Ski negatively regulates erythroid differentiation through its interaction with GATA1

Abstract

The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor beta, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. Here we show that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. We propose that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia.

FIG. 1.

Erythroid differentiation of K562 cells is inhibited by wild-type Ski but not by the L110P mutant. (A) Levels of Ski protein expression in K562 cells. WB, Western blotting. (B, C, and D) The L110P mutation impairs inhibition activity of Ski on K562 erythroid differentiation induced by hemin. Shown are representative data from K562 cells expressing wt-cSki and mt-cSki. (B) Benzidine staining before (− hemin) and after (+hemin) hemin treatment (5 days). (C) Level of hemoglobin production (top panel) as a marker of erythroid differentiation before (lanes 1 to 3) and after (lanes 4 to 6) hemin treatment (5 days). The level of GATA1 protein expression during differentiation is also shown (middle panel). An α-tubulin blot is shown as a loading control (bottom panel). (D) RT-PCR analysis of erythroid-specific genes in K562 cells expressing wt-cSki and mt-cSki. Band intensity was normalized with each value of β-actin as the control. Data from a typical experiment are presented as relative fold induction, with the normalized value for the vector control in the absence of hemin set as 1.0.

FIG. 2.

GATA1 interacts with wild-type Ski but not with the L110P mutant. (A) GATA1-bound Ski was detected by immunoprecipitation (IP). Cell extracts from K562 cells expressing vector control, wt-cSki, or mt-cSki were immunoprecipitated by endogenous GATA1, followed by Western blotting (WB) with anti-T7 (Ski; top panel) and anti-GATA1 (middle panel) antibody. Equivalent amounts of wt-cSki and mt-cSki proteins were presented in the input control (10%; bottom panel). (B) Cell extract from HEL cells expressing wt-cSki was immunoprecipitated by anti-GATA1 antibody or normal rat IgG (negative control), followed by Western blotting as described for panel A. The asterisk indicates a nonspecific signal. (C) Cell extracts from COS-1 cells expressing wt-cSki or mt-cSki were immunoprecipitated by exogenously expressed GATA1, followed by Western blotting as described for panel A. (D) The CF of GATA1 is required for interaction with wt-cSki. (D, top panels) Cell extracts from COS-1 cells expressing wild-type GATA1 (WT), ΔNF, and ΔCF plus wt-cSki were immunoprecipitated by anti-GATA1 antibody or normal rat IgG (negative control), followed by Western blotting as described for panel A. (D, bottom panels) The zinc finger domain of GATA1 (NF plus CF) is sufficient for interaction with wt-cSki. (D, bottom left panel) Cell extract from COS-1 cells expressing wt-cSki and GATA1 (NF plus CF) was immunoprecipitated by anti-Myc antibody or normal mouse IgG (negative control), followed by Western blotting as described for panel A. (D, bottom right panel) Cell extract from COS-1 cells expressing wt-cSki was incubated with bacterially expressed GATA1 (NF plus CF) protein on nickel-nitrilotriacetic acid resin, followed by Western blotting as described for panel A. GATA1 (NF plus CF) protein was detected by Coomassie brilliant blue (CBB) staining. +, present; −, absent.

FIG. 3.

GATA1-mediated transcription is significantly repressed by wild-type Ski but not by the mutant. (A) The L110P mutation significantly reduces repression activity of Ski on GATA1-dependent transactivation. QT6 cells were transiently transfected with pRBGP3-MαP luciferase reporter construct (50 ng/well), together with (black bars) or without (white bars) GATA1 (100 ng/well) plus each effector plasmid (100 ng of vector, wt-cSki, or mt-cSki/well) as indicated. RLU, relative luciferase units. (B) Dose-dependent repression activity of wt-cSki. Transfection was done as described for panel A except that various amounts (in nanograms per well) of effector plasmid (wt-cSki or mt-cSki) were used, as indicated, in the presence of GATA1 plasmid (50 ng/well). (C) Ski overexpression does not repress TK promoter. Transfection was done as described for panel A except for the use of the TK-luciferase reporter construct (50 ng/well).

FIG. 4.

GATA1 DNA binding is blocked by wild-type Ski but not by the L110P mutant. (A) Effect of wild-type Ski and mutant Ski on GATA1 DNA binding. EMSA were performed using COS-1 cell extracts as described for panel B without (lanes 1 to 6) or with (lanes 7 to 12) an excess amount (200 times) of unlabeled probe as indicated. +, present. (B) Western blot of cell extracts prepared from COS-1 cells, which was used for the EMSA described in panel A. Lane 1, nontransfected control; lane 2, GATA1; lane 3, wt-cSki; lane 4, mt-cSki; lane 5, GATA1 plus wt-cSki; lane 6, GATA1 plus mt-cSki. Indicated proteins were equivalently expressed.

FIG. 5.

Ski can displace GATA1 occupancy on promoter and enhancer regions of erythroid-specific genes in vivo. Shown are the results of a ChIP assay of GATA1 binding to promoter and enhancer regions (HS2, ALAS-E, and PBGD genes) in K562 cells expressing vector, wt-cSki, and mt-cSki treated with 40 μM hemin for 48 h. Input, 1% of chromatin lysate subjected to immunoprecipitation; IgG, normal IgG as a negative control; N6, anti-GATA1 antibody.