contributes to GATA1-dependent gene activation
Expression of constructs containing wild type FOG1 ORF (WT) or mutant cDNA (Skoz and WKoz) in 293T cells revealed that alteration of the Kozak sequence had the predicted effect, such that the stronger Kozak resulted in reduced amount of FOG1S, whereas the weakened Kozak led to relatively more FOG1S (Fig. 4C)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Plasmids—Mouse erythroleukemia (MEL) and 293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. BirA-expressing MEL cells were electroporated with plasmid constructs containing FLAG-biotin-tagged wild type or mutant forms of FOG1. To generate constructs containing N-terminally FLAG-tagged FOG1L and FOG1S, forward primers containing a 5Ј BamHI site spanning the start codon of interest were used in conjunction with a common reverse primer to generate a BamHI/PflMI fragment that was cloned into the pEF1␣-FOG1 vector. Total lysates or nuclear extracts were prepared and incubated with anti-FOG1 antibody and with protein G-agarose beads (Roche Applied Science) or with anti-M2 FLAG directly conjugated to agarose beads (Sigma) overnight. Nuclear extracts from MEL cells expressing BirA and biotin-tagged FOG1L were incubated with anti-streptavidin-agarose or anti-M2 FLAG-agarose in a buffer containing 20 mM Tris-HCl, 139 mM KCl, 12 mM NaCl, and 20% glycerol, and 0.5% Nonidet P-40. Molecular mass standards were catalase (240 kDa), ferritin (438 kDa), and thyroglobulin (670 kDa)

RESULTS

MEL Cells

DISCUSSION