GATA2 Germline Mutations Impair GATA2 Transcription, Causing Haploinsufficiency: Functional Analysis of the p.Arg396Gln Mutation

Abstract

Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 mutations appear to cause loss of function of the mutated allele leading to haploinsufficiency; however, this postulate has not been experimentally validated as the basis of these syndromes. We hypothesized that mutations that are translated into abnormal proteins could affect the transcription of GATA2, triggering GATA2 deficiency. Chromatin immunoprecipitation and luciferase assays showed that the human GATA2 protein activates its own transcription through a specific region located at −2.4 kb, whereas the p.Thr354Met, p.Thr355del, and p.Arg396Gln germline mutations impair GATA2 promoter activation. Accordingly, GATA2 expression was decreased to ∼58% in a patient with p.Arg396Gln, compared with controls. p.Arg396Gln is the second most common mutation in these syndromes, and no previous functional analyses have been performed. We therefore analyzed p.Arg396Gln. Our data show that p.Arg396Gln is a loss-of-function mutation affecting DNA-binding ability and, as a consequence, it fails to maintain the immature characteristics of hematopoietic stem and progenitor cells, which could result in defects in this cell compartment. In conclusion, we show that human GATA2 binds to its own promoter, activating its transcription, and that the aforementioned mutations impair the transcription of GATA2. Our results indicate that they can affect other GATA2 target genes, which could partially explain the variability of symptoms in these diseases. Moreover, we show that p.Arg396Gln is a loss-of-function mutation, which is unable to retain the progenitor phenotype in cells where it is expressed.

Decoding disease-causing mechanisms of missense mutations from supramolecular structures

“…For example, missense mutations of GATA2 , encoding a transcription factor protein GATA2, cause familial syndromes with immunodeficiency in a haploinsufficient fashion. GATA2 binds to its own promoter and activates transcription, and the missense mutations at the DNA binding sites impair the promoter binding ability, resulting in decreased transcription of GATA2 24 . Another example would explain dominant-negative mechanisms: mutations in PITX2 , encoding a homeodomain protein PITX2, are responsible for Axenfeld-Rieger syndrome (MIM# 180500).…”

GATA2 deficiency and human hematopoietic development modeled using induced pluripotent stem cells

“…5,21 Haploinsufficiency is likely the reason for the majority of GATA2 deficiency phenotypes, but potential dominant negative effects have been also reported for some mutant GATA2 proteins. [21][22][23] There is no clear link between specific GATA2 mutations and hematologic phenotype. 5 Rodent models only partly recapitulate the human phenotype.…”

GATA2 mutations in myeloid malignancies: Two zinc fingers in many pies

“…The group furthermore described R396Q to be a LOF mutation due to its abrogation in promoter binding, loss of DNA binding ability and inability to retain a progenitor phenotype. They found that R396Q increased the proportion of colony forming unit granulocyte …”

“…They found that R396Q increased the proportion of colony forming unit granulocyte. 28 Ping et al showed that upon coexpression of GATA2 WT with the ZF1 somatic mutant R330X, the transcriptional activity of GATA2 WT was significantly affected and therefore concluded that R330X had a dominantnegative effect over GATA2 WT. The authors did not observe such a dominant-negative effect in ZF1 mutants P304H and L321P.…”