Cofactor-mediated restriction of GATA-1 chromatin occupancy coordinates lineage-specific gene expression

Abstract

GATA-1 and its cofactor FOG-1 are required for the differentiation of erythrocytes and megakaryocytes. In contrast, mast cell development requires GATA-1 and the absence of FOG-1. Through genome-wide comparison of the chromatin occupancy of GATA-1 and a naturally occurring mutant that cannot bind FOG-1 (GATA-1(V205G)), we reveal that FOG-1 intricately regulates the chromatin occupancy of GATA-1. We identified GATA1-selective and GATA-1(V205G)-selective binding sites and show that GATA-1, in the absence of FOG-1, occupies GATA-1(V205G)-selective sites, but not GATA1-selective sites. By integrating ChIP-seq and gene expression data, we discovered that GATA-1(V205G) binds and activates mast cell-specific genes via GATA-1(V205G)-selective sites. We further show that exogenous expression of FOG-1 in mast cells leads to displacement of GATA-1 from mast cell-specific genes and causes their downregulation. Together these findings establish a mechanism of gene regulation whereby a non-DNA binding cofactor directly modulates the occupancy of a transcription factor to control lineage specification.

Figure 1. The GATA-1 FOG-1 interaction is required for megakaryocytic differentiation

(A) Schematic representation of megakaryocytic differentiation of G1ME cells after transduction with GATA-1 or V205G-expressing retroviruses. (B) FACS analysis of CD42 expression and DNA content (DAPI) 48h post infection with empty, GATA-1, or V205G-expressing MigR1 retrovirus. (C) Western blot of nuclear extracts from GFP+ G1ME cells 48h post-infection with empty, GATA-1, or GATA1^V205G-expressing retrovirus and Y10 cells treated with TPA for 48h. (D) ChIP for GATA-1 and GATA-1^V205G in G1ME cells using anti-GATA-1 and anti-HA-tag antibodies at the promoters of Itga2b and Gp1ba and a negative control region upstream of Itga2b. Means ± SD are depicted.

Figure 2. GATA1 and GATA-1^V205G bind distinct sites in chromatin

(A–D) UCSC Genome Browser depiction of GATA1-selective and GATA-1^V205G -selective peaks at representative loci. Red arrows indicate sites of differential binding. (E) ChIP-qPCR with a GATA1-specific antibody for GATA-1 and GATA-1^V205G at a panel of validated GATA-1^V205G -selective and GATA1-selective sites. Means ± SD are depicted. Asterisks mark sites where enrichment is significantly different.

Figure 3. GATA1^V205G-selective sites are devoid of ETS protein binding sites

(A) Informed motif analysis of the ChIP-seq datasets. The enrichment of selected motifs in each peak set was plotted as the −log(p-value). Asterisks mark motifs that are significantly enriched in one peak set relative to the other (p<.01). (B) ChIP for FLI-1 in control and GATA1-transduced G1ME cells at the GATA-1^V205G and GATA1-specific sites. Means ± SD are depicted. Asterisks mark sites where enrichment is significantly greater than control IgG.

Figure 4. Ectopic expression of FOG-1 in FOG-deficient cells displaces wild-type GATA-1 from GATA-1^V205G-selective binding sites

(A) FOG-1 deficient hematopoietic cells were infected with empty or FOG-1 and the occupancy of endogenous GATA-1 was assayed at a panel of GATA-1^V205G-selective and GATA1-selective sites by ChIP-qPCR. Means ± SD are depicted. (B) Western blot analysis on nuclear extracts from sorted GFP+ FOG-1 deficient cells infected with empty or FOG-1 MigR1 expressing retrovirus. (C) FACS analysis for Ter119 and CD41 expression on infected FOG-1 deficient cells 3 days post-infection.

Figure 5. Global gene expression analysis reveals that FOG-1 modulates GATA-1 chromatin occupancy to maintain lineage-specific gene expression

(A) Heatmap of 5123 differentially expressed genes among sorted GFP+ G1ME cells infected with MigR1 GATA-1, or GATA1^V205G-expressing retrovirus. The genes were divided into groups based on hierarchical clustering. (B) Overlap of the genes closest to the GATA1-selective binding sites with the genes differentially expressed by GATA-1 relative to GATA-1^V205G. Chi-square analysis reveals that the differentially bound genes are enriched for differentially expressed genes (p<2.2E-16). (C) Heatmap of the genes that overlap in panel B. The location of each peak relative to the TSS is plotted in an adjacent column and in a pie chart. (D) Overlap of the genes closest to the GATA-1^V205G-selective binding sites with the genes differentially expressed by GATA-1 relative to GATA-1^V205G. Chi-square analysis reveals that the differentially bound genes are enriched for differentially expressed genes (p<2.2E-16). (C) Heatmap of the genes that overlap in panel B. The location of each peak relative to the TSS is plotted in an adjacent column and in a pie chart. (D) Overlap of the genes closest to the GATA-1^V205G-selective binding sites with the genes differentially expressed by GATA-1 relative to GATA-1^V205G. Chi-square analysis reveals that the differentially bound genes are enriched for differentially expressed gene (p=1.0E-5). (E) Heatmap of the genes that overlap in panel D. The location of each peak relative to the TSS is plotted in an adjacent column and in a pie chart. (F) The expression of genes for multiple hematopoietic cell types is plotted for GATA-1 and GATA1^V205G-expressing cells relative to control cells. The presence of a ChIP-Seq peak for GATA-1 or GATA-1^V205G at each gene is included in the boxes below the graph: black box indicates that the gene is bound by the factor, while a yellow star indicates that a GATA1-selective or GATA1^V205G-selective binding site is present.

Figure 6. FOG-1 prohibits GATA-1 occupancy from mast cell genes to maintain lineage fidelity

(A) GSEA for a set of mast cell-specific genes on the GATA-1^V205G samples relative to the MigR1 and GATA-1 samples. (B) Heatmap of expression of mast cell genes from the microarray. The presence of a ChIP-Seq peak for GATA-1 or GATA-1^V205G at each gene is included in the boxes beside the graph: black box indicates that the gene is bound by the factor, while a yellow star indicates that a GATA1-selective or GATA-1^V205G-selective binding site is present. (C) UCSC Genome Browser depiction of ChIP-Seq data at the Ms4a2 and Cpa3 loci. GATA-2 ChIP-Seq data are from uninfected G1ME cells. The red arrows indicate the location of GATA-1^V205G-selective binding sites. (D) ChIP using a GATA1-specific antibody for GATA-1 and GATA-1^V205G in G1ME cells. Primers amplify the GATA-1^V205G-selective binding sites depicted in panel B. (E) ChIP for GATA-1 in G1ER and G1ER-V205M cells induced with β-estradiol for 24h. (F) ChIP for GATA-1 in sorted GFP+ FOG-1 deficient cells 2 days post-infection with empty, FOG-1, or FOG-1^K5A MigR1 expressing retrovirus. (G) ChIP for H3K4-me3 and H3K27-me3 at GATA-1^V205G-selective sites at Cpa3 and Ms4a2. (H) qPCR analysis for expression of the Zfpm1 gene (FOG-1) in G1ME cells 3 days post-infection with empty, GATA-1, or GATA-1^V205G-expressing and in BMMCs. (I) ChIP for GATA-1 in sorted GFP+ BMMCs 2 days post infection with empty, FOG-1, or FOG-1^K5A MigR1 expressing retrovirus. (J) qPCR analysis of gene expression for the indicated genes in sorted GFP+ BMMCs 3 days post infection with empty, FOG-1, or FOG-1^K5A MigR1 expressing retrovirus. (K) FACS analysis of FcεR1 expression on GFP+ BMMCs 3 days post infection with empty, FOG-1, or FOG-1^K5A MigR1 expressing retrovirus. (L) ChIP for GATA-1 in BMMCs at a panel of validated GATA1^V205G-selective and GATA1-selective sites.

Figure 7. FOG-1 modulates GATA-1 chromatin occupancy in a context-dependent manner

(A) An illustration of the three types of GATA-1 binding sites as they relate to dependence on FOG-1. (B) An illustration of FOG-1 prohibiting GATA-1 occupancy at mast cell genes.