Pathogenic GATA2 genetic variants utilize an obligate enhancer mechanism to distort a multilineage differentiation program

Abstract

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

Keywords: GATA; GATA2; differentiation; hematopoiesis; transcription.

Fig. 1.

GATA2 disease variants are defective in regulating differentiation and transcription in a genetic rescue system. (A) Schematic representation of the genetic rescue system. (B) Left: Representative western blot analysis of −77^−/− fetal liver cells expressing HA-GATA2 or variants with anti-HA antibody. Right: Representative western blot analysis of −77^+/+ and −77^−/− fetal liver cells expressing HA-GATA2 or variants with anti-GATA2 antibody. (C) Flow cytometric analysis of GFP⁺ cells stained for monocytic cells (CD11b⁺CD115⁺), granulocytic cells (CD11b⁺CD115⁻), macrophage (F4/80⁺CD11b⁺), mast cells (FcεR1α⁺Kit⁺), and eosinophils (CD11b⁺SiglecF⁺). (D) Quantitation of flow cytometry data (n = 5). (E) Heatmap showing the result of RT-qPCR analysis of erythroid, mast cell, and eosinophil genes (n = 5). (F) Flow cytometric analysis of GFP⁺ cells stained for monocytic cells (CD11b⁺CD115⁺), granulocytic cells (CD11b⁺CD115⁻), macrophages (F4/80⁺CD11b⁺), mast cells (FcεR1α⁺Kit⁺), and eosinophils (CD11b⁺SiglecF⁺). (G) Quantitation of flow cytometry data (n = 4). * < 0.05, ** < 0.01, and *** < 0.001.

Fig. 2.

Context-dependent T354M occupancy of chromatin sites and transcriptional regulation. (A) Venn diagram depicting overlap between GATA2- and disease variant-regulated genes. (B) Heatmap depicting RT-qPCR analysis of GATA2-regulated genes (n = 5). (C) RT-qPCR analysis of GATA2-regulated mRNAs and primary transcripts in GATA2- or T354M-expressing cells (n = 5). (D) Representative western blot analysis of hi-77^−/− cells expressing HA-GATA2 or variants with anti-HA antibody. (E) Representative image of Giemsa-stained cells. Cells were cultured for 3 d. (F) Heatmap depicting RT-qPCR analysis of GATA2-regulated mRNAs identified with GATA2- or disease variant-expressing hi-77^−/− cells (n = 6). (G) Quantitative ChIP analysis at GATA2-occupied sites in target genes with anti-HA antibody (n = 5). (H) Quantitative ChIP analysis at GATA2-occupied sites in target genes with anti-GATA2 antibody (n = 4). (I) CUT&RUN profile of GATA2 at Ms4a3 locus (GSE171384). (J) PCR-genotyping assay for −77^−/−Ms4a3+27.6^−/− allele. (K) RT-qPCR analysis of Ms4a3 mRNA levels (n = 4). * < 0.05, ** < 0.01, and *** < 0.001.

Fig. 3.

Zinc finger requirements for T354M-mediated transcriptional regulation. (A) Schematic representation of zinc finger structures. (B) Left, Representative western blot analysis of HA-GATA2 or variants using anti-HA antibody. Right, RT-qPCR analysis of GATA2-regulated mRNAs in cells expressing GATA2 or variants (n = 5). (C) Left, Representative western blot analysis of HA-GATA2 or variants using anti-HA antibody. Right, RT-qPCR analysis of GATA2-regulated mRNAs in cells expressing GATA2 or variants (n = 5). (D) RT-qPCR analysis of T354M-regulated mRNAs in cells expressing GATA2 or variants (n = 5). (E) Quantitative ChIP analysis at GATA-occupied sites in target genes with anti-HA antibody (n = 3). (F) RT-qPCR analysis of Irf8 mRNA in cells expressing GATA2 or variants (n = 5). (G) Left, Representative western blot analysis of HA-GATA2 or variants using anti-HA antibody. Right, RT-qPCR analysis of GATA2-regulated mRNAs in GATA2- or variant-expressing cells (n = 4). * < 0.05, ** < 0.01, and *** < 0.001.

Fig. 4.

Genetic ablation of TAL1 reveals its context-dependent requirement for GATA2 and T354M function. (A) Quantitative ChIP analysis with anti-TAL1 antibody at GATA2-occupied sites in target genes (n = 4). (B) Quantitative ChIP analysis with anti-LDB1 antibody at GATA2-occupied sites in target genes (n = 4). (C) PCR-based genotyping assay for −77^−/−Tal1^−/− allele. (D) Top, Representative western blot analysis of TAL1. Bottom, RT-qPCR analysis of Tal1 mRNA level in −77^−/− and −77^−/−Tal1^−/− cells. (E) RT-qPCR analysis of GATA2 target gene mRNAs in cells expressing GATA2 or variants (n = 5). (F) Quantitative ChIP analysis at GATA2-occupied sites in target genes with anti-HA antibody (n = 3). (G) Model of collaborative transcriptional regulation by GATA2/T354M and TAL1. * < 0.05, ** < 0.01, and *** < 0.001.

Fig. 5.

Genetic ablation of a Cebpe enhancer reveals the vital importance of C/EBPε for context-dependent GATA2 and T354M function. (A) Left, Comparison of protein levels [normalized using MaxLFQ from the prior analysis (43)] in CMP/GMP. Center, mRNA levels (TPM) in Lin⁻ progenitors calculated using RSEM (68). Right, mRNA levels (TPM) in hi-77^+/+ or hi-77^−/− cell lines. (B) RT-qPCR analysis of Cebpe mRNA in cells expressing GATA2 or variants (n = 5). (C) ChIP-Seq profile of transcription factors at Cebpe locus in hi-77^+/+ cells mined from dataset GEO:GSE224904. (D) PCR-genotyping assay for −77^−/−Cebpe+6^−/− allele. (E) Left, Representative western blot analysis of C/EBPε. Right: Quantification of C/EBPε protein in hi-77^−/− cells expressing HA-GATA2 or variants (n = 4). (F) RT-qPCR analysis of GATA2 target gene mRNAs in cells expressing GATA2 or variants (n = 4). (G) ChIP-Seq profile of transcription factors at Prg2/3, Epx, Ms4a2/3, Csf2rb, and Anxa1 in mouse myelocytes mined from dataset GEO:GSE73844. (H) RT-qPCR analysis of GATA2 target gene mRNAs in hi-77^+/+ cells and hi-77^+/+Cebpe+6^−/− cells (n = 6). (I) Representative western blot analysis of HA-C/EBPε by using anti-HA antibody. Right, RT-qPCR analysis of C/EBPε target gene mRNAs (n = 5). (J) Top, Flow cytometric analysis of GFP⁺ cells stained with CD11b and CD115. Bottom, Quantitation of flow cytometry data (n = 5). * < 0.05, ** < 0.01, and *** < 0.001.

Fig. 6.

Pathogenic GATA2 genetic variants utilize an obligate enhancer-dependent feedforward mechanism to distort a multilineage differentiation program. GATA2 and T354M utilize C/EBPε and TAL1 to regulate a target gene cohort and multilineage differentiation. The fragmented activity of T354M generates a lineage-distorted differentiation program involving increased eosinophil, but not erythroid or mast cell, differentiation. As other GATA2 pathogenic variants share this attribute, this may represent a hallmark of GATA2 pathogenic variants. Considering the involvement of C/EBPε as a suppressor of MLL-rearrangement AML (70), and GATA2 as a suppressor of MDS/AML, the GATA2-C/EBPε paradigm links two vital systems that ensure the normal development and function of the hematopoietic system.