The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum

ABSTRACT GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Sclerotinia sclerotiorum Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in S. sclerotiorum. Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of Ssfdh1; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors. IMPORTANCE S. sclerotiorum is a pathogenic fungus with sclerotium and infection cushion development, making S. sclerotiorum one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of Ssfdh1. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.

Differential Contribution of the Gata1 Gene Hematopoietic Enhancer to Erythroid Differentiation

ABSTRACT GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type–specific expression of the Epo gene.

The Presence of FYAnull Alele of Duffy Blood Group System in Blood Donors and Individuals from a Malarial Endemic Region of Brazil.

BACKGROUND: Malaria is a virulent disease caused by the Plasmodium parasite. Innate resistance to malaria infections in humans is conferred by various blood group polymorphisms. The Duffy blood group system consists of Fya and Fyb antigens which are encoded by codominant alleles FYA and FYB. Four phenotypes are defined: Fy(a+b+), Fy(a+b−), Fy(a−b+) and Fy(a−b−). Erythrocytes of Duffy-negative individuals are resistant to invasion by P. vivax. In Blacks the Fy(a−b−) phenotype is associated with a single point mutation (-33T-C) in the GATA-1 binding motif for the erythroid promoter of FYB. STUDY DESIGN AND METHODS: We investigated the phenotypes and the genotypes of Duffy blood group system of 250 individuals living in a malarial endemic region (MER) in the state of Amazon (Brazil), and of 199 blood donors (BD) from a non-endemic region. The phenotyping was done by agglutination gel tests (DiaMed-Latino América) using anti-Fya and anti-Fyb reagents. The molecular analysis for FYA, FYB, FYBES (GATA box mutation nt -33T-C), and FYBWeak (mutations 265 C-T, and 298 G-A) alleles, were performed by PCR-RFLP. The PCR products were digested by Ban I for FYA and FYB identifications; by Sty I for GATA box mutation; Acy I and Mwo I for 265 C-T and 298 G-A mutations, respectively. Some samples that showed discrepancy between the phenotype and genotype results were examined by sequence analysis using the ABI PrismâBig Dyeä Terminator Cycle Sequencing Ready Reaction Kit” (Perkin Elmer), and the interpretation by the software ABI PRISMä 377 DNA Sequencer”, 3.3 version (Perkin Elmer). RESULTS: We found that 34/250 (13.6%) of 250 persons living in the MER and 37/199 (18.6%) of BD had phenotype and genotype discrepant results [Fy(a+b−) FYA/FYB]. In addition, we found that 16/34 (47%) of people living in the MER, and 4/37 (10.8%) of BD did not present the -33T-C mutation, the 265 C-T, or the 298 G-A mutations. The sequence analysis of 2 samples from persons from MER indicated the presence of -33T-C mutation in the FYA allele in one individual (1 FYA/FYB and W/M; FYA/FYB and M/M). Additionally, we detected that 18/34 (53%) of people living in the MER, and 33/37 (89.2%) of BD presented the -33T-C mutation. The sequence analysis of 5 samples indicated the presence of -33T-C mutation in the FYA allele in 4 cases [2 persons from MER and 2 from BD (FYA/FYB e M/M)]. CONCLUSION: Recently the mutation responsible for erythrocyte Duffy antigen-negativity [Fy(a−b−)] was demonstrated in FYA allele in a malarial endemic region of Papua New Guinea. The present data demonstrated the presence of the FYAnull allele not only in persons living in a malarial endemic region but also in Brazilian blood donors from non-endemic areas. In contrast with that which happens with the FYB allele, our results indicated that the presence of the -33T-C mutation in the FYA allele does not abolish the expression of the Fya antigen in the erythrocyte.

A GATA Box in the GATA-1 Gene Hematopoietic Enhancer Is a Critical Element in the Network of GATA Factors and Sites That Regulate This Gene

ABSTRACT A region located at kbp −3.9 to −2.6 5′ to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenousGATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL−/− embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.