GATA Family Of Transcription Factors Research Articles

Recovery of synaptic loss and depressive-like behavior induced by GATA1 through blocking of the neuroinflammatory response.

GATA1, a member of the GATA transcription factor family, is a critical factor in hematopoietic system development. In a previous study, we demonstrated the increased expression of GATA1 in the dorsolateral prefrontal cortex (dlPFC) of patients suffering from depression and described its role as a transcriptional repressor of synapse-related genes. In this study, we investigated how GATA1 globally altered gene expression using multi-omics approaches. Through the combined analyses of ChIPseq, mRNAseq, and small RNAseq, we profiled genes that are potentially affected by GATA1 in cultured cortical neurons, and Gene Ontology (GO) analysis revealed that GATA1 might be associated with immune-related functions. We hypothesized that GATA1 induces immune activation, which has detrimental effects including synapse loss and depressive-like behavior. To test this hypothesis, we first performed a microglial morphometric analysis of a brain having overexpression of GATA1 because microglia are the resident immune cells of the central nervous system. Fractal analysis showed that the ramification and process length of microglia decreased in brains having GATA1 overexpression compared to the control, suggesting that GATA1 overexpression increases the activation of microglia. Through flow cytometry and immunohistochemical analysis, we found that activated microglia showed pro-inflammatory phenotypes characterized by the expression of CD86 and CD68. Finally, we demonstrated that the effects of GATA1 overexpression including synapse loss and depressive-like behavior could be blocked by inhibiting microglial activation using minocycline. These results will elucidate the regulatory mechanisms of GATA1 that affect pathophysiological conditions such as depression and provide a potential target for the treatment of depression.

TRPS1 modulates chromatin accessibility to regulate estrogen receptor alpha (ER) binding and ER target gene expression in luminal breast cancer cells.

Common genetic variants in the repressive GATA-family transcription factor (TF) TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over 80 percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes estrogen receptor alpha (ER) to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the genomic distribution of ER, both activating and repressing transcription of genes related to cancer cell fitness.

GATA family transcription factors in alga Chlamydomonas reinhardtii.

GATA family transcription factors (GATA-TFs) are metalloproteins that regulate many metabolic pathways. These conserved proteins recognize the consensus sequence (A/T)GATA(A/G) in the promoter regions of many genes and regulate their transcription in response to environmental signals. Currently, the study of GATA-TFs is of increasing interest. GATA genes and their proteins are most actively studied in vascular plants and fungi. Based on the results of numerous studies, it has been shown that GATA factors regulate the metabolic pathways of nitrogen and carbon, and also play a major role in the processes induced by light and circadian rhythms. In algae, GATA-TFs remain poorly studied, and information about them is scattered. In this work, all known data on GATA-TFs in the unicellular green alga Chlamydomonas reinhardtii has been collected and systematized. The genome of this alga contains 12 GATA coding genes. Using the phylogenetic analysis, we identified three classes of GATA factors in C. reinhardtii according to the structure of the zinc finger domain and showed their difference from the classification of GATA factors developed on vascular plants.

Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla.

Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the charactertics of GATA TFs in U. rhynchophylla and how different light qualities affect the expression of GATA and key enzyme genes, thereby affecting the changes in U. rhynchophylla alkaloids have not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIAs content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content. In this research, we comprehensively analyzed the UrGATA TFs, and offered insight into the involvement of UrGATA TFs from U. rhynchophylla in TIAs biosynthesis.

Role of the pioneer transcription factor GATA2 in health and disease.

The transcription factor GATA2 is involved in human diseases ranging from hematopoietic disorders, to cancer, to infectious diseases. GATA2 is one of six GATA-family transcription factors that act as pioneering transcription factors which facilitate the opening of heterochromatin and the subsequent binding of other transcription factors to induce gene expression from previously inaccessible regions of the genome. Although GATA2 is essential for hematopoiesis and lymphangiogenesis, it is also expressed in other tissues such as the lung, prostate gland, gastrointestinal tract, central nervous system, placenta, fetal liver, and fetal heart. Gene or transcriptional abnormalities of GATA2 causes or predisposes patients to several diseases including the hematological cancers acute myeloid leukemia and acute lymphoblastic leukemia, the primary immunodeficiency MonoMAC syndrome, and to cancers of the lung, prostate, uterus, kidney, breast, gastric tract, and ovaries. Recent data has also linked GATA2 expression and mutations to responses to infectious diseases including SARS-CoV-2 and Pneumocystis carinii pneumonia, and to inflammatory disorders such as atherosclerosis. In this article we review the role of GATA2 in the etiology and progression of these various diseases.

Genome-wide identification, evolution and expression pattern analysis of the GATA gene family in Sorghum bicolor.

The GATA family of transcription factors is zinc finger DNA binding proteins involved in a variety of biological processes, including plant growth and development and response to biotic/abiotic stresses, and thus play an essential role in plant response to environmental changes. However, the GATA gene family of Sorghum (SbGATA) has not been systematically analyzed and reported yet. Herein, we used a variety of bioinformatics methods and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to explore the evolution and function of the 33 SbGATA genes identified. These SbGATA genes, distributed on 10 chromosomes, are classified into four subfamilies (I-IV) containing one pair of tandem duplications and nine pairs of segment duplications, which are more closely related to the monocot Brachypodium distachyon and Oryza sativa GATA genes. The physicochemical properties of the SbGATAs are significantly different among the subfamilies, while the protein structure and conserved protein motifs are highly conserved in the subfamilies. In addition, the transcription of SbGATAs is tissue-specific during Sorghum growth and development, which allows for functional diversity in response to stress and hormones. Collectively, our study lays a theoretical foundation for an in-depth analysis of the functions, mechanisms and evolutionary relationships of SbGATA during plant growth and development.

Interactive effects of light quality and nitrate supply on growth and metabolic processes in two lettuce cultivars (Lactuca sativa L.)

Light quality and nutrient parameters alter plant growth and metabolic pathways in leafy greens, therefore being primarily optimized in indoor precision agriculture. While their individual effects were studied extensively with large-scale phenomics and transcriptomics, the combinatorial effects remain poorly understood. Here, we explored the hidden interactions among light- and nitrate-responsive transcriptomes and their associations with six growth and biochemical traits, using the full factorial experimental design with three lights (red (R), blue (B) and a 1:1 mixture of red and blue (RB)), two nitrate concentrations and two cultivars (Green oak-leaf and Red oak-leaf). Multifactor analysis of 36 RNA-sequencing data revealed their largely independent but additive effects that are associated with MYB, bHLH and GATA family transcription factors. Red light and sufficient nitrate supply additively increased the primary metabolite pathway and bHLH and GATA genes, leading to a high growth rate. Blue and RB lights, together with nitrate deficiency, induced flavonoid pathway and MYB genes. Variety-specific deviations were detected in late anthocyanin biosynthetic genes and specific MYB transcription factors that would explain anthocyanin accumulations in the Red oak-leaf cultivar. Taken together, our large-scale transcriptomic and physiologic results suggest that the two environmental parameters (i.e., light quality and nitrate level) (1) utilize independent transcriptional regulatory mechanisms to achieve similar phenotypic and metabolic changes, and (2) induce the variety-specific anthocyanin accumulations via a small number of genes that show different gene expression profiles between the two cultivars.

Transcriptional mechanism of the mouse β4-galactosyltransferase 6 gene in mouse neuroblastoma cell line Neuro-2a.

Lactosylceramide (Lac-Cer) constitutes the backbone structure of various gangliosides whose abnormal expression is associated with malignancy of neuroblastoma. The understanding of the regulatory mechanism of Lac-Cer contributes to the development of neuroblastoma therapy. In this study, the transcriptional mechanism of mouse β4-galactosyltransferase (β4GalT) 6, which is one of Lac-Cer synthase, was analyzed using mouse neuroblastoma cell line Neuro-2a. The -226 to -13 region relative to the most downstream transcriptional start site was determined to be the promoter region by luciferase assay using the 5'-deletion constructs. The mutation into the activating protein (AP) 4-binding site -110/-101 drastically decreased the promoter activity, indicating that this site is mainly implicated in the transcription. Furthermore, the mutation into the GATA-binding site -210/-201 or another AP4-binding site -202/-193 partially decreased the promoter activity. The study suggests that the mouse β4GalT6 gene is transcriptionally regulated by AP4 in cooperation with GATA family transcription factor in neuroblastoma.

Determination of Complex Formation between Drosophila Nrf2 and GATA4 Factors at Selective Chromatin Loci Demonstrates Transcription Coactivation.

Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila. Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.

Endothelial-to-osteoblast transition in normal mouse bone development

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone EC-to-osteoblast (OSB) transition. Here, we investigated whether EC-to-OSB transition also occurs during normal bone formation. We developed an EC and OSB dual-color reporter mouse (DRM) model that marks EC-OSB hybrid cells with red and green fluorescent proteins. We observed EC-to-OSB transition (RFP and GFP co-expression) in both endochondral and intramembranous bone formation during embryonic development and in adults. Co-expression was confirmed in cells isolated from DRM. Bone marrow- and lung-derived ECs underwent transition to OSBs and mineralization in osteogenic medium. RNA-sequencing revealed GATA family transcription factors were upregulated in EC-OSB hybrid cells and knockdown of GATA3 inhibited BMP4-induced mineralization. Our findings support that EC-to-OSB transition occurs during normal bone development and suggest a new paradigm regarding the endothelial origin of OSBs.

SlGATA17, A tomato GATA protein, interacts with SlHY5 to modulate salinity tolerance and germination

The GATA transcription factor family participates in plant biological processes and abiotic stress across various crop species, but their function in response to salinity stress remains unexplored, particularly in tomato. Here, a tomato (Solanum lycopersicum) GATA gene, SlGATA17, was characterized in the tolerance to salinity stress. SlGATA17 had transcription activity in yeast, and the transcriptional activation domain was location on 32–60 amino acids of the N-terminal. SlGATA17 expression was induced by heat, cold, ABA, ACC and IAA treatments, but inhibited by NaCl and PEG treatments. In this study, the Slgata17 mutant plants showed slight injury symptoms compared to wild-type plants under salinity stress, and higher expression levels of stress-related marker genes (SlDREB2A, SlAREB1, SlRD29A and SlP5CS1). Heterologous expression of SlGATA17 in Arabidopsis promoted seed germination under NaCl, ABA and mannitol conditions, and inhibited ABA content and the expression levels of ABA related genes (AtNCED3, AtNCED5, AtNCED9, AtABI3, AtEM1 and AtEM6). However, SlGATA17 transgenic Arabidopsis were hypersensitive to NaCl at the post-germination stage. Yeast two assay and BiFC assay showed that tomato transcription factor ELONGATED HYPOCOTYL (SlHY5) interacts with SlGATA17. Yeast one assay and Dual-Luc assay showed that SlHY5 negatively regulated SlGATA17 expression via binding to the GATA-box of SlGATA17 promoter. SlHY5-SlGATA17 might function as a regulatory module in response to abiotic stress.

MeGATAs, functional generalists in interactions between cassava growth and development, and abiotic stresses.

The proteins with DNA-binding preference to the consensus DNA sequence (A/T) GATA (A/G) belong to a GATA transcription factor family, with a wide array of biological processes in plants. Cassava (Manihot esculenta) is an important food crop with high production of starch in storage roots. Little was however known about cassava GATA domain-containing genes (MeGATAs). Thirty-six MeGATAs, MeGATA1 to MeGATA36, were found in this study. Some MeGATAs showed a collinear relationship with orthologous genes of Arabidopsis, poplar and potato, rice, maize and sorghum. Eight MeGATA-encoded proteins (MeGATAs) analysed were all localized in the nucleus. Some MeGATAs had potentials of binding ligands and/or enzyme activity. One pair of tandem-duplicated MeGATA17-MeGATA18 and 30 pairs of whole genome-duplicated MeGATAs were found. Fourteen MeGATAs showed low or no expression in the tissues. Nine analysed MeGATAs showed expression responses to abiotic stresses and exogenous phytohormones. Three groups of MeGATA protein interactions were found. Fifty-three miRNAs which can target 18 MeGATAs were identified. Eight MeGATAs were found to target other 292 cassava genes, which were directed to radial pattern formation and phyllome development by gene ontology enrichment, and autophagy by Kyoto Encyclopaedia of Genes and Genomes enrichment. These data suggest that MeGATAs are functional generalists in interactions between cassava growth and development, abiotic stresses and starch metabolism.

Escape from Type II JAK2 Inhibition in MPN Depends on AXL/MAPK Activation and Is Targetable

Myeloproliferative neoplasms (MPN) are characterized by dysregulated JAK2 signaling. Clinical type I JAK2 inhibitors as ruxolitinib are hampered by limited disease-modifying effects and occurrence of resistance. Based on pre-clinical data, novel type II JAK2 inhibitors as CHZ868 effectively stabilize inactive JAK2, reduce MPN clones and overcome resistance to ruxolitinib. We study whether MPN cells could escape from type ll JAK2 inhibition, and characterize therapeutic approaches. To assess potential escape from type II JAK inhibition, JAK2V617F MPN cells incl. SET2, UKE-1 and Ba/F3 EPOR Jak2V617F were exposed long-term to CHZ868. Outgrowing cells showed significantly increased IC50 to CHZ868 and reduced susceptibility for apoptosis induction. No second-site mutations in JAK2 were detected. Phosphoproteomic analysis identified >2500 differentially phosphorylated sites with most prominent activation of the MAPK pathway in JAK inhibitor resistant cells. Type II JAK inhibition persistently suppressed JAK2 - STAT3/5 signaling, while MAPK target activation incl. MEK and ERK was prominent also by phospho-immunoblot suggesting MAPK pathway signaling as a driver of escape from type II JAK inhibition. To characterize the molecular basis, we profiled differential gene expression and chromatin accessibility by RNA-sequencing and ATAC-sequencing in MPN cells escaping JAK inhibition. Paired analysis of RNA-seq/ATAC-seq revealed enrichment of H3K27me3 and PRC2 signatures with overall more compacted chromatin. Expression of AXL tyrosine kinase known to signal through the MAPK pathway, was significantly increased along with enriched RAS signaling target gene patterns by GSEA. Motif analysis suggested differential transcriptional programs with loss of ETS and increased accessibility of GATA transcription factor family binding sites. Enhanced AXL expression was confirmed by immunoblotting and depletion of AXL by AXL-specific shRNA knock-down resensitized resistant cells to CHZ868 with decreased IC50. Since these findings pointed towards a role of AXL activation for escape of MPN cells from type II JAK inhibition, we targeted AXL activation using respective inhibitors. AXL inhibitor bemcentinib and AXL/FLT3 inhibitor gilteritinib reduced proliferation of resistant cells with up to 5.6-fold and 3.4-fold decreased IC50, respectively. Given that type II JAK inhibition with CHZ868 maintained suppression of JAK2-STAT3/5 activation in resistant cells, we combined JAK2 and AXL inhibition. Notably, dual JAK2 / AXL inhibition with CHZ868 / bemcentinib or CHZ868 / gilteritinib showed most pronounced inhibitory effects with IC50 similar to values seen in JAK inhibitor sensitive cells and effective induction of apoptosis. Resistant MPN cells engrafted subcutaneously into immunocompromised NSG mice and resulted in robust tumor growth despite JAK2 inhibitor treatment. However, AXL inhibition with bemcentinib reduced progression of type II JAK inhibitor resistant tumors. Combined JAK2 and AXL inhibition showed most pronounced suppression of subcutaneous tumor growth with minimal progression. Given that inhibitors of MAPK signaling targeting e.g. MEK-ERK activation, are in clinical use for other malignancies with favorable tolerability profiles, we then characterized combined JAK2 / MAPK inhibition to overcome AXL-MAPK driven escape of MPN cells from type II JAK inhibitors. Combination of CHZ868 and MEK inhibitor trametinib significantly reduced subcutaneous tumor growth in NSG mice similarly to JAK2 / AXL inhibition. Expression of MAPK pathway effectors as DUSP6 was substantially reduced. Furthermore, intravenous engraftment of JAK inhibitor resistant cells into NSG mice led to substantial bone marrow (BM) infiltration shown by luciferase imaging and hCD45 immunohistochemistry despite treatment with CHZ868. Combined JAK2 / MAPK inhibition with CHZ868 and trametinib significantly reduced BM infiltration by luciferase imaging and histology supporting that escape from type II JAK inhibition is dependent on AXL-MAPK activation. In summary, we report on a novel mechanism of AXL-MAPK driven escape from type II JAK2 inhibition which is targetable in vitro and in vivo. Our findings highlight that rationally designed combination therapy approaches can restore sensitivity to JAK inhibitor therapy in MPN, which may have potential implications for other malignant settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 11984: A Novel Pathogenic Gata6 Variant Identified in a Family With Persistent Truncus Arteriosus, Childhood-Onset Diabetes Mellitus and Spontaneous Intestinal Perforation

GATA6 is a member of the GATA family of transcription factors, among which GATA4, 5 and 6 are known to play critical roles during heart development. Pathogenic variants in GATA6 have been primarily associated with congenital heart disease (CHD) and pancreatic agenesis in humans. Although the phenotypic spectrum associated with pathologic variation in GATA6 has expanded to include developmental delay along with congenital malformations affecting the biliary system and diaphragm, the full disease spectrum of GATA6 pathogenic variation and genotype-phenotype correlations are unclear. Here, we report a family where the father had persistent truncus arteriosus (PTA) and three children had CHD, 2 with PTA and 1 with an atrial septal defect. Notably, the father had childhood-onset diabetes mellitus and one child had spontaneous intestinal perforation (SIP). Using exome sequencing, we identified a novel, heterozygous missense variant in GATA6 (c.1403G>A; p.Cys468Tyr) in affected members. This variant is absent from the gnomAD and ExAC databases and causes a missense change predicted to be damaging by all in silico bioinformatic tools. Sanger sequencing confirmed the exome sequencing results and segregation of this variant. GATA6 p.Cys468Tyr variant affects the highly conserved zinc-finger domain of GATA6 and impedes its ability to bind DNA. Using wild-type and mutant GATA6 constructs driving atrial natriuretic peptide promoter reporter, in vitro luciferase assays showed that p.Cys468Tyr mutant protein exhibited significantly decreased luciferase transcriptional activity. Furthermore, immunofluorescence revealed that p.Cys468Tyr mutant demonstrated an impaired localization pattern in the nucleus and induced protein aggregation. Our findings expand the variant and phenotypic spectrum for GATA6. Our study highlights that GATA6 variants can cause not only cardiac and pancreatic malformations but also gastrointestinal abnormalities, including neonatal SIP. Further investigation is needed to define the mechanisms underlying the full phenotypic spectrum associated with pathogenic variation in GATA6 .

Deleting Gata4 in hepatocytes promoted the progression of NAFLD via increasing steatosis and apoptosis, and desensitizing insulin signaling

Gata4 is a member of the zinc finger GATA transcription factor family and is required for liver development during the embryonic stage. Gata4 expression is repressed during NAFLD progression, however how it functions in this situation remains unclear. Here, Gata4 was deleted specifically in hepatocytes via Cre recombinase driven by the Alb promoter region. Under a high-fat diet (HFD) or methionine and choline deficient diet (MCD), Gata4 knockout (KO) male, but not female, mice displayed more severe NAFLD or NASH, evidenced by increased steatosis, fibrosis, as well as a higher NAS score and serum ALT level. The Gata4KO male liver exposed to a HFD or MCD had a reduced ratio of pACC/ACC, similar to the Gata4KO hepatocytes treated with palmitic acid. More cell apoptosis, which is associated with activated JNK signaling and inhibited NFκB signaling, was observed in the Gata4KO male liver and isolated hepatocytes. However, the inflammatory status in the Gata4KO male liver was similar to the control liver. Importantly, lower activation of AKT signaling in the liver, which is consistent with de-sensitized insulin signaling in isolated hepatocytes, was found in the Gata4KO male. In summary, our data demonstrated that loss of Gata4 in hepatocytes promoted NAFLD progression in male mice.

An NmrA-Like Protein, Lws1, Is Important for Pathogenesis in the Woody Plant Pathogen Lasiodiplodia theobromae.

The NmrA-like proteins have been reported to be important nitrogen metabolism regulators and virulence factors in herbaceous plant pathogens. However, their role in the woody plant pathogen Lasiodiplodia theobromae is less clear. In the current study, we identified a putative NmrA-like protein, Lws1, in L. theobromae and investigated its pathogenic role via gene silencing and overexpression experiments. We also evaluated the effects of external carbon and nitrogen sources on Lws1 gene expression via qRT-PCR assays. Moreover, we analyzed the molecular interaction between Lws1 and its target protein via the yeast two-hybrid system. The results show that Lws1 contained a canonical glycine-rich motif shared by the short-chain dehydrogenase/reductase (SDR) superfamily proteins and functioned as a negative regulator during disease development. Transcription profiling revealed that the transcription of Lws1 was affected by external nitrogen and carbon sources. Interaction analyses demonstrated that Lws1 interacted with a putative GATA family transcription factor, LtAreA. In conclusion, these results suggest that Lws1 serves as a critical regulator in nutrition metabolism and disease development during infection.

O-216 CRISPR/Cas9 mediated knock-out (KO) reveals a divergent role for trophectoderm markers GATA2/3 in the mouse and human preimplantation embryo

Abstract Study question What is the effect of CRISPR/Cas9-mediated KO of trophectoderm (TE) markers GATA2/3 on embryo development and lineage commitment in both mouse and human preimplantation embryos? Summary answer CRISPR/Cas9-mediated KO of GATA2/3 points to interspecies differences in TE regulation and potential cross-talk between trophectoderm and inner cell mass (ICM) in mouse preimplantation embryos. What is known already GATA3 and its isoform GATA2 are major TE markers regulating the first lineage segregation, operating downstream of the HIPPO-pathway. Although recent evidence suggests that the HIPPO-pathway is conserved across mouse and human, it is unknown whether GATA2/3 share similar interspecies function during preimplantation development. GATA3 RNA depletion experiments in mouse embryos revealed a compensatory upregulation of GATA2, that potentially masked the observed phenotype. Upon double KO (DKO), the phenotype appeared more severe, as embryos were unable to cavitate. However, the precise effect of the (D)KO on embryo development was not investigated thoroughly, and should be expanded towards human preimplantation embryos. Study design, size, duration Mouse embryonic stem cells (mESCs), mouse zygotes and donated human spare oocytes were targeted. CRISPR/Cas9 ribonucleoprotein complexes, either targeting Gata3/GATA3, Gata2 or both, were delivered via nucleofection, electroporation or co-injected with sperm, in mESCs, mouse zygotes or human oocytes, respectively. Appropriate non-targeted control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing were applied to check for gene editing efficiency and the impact of KO on embryonic development. Participants/materials, setting, methods The targeted embryos and controls were cultured for 4.5 (mouse) or 6.5 days (human) in vitro. They were stained for different developmental markers, including TEAD4 and CDX2 (TE), OCT4 and SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE). Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency, while in silico predicted off-target sites were evaluated in targeted mESCs. Main results and the role of chance GATA3 KO mouse embryos exhibited morula arrest (94%; n = 16). All GATA3-edited mouse embryos exhibited a reduction of CDX2-positive cells. From the 12 full KO embryos, four showed a decreased number of NANOG-positive blastomeres. No effect was observed for TEAD4 and OCT4. Complete KO morulas were devoid of SOX2 expression. GATA2 KO mouse embryos could still form blastocysts (19% morula arrest, n = 21 embryos), even when harboring 100% frameshift mutations. KO did not noticeably influence cell number nor the expression of GATA3 or NANOG. GATA2/3 DKO mouse embryos could still form blastocysts (38% morula arrest, n = 21), showing a milder phenotype compared to GATA3 KO embryos. In the presumed DKO blastocysts, the whole ICM is NANOG-positive and increased in cell number. SOX2 expression was still retained in the ICM, but presumed polar TE was also SOX2-positive. Three out of four GATA3 KO human embryos harboring 100% frameshift mutations, were surprisingly able to form blastocysts. In one embryo, no morphological TE could be formed, while the others showed one or two CDX2-positive cells in TE. No effect was observed for TEAD4 and OCT4. In addition, all KO embryos displayed an increase in ICM/TE fraction. Some outer cells in KO blastocysts were NANOG-positive. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mESCs. The observations of the study will be consolidated by increasing the sample size, especially in human. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the GATA transcription factor family could significantly improve our understanding of several pregnancy-related complications related to trophectoderm specification, such as early miscarriage or preeclampsia. Trial registration number NA

Meta-Analysis of Immune Induced Gene Expression Changes in Diverse Drosophila melanogaster Innate Immune Responses

Simple SummaryOrganisms can be infected by a wide range of pathogens, including bacteria, viruses, and parasites. Following infection, the host mounts an immune response to attempt to eliminate the pathogen. These responses are often specific to the type of pathogen and mediated by the expression of specialized genes. We have characterized the expression changes induced in host Drosophila fruit flies following infection by multiple types of pathogens, and identified a small number of genes that show expression changes in each infection. This includes genes that are known to be involved in pathogen resistance, and others that have not been previously studied as immune response genes. These findings provide new insight into transcriptional changes that accompany Drosophila immunity. They may suggest possible roles for the differentially expressed genes in innate immune responses to diverse classes of pathogens, and serve to identify candidate genes for further empirical study of these processes.Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, we performed meta-analysis of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. We identified 62 genes that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, we also identified 21 genes that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. We further identified 31 genes with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. Our study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction.

Preliminary Interpretations of Epigenetic Profiling of Cord Blood in Preeclampsia

Preeclampsia (PE) is characterized by new-onset hypertension after 20 weeks of pregnancy and results in high maternal and fetal mortality worldwide. It has been reported that PE is associated with abnormalities in the umbilical cord and cord blood. However, previous studies were focused primarily on the transcriptomics level, while the underlying gene regulatory landscapes are still unclear. Thus, we performed the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) using the umbilical cord blood samples collected from a patient with superimposed PE and three healthy donors to uncover the chromatin accessibility changes attributed to PE. We have identified genes associated with immunomodulation and hypoxia response that have higher chromatin accessibility close to their transcription start sites. Motif analysis indicated that the GATA family transcription factor binding was enriched in PE and may play an essential regulatory role in the disease progression. Overall, our findings provide an overview of gene regulatory programs and the corresponding downstream pathways associated with PE that may influence the placenta function and fetal growth.

Distinct expression and prognostic values of GATA transcription factor family in human ovarian cancer

Accumulated studies have provided controversial evidences of expression patterns and prognostic value of the GATA family in human ovarian cancer. In the present study, we accessed the distinct expression and prognostic roles of 7 individual members of GATA family in ovarian cancer (OC) patients through Oncomine analysis, CCLE analysis, Human Protein Atlas (HPA), Kaplan–Meier plotter (KM plotter) database, cBioPortal and Metascape. Our results indicated that GATA1, GATA3, GATA4 and TRPS1 mRNA and protein expression was significantly higher in OC than normal samples. High expression of GATA1, GATA2, and GATA4 were significantly correlated with better overall survival (OS), while increased GATA3 and GATA6 expression were associated with worse prognosis in OC patients. GATA1, GATA2, GATA3 and GATA6 were closely related to the different pathological histology, pathological grade, clinical stage and TP53 mutation status of OC. The genetic variation and interaction of the GATA family may be closely related to the pathogenesis and prognosis of OC, and the regulatory network composed of GATA family genes and their neighboring genes are mainly involved in Notch signaling pathway, Th1 and Th2 cell differentiation and Hippo signaling pathway. Transcriptional GATA1/2/3/4/6 could be prognostic markers and potential therapeutic target for OC patients.