Transcriptome and DNA Methylome Dynamics during Triclosan-Induced Cardiomyocyte Differentiation Toxicity

Cardiac development is a dynamic process and sensitive to environmental chemicals. Triclosan is widely used as an antibacterial agent and reported to transport across the placenta and affect embryonic development. Here, we used human embryonic stem cell- (hESC-) derived cardiomyocytes (CMs) to determine the effects of TCS exposure on cardiac development. After TCS treatment, the differentiation process was significantly blocked and spontaneous beating rates of CMs were also decreased. Transcriptome analysis showed the dysregulation of genes involved in cardiogenesis, including GATA4 and TNNT2. Additionally, DNA methylation was also altered by TCS exposure, especially in those regions with GATA motif enrichment. These alterations of transcriptome and DNA methylation were all associated with signaling pathways integral to heart development. Our findings indicate that TCS exposure might cause cardiomyocyte differentiation toxicity and provide the new insights into how environmental factors regulate DNA methylation and gene expressions during heart development.

Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system

Key Points Expression of the Xg blood group protein is governed by rs311103, and its minor allele disrupts a GATA motif to cause the Xg(a−) phenotype. These data elucidate the genetic basis of the last unresolved blood group system and make genotyping for Xga status possible.

GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis

GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution. In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. Disclosures No relevant conflicts of interest to declare.

Progenitor Stage-Specific Activity of acis-Acting Double GATA Motif forGata1Gene Expression

GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discretecis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using aGata1bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1ΔdbGATAmice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossedGata1ΔdbGATAmice withGata2hypomorphic mutant mice (Gata2fGN/fGNmice), theGata1ΔdbGATA::Gata2fGN/fGNcompound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion onGata1expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATAlocus. These results thus reveal a novel role of the dbGATA motif in the maintenance ofGata1expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.

DNA Binding Diversity Achieved Through the Interaction of GATA1 N-Finger and GATA Motif Is Important for Embryonic Erythropoiesis

Abstract 3441 Transcription factor GATA1 regulates a set of genes essential for the erythroid and megakaryocytic cell differentiation through the interaction with GATA motifs (consensus sequence: A/TGATAA/T). Two zinc fingers within GATA1 have been identified to be important in the DNA binding of GATA1, which are referred to as C-finger (CF) and N-finger (NF) domains. It has been shown that transactivation activity of GATA1 is completely abolished upon deletion of the CF domain, indicating that the CF domain is a requisite for the DNA binding of GATA1. While conventional reporter transactivation analyses hardly clarified the importance of the NF domain for the DNA binding, substitution mutations on 216th arginine (R216) located in the DNA-interacting surface of the NF domain have been identified to cause familial diseases of thrombocytopenia, thalassemia, and porphyria. As a consequence of the substitution of R216 to glutamine (Q) or tryptophan (W), DNA binding activity of GATA1 to a palindromic configuration of two GATA motifs (palindromic GATA) was largely diminished, while that to a single GATA motif was maintained. In this study we have examined the DNA binding diversity of GATA1 caused by the difference in the configuration of GATA motifs. We performed surface plasmon resonance (SPR) analyses of GATA1 to a single GATA, a palindromic GATA, and a repeating configuration of two GATA motifs (tandem GATA). We found that GATA1 binds to the palindromic GATA motif in a bivalent way, while it binds to the single GATA motif in a monovalent mode. We also found that a double quantity of GATA1 is associated with the tandem GATA motif and GATA1 lacking the NF domain binds to any configurations of GATA motif in a monovalent way. To further investigate contribution of the NF domain to the binding mode of GATA1, we have constructed two types of GATA1 mutants; one type was the substitution mutations on R216 (R216Q and R216W) that were mouse homologues of the human mutations, while the other type was the alanine substitution mutation on three lysine residues (K245, K246 and K312; referred to as 3KA mutant), whereby dimerization potential of GATA1 was reduced to trace level similar to the case for GATA1 lacking the NF domain. Impotantly, R216Q and R216W mutants bind the palindromic GATA motif in a monovalent way, while these mutants bind normally to the other configuration of GATA motifs. In contrast, we found that one molecule of 3KA mutant bound to the tandem GATA motif and this observation seems to explain well the fact that dimerization potential of GATA1 is an important requisite for the full-function of GATA1 in embryos. The binding modes of this 3KA mutant to the other configurations were not influenced. These results thus demonstrate that the both NF and CF domains recognize the multiple configurations of GATA motifs and specify the binding modes of GATA1. Importantly, GATA1-deficient mice rescued with R216Q were lethal during late gestation period due to abnormality in erythroid differentiation, indicating that the contribution of the NF domain to the recognition of the palindromic GATA motif configuration indeed functions in vivo. These results thus support our contention that the NF domain acts to regulate a proper spatio-temporal gene expression of a subset of GATA1 target genes utilizing the variations in the GATA motif configuration. Disclosures: No relevant conflicts of interest to declare.

Context-dependent function of “GATA switch” sites in vivo

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the −2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring −1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the −2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.