SUMOylation Regulates Growth Factor Independence 1 in Transcriptional Control and Hematopoiesis

Abstract

Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers. We show a binding interface between the GFI1 linker and the SP-RING domain of PIAS3, an E3-SUMO (small ubiquitin-related modifier) ligase. The PIAS3 binding region in GFI1 contains a conserved type I SUMOylation consensus element, centered on lysine-239 (K239). In silico prediction algorithms identify K239 as the only high-probability site for SUMO modification. We show that GFI1 is modified by SUMO at K239. SUMOylation-resistant derivatives of GFI1 fail to complement Gfi1 depletion phenotypes in zebrafish primitive erythropoiesis and granulocytic differentiation in cultured human cells. LSD1/CoREST recruitment and MYC repression by GFI1 are profoundly impaired for SUMOylation-resistant GFI1 derivatives, while enforced expression of MYC blocks granulocytic differentiation. These findings suggest that SUMOylation within the GFI1 linker favors LSD1/CoREST recruitment and MYC repression to govern hematopoietic differentiation.

FIG 1

The C-terminal half of the GFI1 linker binds the PIAS3 SP-RING domain. (A) PIAS3 binds GFI1. Myc-tagged GFI1 and Flag-tagged PIAS3 were expressed as shown. PIAS3 was immunopurified from whole-cell lysates (WCL), and coprecipitation of GFI1 was determined by immunoblot (Western blotting [WB]) analysis of immune complexes (IC). Green fluorescent protein (GFP) and tubulin were employed as controls for transfection efficiency and gel loading, respectively. (B) Structures of GFI1 and PIAS3 derivatives employed in the experiments shown in panels C and D. SNAG and ZnF regions are shown for GFI1. SAP, SP-RING, AD, and S/T-rich domains are shown for PIAS3. Amino acid residues corresponding to fragment boundaries are indicated in parentheses. Hatched boxes represent regions forming the GFI1-PIAS3 binding interface. Open circles indicate the 6×myc tag. (C and D) Mapping the GFI1-PIAS3 binding interface. PIAS3 and GFI1 forms, with accompanying epitope tags, were expressed as shown. Coprecipitating GFI1 (C) and PIAS3 (D) forms were identified by Western blotting. WT, wild type; FL, Flag epitope tag; IP, immunoprecipitation.

FIG 2

GFI1 is SUMOylated and ubiquitinated. (A) High-molecular-weight (HMW) derivatives of GFI1 identified under instantly denaturing conditions. COS7L cells were transfected with empty vector or expression constructs for Flag-tagged GFI1, HDAC1, and β-catenin. Cells were collected in-ice cold PBS, snap-frozen in liquid nitrogen, and suspended in 20% TCA. Precipitated proteins were solubilized by boiling in solubilizing buffer. Aliquots were analyzed by immunoblotting with anti-Flag antibody M2. (B) HMW derivatives of GFI1 contain SUMO and ubiquitin modifications. Flag-tagged GFI1 was expressed in COS7L cells, collected, and processed as described for panel A. Anti-Flag immune complexes (IC) and whole-cell lysate (WCL) were analyzed by SDS-PAGE and Western blotting (WB) as shown. (C) Each SUMO paralog can conjugate GFI1. Flag-tagged SUMO1, SUMO2, or SUMO3 was expressed in COS7L cells with myc-tagged GFI1 as shown. Cells were harvested as described for panel A. SUMOylated proteins were collected by anti-Flag immune precipitation. Immune complexes and whole-cell lysates were analyzed by Western blotting (WB). (D) Endogenously expressed GFI1 is SUMOylated. HL-60 cells in the quantities shown, stably expressing Flag-tagged SUMO2, SUMO3, or vector control, were collected and processed as described for panel A and then immunopurified via the Flag epitope tag (FL). Immune complexes (IC) were probed for GFI1 by Western blotting (WB). Expression of GFI1 and SUMO proteins was confirmed in whole-cell lysates (WCL) by Western blotting. Ub, ubiquitin.

FIG 3

GFI1 SUMOylation requires a type I SUMOylation consensus element in the GFI1 linker. (A) Alignment and conservation of a type I SUMOylation consensus sequence in the GFI1 linker in selected mammalian species. The putative SUMO acceptor lysine (K239 in rat) is indicated by an asterisk. (B) SUMO modification of GFI1 is abolished by K239R or E241Q substitution. Flag-tagged GFI1 or its K239R or E241Q derivative was expressed and then isolated by anti-Flag immune precipitation (IP) from total cellular protein harvested under instantly denaturing conditions as described in the legend to Fig. 2. Immune complexes (IC) and whole-cell lysates (WCL) were subjected to immunoblotting with anti-SUMO or anti-Flag antibodies as shown. (C) GFI1 and its K239R or E241Q derivative display PIAS3 binding. A 6×myc-tagged GFI1 or its K239R or E241Q derivative was expressed with Flag-tagged PIAS3. Presence of GFI1, GFI1-K239R, or GFI1-E241Q in anti-Flag immune complexes was determined by Western blotting. (D) GFI1 and its K239R derivative localize to the nucleus. NIH 3T3 cells were transduced with retrovirus expressing Flag-tagged GFI1 or its K239R derivative, and then subcellular localization was determined in stable polyclonal populations by epifluorescence detection. Nuclei were counterstained with To-Pro-3-iodide.

FIG 4

GFI1 SUMOylation on K239 is required for zebrafish primitive erythropoiesis. (A) One-cell-stage zebrafish embryos were injected with gfi1aa splice-blocking morpholino, alone or in combination with RNA expressing wild-type GFI1, GFI1-K239R, or GFI1-E241Q, each with a C-terminal Flag epitope tag. At 48 h postfertilization (hpf), primitive erythropoiesis was revealed by o-dianisidine staining. Four representative embryos are shown out of 200 embryos scored for each knockdown/rescue combination. Arrows in uninjected controls show the zone of primitive erythropoiesis. Primitive erythropoiesis was quantified using a graded scoring system. Scores of 1 to 4 were assigned for each embryo to indicate none, modest, moderate, or complete hemoglobinization, respectively. Statistical significance was assessed using a Wilcoxon-Mann-Whitney test (***, P < 0.0005). (B) Morpholino-induced splicing blockade of gfi1aa in zebrafish. Total RNA was isolated from uninjected (U) zebrafish embryos or after injection of a scrambled control (Sc) or gfi1aa splice-blocking morpholino (SB) targeting the boundary between intron 1 and exon 2, as shown (thick line). Unspliced gfi1aa cDNA was amplified with primers a and b spanning the intron 1-exon 2 boundary to yield a 169-bp amplimer. Spliced gfi1aa mRNA was amplified using primers c and b to yield a 263-bp amplimer. (C) Expression of Flag-tagged GFI1, GFI1-K239R (K>R), and GFI1-E241Q (E>Q) derivatives in extracts prepared from the equivalent of eight zebrafish embryos at 24 h postinjection.

FIG 5

GFI1 SUMOylation supports HL-60 cell granulocytic differentiation in response to all-trans retinoic acid (ATRA). (A) GFI1 depletion from HL-60 cells. Whole-cell extracts (WCLs) were prepared from either naive HL-60 cells or those infected with retrovirus expressing small hairpin RNA targeting GFI1 (sh-Gfi1) or a content-matched scrambled control shRNA (Scr. Ctl.). GFI1 levels were determined by Western blotting. Actin levels were used to confirm equal loading. (B) CD11B expression following stimulation with ATRA. HL-60 cells were treated with 0.1 μM ATRA or vehicle following shRNA-mediated depletion of GFI1 versus a content-matched scrambled control shRNA. CD11B⁺ cells were determined by flow cytometry. (C) Granulocytic differentiation of HL-60 cells requires GFI1. HL-60 cells were treated with ATRA or vehicle for 4 days following shRNA-mediated depletion of GFI1 or a scrambled control. One thousand cells from randomly selected fields for each condition were visually scored as immature (promyelocyte, myelocyte, or metamyelocyte morphology) or mature (band form or multisegmented nuclei). (D) Morphology of immature versus mature HL-60 cells following ATRA treatment. Arrows indicate mature cells with segmented nuclei reminiscent of granulocyte differentiation. (E) GFI1-K239R expression fails to complement the granulocyte maturation defect brought on by GFI1 depletion. HL-60 cells were subjected to scrambled control or GFI1-depleting shRNA, then rescued with expression constructs for Flag-tagged wild type (WT:FL) or GFI1-K239R (K>R:FL) as shown. After 4 days of ATRA exposure, cells were scored visually for granulocyte maturation as described for panel C. Expression of Flag-tagged GFI1 variants was confirmed by Western blotting.

FIG 6

SUMOylation modulates GFI1-dependent MYC expression to direct ATRA-mediated granulocyte maturation in HL-60 cells. (A) GFI1 modulates MYC expression in ATRA-mediated granulocytic differentiation. GFI1-targeted shRNA (sh-Gfi1) or a content-matched scrambled control (Scr) was used to deplete GFI1 in HL-60 cells, followed by rescue with a vector control (Vect) or GFI1 as shown. Levels of CEBPA, MYC, and AZU (azurocidin) mRNAs were determined by qRT-PCR relative to that of the GUS internal control. Fold change in expression is shown relative to that of the untreated, scrambled or vector control cells. (B) GFI1 was depleted from HL-60 cells using GFI1-targeted shRNA, followed by restored expression of GFI1, GFI1-K239R, or GFI1-E241Q and then treatment with vehicle or ATRA as shown. Expression of CEBPA, MYC, and AZU was measured as described for panel A. Statistical significance was determined by Wilcoxon-Mann-Whitney testing (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

FIG 7

Enforced MYC expression blocks granulocytic differentiation of HL-60 cells in response to ATRA. Naive HL-60 cells and those transduced with a MYC-expressing retrovirus or with a control vector were treated with 0.1 μM ATRA for 4 days and scored for immature versus mature granulocytic morphology. The Western blot shows c-Myc expression in transduced cells relative to that in vector control cells (inset) using anti-myc rabbit polyclonal antibody, A-14. Tubulin serves as a loading control. Statistical significance was determined by Wilcoxon-Mann-Whitney testing (*, P < 0.05).

FIG 8

SUMOylation supports LSD1/CoREST binding and transcriptional repression by GFI1. (A) SUMOylation contributes to GFI1-mediated repression. GFI1-Δ1C, the SNAG domain, or GFI1-Δ1C with the K239R substitution was expressed as a fusion with Gal4 in HEK293-T-REx-5×Gal-luciferase cells grown in six-well plates. Firefly luciferase activity was determined relative to that of Renilla luciferase using a dual-luciferase assay kit. (B and C) LSD1 activity contributes to transcriptional repression by GFI1. GFI1-Δ1C, the SNAG domain, or GFI1-Δ1C/K239R was expressed as a Gal4 fusion protein in the presence or absence of LSD1 inhibitor, HCI-2509 (LSD1i). Luciferase activity was scored as described for panel A. Similarly, transcriptional repression by GFI1-Δ1C or the SNAG domain, each harboring the P2A mutation that abolishes LSD1 binding, was compared to repression produced by its wild-type configurations at this position. (D) GFI1-K239R displays impaired LSD1/CoREST binding. Flag-tagged GFI1 or GFI1-K239R was expressed in COS7L cells and immunopurified from nuclear extracts (NE) by anti-Flag immune precipitation. Endogenously expressed LSD1 and CoREST coprecipitating with GFI1 or GFI1-K239R were determined by Western blotting (WB) of anti-Flag immune complexes (IC). (E) A model hypothesizing how GFI1 SUMOylation could influence transcriptional repression by the GFI1-LSD1/CoREST complex. RLU, relative light units.