GATA2-TGF beta-TAL1 pathway as a critical mediator of NK Cell development - ABSTRACT (Significance) GATA2 is an obligatory transcription factor for human NK cell development, and mutations in GATA2 causes a spectrum of diseases that include uncontrolled immune system activation and bone marrow failure. Here, we uncover a novel, previously unknown GATA2-TGF-b1-TAL1 transcriptional network essential for human NK cell development and functions. (Current field) GATA2 is necessary for hematopoietic stem cell (HSC) homeostasis and contains two zinc finger, two transactivation, and one negative regulatory domain. GATA2 forms a core heptad complex with TAL1, FLI1, RUNX1, LYL1, LMO2, and ERG to directly impact the survival and differentiation of HSCs by regulating more than 1000 target genes. Outside of HSCs, GATA2 haploinsufficiency results in dysfunctional NK, B cell, and myeloid cell development. Mechanisms by which GATA2 regulates human NK cell development and functions are unknown and likely related to the disease phenotype. (Preliminary data) We analyzed the scRNA-seq data of NK cells from 10 patients with GATA2 mutations and compared them with six healthy controls. Among the upregulated transcripts, we found an increase in the transcript and protein levels of TAL1 in NK cells from the patients and cells expressing GATA2T354M mutant but not WT GATA2. Among the down-regulated transcripts, we found that TGFB1 is one of the top five in the NK cells from GATA2 mutant patients. This link between GATA2 and TGF-1 was validated by our finding of two functional GATA motifs in the promoter region of TGF-1, which was confirmed by promoter-reporter assay. Independent of GATA2, TAL1 forms heterodimers with HEB, E2.2, and E2A, which counter ID2 function. Importantly, the transcript level of ID2, obligatory for acquiring functional capabilities, is considerably reduced in NK cells derived from GATA2T354M patients. We find that HEB transcript level is increased in GATA2T354M patients. We also found a significant decrease in ZEB2 transcript in NK cells from GATA2T354M patients, which could be potentially repressed by augmented TAL1/HEB complexes. Based on these, we propose to test the hypothesis that an interplay between the GATA2, TGF-b1, and TAL1 is required for the development and functions of human NK cells. (Approaches) We have employed scRNA/bulk RNA seq, novel lipid nanoparticle (LNP)-based transfer of mRNA and siRNA into primary NK cells, ATAC, CUT&Tag, in vitro expression of GATA2 and GATA2T354M constructs, and western blot to obtain preliminary data. We will continue employing these techniques. Importantly, we are generating conditional knockout of the TAL1 gene by breeding the Tal1fl/fl mice (from Dr. Orkin's lab) with CD122Cre and NCR1Cre mice. (Expected contributions of the study to the field) GATA2-TGF-b1-TAL1 axis is novel. This knowledge is essential to understanding disease phenotypes such as monoMAC syndrome. In addition, the outcomes of this proposal will define the transcriptional regulation of human NK cell development.
