Spacing Constraints of Neighboring Zinc Finger Modules within GATA2

Genomic analyses in clinical and experimental contexts have accelerated discoveries of human genetic variants. While elucidating the consequences of conspicuously loss-of-function variants is highly tractable, decoding the impact of missense or non-coding variants is considerably more challenging. Previously, we described a germline variant in GATA2 in a patient with GATA2-deficiency syndrome, which inserts nine amino acids between the two zinc fingers (9aa-Ins), one of which mediates sequence-specific DNA binding (Cavalcante de Andrade Silva M. et al., Leukemia, 2021). Unlike other GATA2 coding region and enhancer variants identified (Bresnick E.H. et al., Blood Adv., 2020), it was unclear whether the altered zinc finger spacing would be inhibitory, stimulatory or of no consequence. The 9aa-Ins variant was defective in activating several target genes (Hdc, Ear2 and Tpsb2) in a genetic complementation assay with Gata2 -77 enhancer-mutant (-77 ^-/-) primary hematopoietic progenitor cells. As only several target genes were tested, we used RNA-seq to conduct a genome-wide comparison of the capacity of GATA2 and 9aa-Ins to activate and repress transcription. To elucidate mechanisms, we considered the following models: 1) 9aa-Ins fails to regulate all genes normally controlled by GATA2; 2) 9aa-Ins fails to repress all genes normally controlled by GATA2; 3) 9aa-Ins fails to activate genes normally controlled by GATA2; 4) 9aa-Ins ectopically regulates genes not controlled by GATA2. Using a genetic complementation approach with -77 ^-/- cells that were immortalized by the Hoxb8 transcription factor (hi-77 ^-/-) (Wang G.G. et al., Nat. Methods, 2006; Johnson K.D. et al., JEM, 2020), we compared GATA2 and 9aa-Ins activities when expressed at a comparable level. This analysis revealed 2,138 GATA2-regulated, 525 GATA2 and 9aa-Ins-regulated, and 414 ectopically-regulated genes (at least two-fold change, adjusted P-value <0.05). A similar number of genes were GATA2-activated (1,061) and repressed (1,077). Only 144 out of the 1,061 (14%) were 9aa-Ins-activated and 381 out of 1,077 (35%) were 9aa-Ins-repressed, illustrating the severe consequences of this mutation and a greater impact on activation versus repression. Statistical analysis with a range of P-values constraints (0.01 to 0.1) verified that activation by 9aa-Ins was more significantly impaired than repression (86% were no longer activated, and 65% were no longer repressed, P = 5.4 x 10 ^-6). Gene ontology analysis revealed that the 9aa insertion impaired GATA2-mediated activation of genes related to GPCR signaling and GATA2-mediated repression of genes related to innate immune machinery. The ectopically-regulated genes did not conform to a particular mechanism or pathway.

Since it was unclear whether the transcriptional defects of the 9aa-Ins mutant reflect a unique inhibitory activity imparted by the 9aa sequence, we systematically varied the inter-zinc finger spacer length to establish whether any alterations can be tolerated. Using the genetic complementation assay, 2, 4, 6, 8, and 9aa spacer variants were compared with GATA2 for their capacity to regulate GATA2-activated genes (Hdc, Il1rl1, Gata1 and S1pr1) and repressed genes (Irf8, Il6st, Il6ra and Tifab). GATA2-mediated activation was compromised by insertions of two amino acids or more, whereas repression tolerated two and four amino acid insertions; 6, 8 and 9 were more inhibitory. Quantitative analyses revealed that a 6aa insertion reduced activation of the GATA2-activated genes by >50% of the wild type value, whereas the GATA2-repressed genes were still repressed by at least 50% (18% retention of activation and 83% retention of repression, P = 0.001). Thus, zinc finger spacing alterations differentially impacted activation versus repression. These results provide a rigorous foundation for interpreting variants that alter zinc finger spacing without disrupting vital finger residues. In vitro and in vivo functional analyses and molecular modeling are ongoing to further dissect the underlying mechanisms and ascertain the importance of genetic networks and circuits that are sensitive or resistant to human disease variants.