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, 285 (36), 28064-75

Sumoylation Regulates Interaction of FOG1 With C-terminal-binding Protein (CTBP)

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Sumoylation Regulates Interaction of FOG1 With C-terminal-binding Protein (CTBP)

Jonathan W Snow et al. J Biol Chem.

Abstract

Erythropoietic and megakaryocytic programs are specified from multipotential progenitors by the transcription factor GATA1. FOG1, a GATA1-interaction partner, is critical for GATA1 function in several contexts by bringing multiple complexes into association with GATA1 to facilitate activation or repression of target genes. To further elucidate regulation of these associations by cellular and extracellular cues, we examined FOG1 for post-translational modifications. We found that FOG1 is SUMOylated and phosphorylated in erythroid cells in a differentiation-dependent manner. Removal of the SUMOylation sites in FOG1 does not impair nuclear localization, protein stability, or chromatin occupancy. However, SUMOylation of FOG1 modulates interactions with C-terminal binding protein family members, specifically promoting CTBP1 binding. Phosphorylation of FOG1 modulates SUMOylation and, therefore, indirectly regulates the CTBP interaction. Post-translational modification of FOG1 may contribute to control of co-occupancy by CTBP family members, the NuRD complex, and GATA1 at differentially regulated genes.

Figures

FIGURE 1.
FIGURE 1.
FOG1 is SUMOylated in vivo and in primary erythroid cells. A, two novel bands are illuminated by a FOG1 antibody in MEL cell nuclear extracts selectively in the presence of NEM. B, FLAG immunoprecipitation (IP) of a FLAG-tagged FOG1 stably expressed in MEL cells pulls down the two novel bands as detected by FOG1 antibody. C, novel bands are found in nuclear extracts of E14.5 fetal livers. D, FOG1 can be SUMOylated in vivo using a heterologous cell system. Constructs containing either a HA-hSUMO1 fusion protein or vector alone were co-expressed in 293T with a construct containing FLAG-Bio-tagged FOG1. After immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates were run on a Western blot with antibodies against FLAG or HA.
FIGURE 2.
FIGURE 2.
FOG1 is SUMOylated in multiple hematopoietic lineages in a differentiation-dependent manner and are predominantly nuclear. Nuclear and cytoplasmic extracts from MEL cells (A) and L8057 cells (B) were prepared. Equal amounts of protein were run on SDS-PAGE gels for Western blot analysis using anti-FOG1 antibody, and fraction purity was demonstrated using antibodies against MTA2 (nuclear (Nuc)) and Hsp90 (cytoplasmic (Cyt)). Nuclear extracts from G1E and G1ER cells were prepared (C). Equal amounts of protein were run on SDS-PAGE gels for Western blot analysis using antibodies directed toward FOG1, GATA1, GATA2, and MTA2.
FIGURE 3.
FIGURE 3.
FOG1 SUMOylation is regulated by SENP1 and GATA factors. Constructs containing either a GFP-SUMO1 fusion protein or a GFP-SUMO1 fusion in which a G97A mutation (to abrogate SUMO1 attachment) was engineered were co-expressed in 293T cells with a construct containing FLAG-Bio-tagged FOG1. After immunoprecipitation (IP) was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates were run for Western blot with anti-FLAG or anti-GFP (A). A construct containing a GFP-SUMO1 fusion protein was co-expressed with a construct containing FLAG-Bio-tagged FOG1 in the presence or absence of the FLAG-tagged SUMO-isopeptidase SENP1. After immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates were run for Western blot with anti-FLAG and anti-GFP (B). A construct containing a GFP-SUMO1 fusion protein was co-expressed with a construct containing HA-tagged FOG1 alone or with FLAG-tagged GATA1 or FLAG-tagged GATA2. After immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates were run for Western blot with anti-HA, anti-FLAG, and anti-GFP (C).
FIGURE 4.
FIGURE 4.
FOG1 is SUMOylated on lysines K469R and K507R. Constructs in which all six putative SUMOylated lysines were mutated to arginine in FOG1 cDNA were co-expressed along with a GFP-SUMO1 fusion protein. Immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, and input and immunoprecipitates (IP) were run for Western blot with anti-FOG1 or anti-GFP (A). After identification of lysines 469 and 507 as the SUMOylated lysines, a construct containing mutations in which both lysines were mutated to arginine was made and used in immunoprecipitation experiments along with constructs for wild-type FOG1 and both single lysine 469 and 507 single mutants. Western blot analysis using antibodies against FOG1 or GFP was performed for these FOG1 versions (B).
FIGURE 5.
FIGURE 5.
FOG1 SUMOylation is regulated by Phosphorylation. Phosphatase treatment of MEL nuclear extracts with calf intestinal phosphatase or λ-phosphatase results in faster migration of FOG1 in SDS-PAGE as detected by Western blot using anti-FOG1 antibody (A). Nuclear extracts from G1E and G1ER cells differentiated with β-estradiol for 24 h were treated with λ-phosphatase and blotted with an antibody directed against FOG1 (B). Cellular extracts from 293T cells transfected with a construct containing HA-tagged FOG1 alone or with FLAG-tagged GATA1 or FLAG-tagged GATA2 were treated with λ-phosphatase or mock-treated before being run for Western blot with anti-HA (C). We generated constructs with either a FLAG-tagged wild-type FOG1 (WT) or a FLAG-tagged mutant version of FOG (2D) in which three residues (two serine and one threonine) C-terminal to the second SUMOylated lysines were mutated to aspartic acid in a FOG1 cDNA and performed immunoprecipitation experiments as above. Western blot analysis using antibodies against FOG1 or GFP for these single mutants was performed (D). A schematic shows the location of SUMOylated lysines and phosphorylation sites relative to other structural features of FOG1 (E).
FIGURE 6.
FIGURE 6.
KR mutant FOG1 is not altered in cellular localization, stability, or chromatin occupancy. Nuclear extracts from MEL cells expressing either a wild-type FL-Bio-tagged version of FOG1 (WT), a FL-Bio-tagged version of FOG1 containing both the K469R and K507R mutations (KR), or no tagged FOG1 (MB), were immunoprecipitated using anti-FLAG antibody (M2) conjugated to agarose. These extracts were blotted with streptavidin-HRP (A). Whole cell lysates were prepared from MEL cells expressing either a wild-type FOG1 (WT) or a mutant FOG1 (KR) after 0 or 16 h of cycloheximide treatment. Tagged FOG1 protein levels were assessed by blotting with Streptavidin-HRP (B). Nuclear (Nuc) and cytoplasmic (Cyt) extracts were prepared from MEL cells expressing either a wild-type FOG1 (WT) or a mutant FOG1 (KR), and tagged FOG1 protein levels were assessed by blotting with Streptavidin-HRP (C). Chromatin occupancy by FOG1 at select genes using biotin ChIP of wild-type (WT) and KR mutant FOG1 (KR) tagged FOG1 relative to cells expressing no tagged FOG1 (MB). Quantitative PCR of select genes (Zfpm1, Hba-a1, and Car2, with Sox2 as the negative control) in undifferentiated MEL cells (D).
FIGURE 7.
FIGURE 7.
SUMOylation of FOG1 alters its interaction with CTBP family members. A construct containing a GFP-SUMO1 fusion protein was co-expressed with constructs containing FOG1, either wild type (WT), or a version containing both the K469R and K507R mutations (known as KR), GATA1, CTBP1, and CTBP2. After immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates (IP) were run for Western blot with antibodies to FOG1 and interaction partners MTA2 and GATA1 (A). Western blot analysis was performed for the interaction partners CTBP1 and CTBP2, first using an anti-FLAG antibody recognizing both members and then using antibodies specifically recognizing CTBP1 and CTBP2 (B). RNA was purified from cells sorted from different stages of differentiation and made into cDNA for quantitative PCR analysis of Gata2, c-kit, Gata1, Hba-a1, Fog1, Ctbp family members, Lsd1, and Senp1 and Senp2 (expression relative to β-actin levels) (C). A reporter construct consisting of 1992 bp of the murine c-mpl promoter linked to the firefly luciferase cDNA was cotransfected with an empty expression plasmid, a plasmid expressing FLAG-GATA-1, or plasmids expressing FLAG-GATA-1 with plasmids expressing wild-type HA-FOG-1 (WT) or KR mutant HA-FOG-1 (KR) as indicated. The positions of various binding sites relative to the natural transcriptional start site are indicated in the diagram of the reporter construct. Luciferase activity (RLU) was measured for triplicate samples after 24 h and is indicated on the y axis as the average ±S.E. D, shown is a model depicting increased post-translational modification of FOG1 during erythroid development and the effects on its CTBP binding (E).

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