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, 284 (43), 29310-9

Translational Isoforms of FOG1 Regulate GATA1-interacting Complexes

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Translational Isoforms of FOG1 Regulate GATA1-interacting Complexes

Jonathan W Snow et al. J Biol Chem.

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.

Figures

FIGURE 1.
FIGURE 1.
FOG1 exists as two isoforms in an erythroid cell line and in primary erythroid cells. A, two bands are recognized by α-FOG1 antibody in MEL cell nuclear extracts: the canonical FOG1 and a faster migrating form, delineated as FOG1L and FOG1S, respectively. B, both bands are found in nuclear extracts of embryonic day 14.5 fetal livers and in the spleen of phenylhydrazine-treated mice.
FIGURE 2.
FIGURE 2.
FOG1S lacks an N-terminal domain, and both isoforms are produced from a single transcript. A, schematic showing location of epitopes recognized by various α-FOG1 antibodies relative to other structural features of FOG1. B, isoforms recognized by these antibodies in MEL cell nuclear extracts. C, antibodies are used to illuminate FOG1 exogenously expressed in 293T cells transfected with FOG1 cDNA or vector alone.
FIGURE 3.
FIGURE 3.
FOG1S is translated from the second in-frame ATG. A, table showing the first four in-frame ATGs from the mouse FOG1 ORF, along with Exon location, predicted molecular mass of the product, Kozak sequence and strength, and mouse and human conservation. B, vectors expressing the wild type FOG1 ORF (WT) or truncations of FOG1 starting from the second (ATG2), third (ATG3), or fourth (ATG4) internal ATG were transfected into 293T cells and Western blotted with α-FOG1 (A-20) antibody. C, vectors expressing wild type FOG1 (WT) or FOG1 mutated at the first ATG to ACG (ATG1M) were transfected into 293T cells and Western blotted with FOG1 (A-20) antibody. D, vectors expressing wild type FOG1 ORF (WT) or FOG1 mutated at the second ATG (ATG2M) were transfected into 293T cells and Western blotted with α-FOG1 (A-20) antibody. E, vectors expressing wild type FOG1 ORF (WT) or FOG1 mutated at the second ATG (ATG2M), third ATG (ATG3M), or both the second and third ATG (ATG2/3M) were transfected into 293T cells and Western blotted with α-FOG1 (A-20) antibody.
FIGURE 4.
FIGURE 4.
Conserved 5′-UTR and Kozak sequence modulate isoform production. A, sequence alignments of the human (h) and mouse (m) 5′-UTR from the FOG1 cDNA. The conserved upstream ORF sequence in the 5′-UTR is underlined. B, vectors expressing wild type FOG1 cDNA or FOG1 constructs in which the 5′-UTR, 3′-UTR, or both (5′3′-UTR) were replaced by the generic UTR found in the pEF1α vector were transfected into 293T cells and Western blotted with α-FOG1 (A-20) antibody. C, vectors expressing wild type FOG1 cDNA or FOG1 cDNA with the Kozak sequence mutated from GGAGACATGTCC (WT) to a stronger (GGAGACATGgCC) (SKoz) or a weaker version (tGAtACATGTCC) (WKoz) were transfected into 293T cells and Western blotted with α-FOG1 (A-20) antibody.
FIGURE 5.
FIGURE 5.
FOG1L and FOG1S form distinct complexes with known FOG1 interaction partners. A, native complexes were fractionated from MEL cell nuclear extracts using a Sephacryl S400 26/60 column (every fourth fraction of 1-ml fractions is shown on the blot). Larger to smaller molecular mass fractions are shown from left to right (fractions 48, 68, and 84 correspond to sizes of ∼670, 438, and 240 kDa, respectively). B, constructs containing either FOG1L or FOG1S with an N-terminal FLAG-Bio tag were transfected into 293T cells. After immunoprecipitation (IP) with the α-FOG1 antibody recognizing the C terminus (A-20), input and immunoprecipitates were run for Western blot with the FOG1 antibody specific for the N terminus (M-20), followed by blotting with anti-FLAG (FOG1L and FOG1S). C, constructs containing either the tagged FOG1L or FOG1S were transfected into 293T cells. After immunoprecipitation with α-FOG1 antibody, input and immunoprecipitates were run for Western blot with antibodies directed to FLAG (FOG1L and FOG1S) and MTA2. D, constructs containing either the tagged FOG1L or FOG1S were cotransfected into 293T cells with a construct expressing GATA1. After immunoprecipitation with α-FOG1 antibody, input and immunoprecipitates were run for Western blot with antibodies to FLAG (FOG1L and FOG1S) and GATA1.
FIGURE 6.
FIGURE 6.
FOG1 interacts with LSD1-containing CTBP complex. A, table showing number of peptides recovered by tandem affinity purification using FLAG-Bio-tagged WT or N-truncated (N67) FOG1. B, coimmunoprecipitation (IP) of endogenous LSD1 from MEL cells expressing FLAG-Bio-Tagged FOG1 immunoprecipitated using α-FLAG-agarose. C, coimmunoprecipitation of LSD1 and CoREST by FOG1 using antibody directed to FOG1 or normal goat Ig, after cotransfection of 293Ts with constructs expressing HA-tagged FOG1, V5-tagged LSD1, and FLAG-tagged CoREST. D, constructs containing either FOG1L or FOG1S with an N-terminal FLAG-Bio tagged FOG1 were cotransfected with constructs expressing V5-tagged LSD1, FLAG-tagged CTBP1, and FLAG-tagged CTBP2. After immunoprecipitation was performed on whole cell lysates with a FOG1 antibody, input and immunoprecipitates were run for Western blot with an antibodies to FLAG (FOG1L or FOG1S, and both CTBP family members) and V5 (LSD1).
FIGURE 7.
FIGURE 7.
FOG1 isoforms possess differential nuclear localization patterns. A and B, nuclear (Nuc) and cytoplasmic (Cyt) extracts from MEL cells (A) and primary fetal liver cells (B) were prepared. Equal amounts of protein were run on SDS-PAGE gels for Western blot analysis antibody against FOG1, as well as antibodies to MTA2 and Hsp90 to demonstrate purity of cellular fractions. C, model of differential FOG1L and FOG1S regulation and complex formation in erythroid cells.

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References

    1. Tsai S. F., Martin D. I., Zon L. I., D'Andrea A. D., Wong G. G., Orkin S. H. (1989) Nature 339, 446–451 - PubMed
    1. Evans T., Felsenfeld G. (1989) Cell 58, 877–885 - PubMed
    1. Weiss M. J., Orkin S. H. (1995) Exp. Hematol. 23, 99–107 - PubMed
    1. Orkin S. H. (1992) Blood 80, 575–581 - PubMed
    1. Pevny L., Simon M. C., Robertson E., Klein W. H., Tsai S. F., D'Agati V., Orkin S. H., Costantini F. (1991) Nature 349, 257–260 - PubMed

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