Association of GATA3 expression in triple-positive breast cancer with overall survival and immune cell infiltration.

Breast cancer remains a leading cause of cancer-related mortality among women, with triple-positive breast cancer (TPBC) being a particularly aggressive subtype. GATA binding protein 3 (GATA3) plays a crucial role in the luminal differentiation of breast epithelium and T-cell differentiation. However, the relationship between GATA3 and immune infiltration in TPBC remains unclear. This study collected and analyzed TPBC data from The Cancer Genome Atlas (TCGA), METABRIC, and GSE123845 databases. Univariate and multivariate Cox regression analyses, along with Kaplan-Meier survival analyses, were employed to assess the prognostic value of GATA3 and other clinical features. Subsequently, Gene Set Enrichment Analysis (GSEA) was conducted to explore the potential biological functions and regulatory mechanisms of GATA3 in TPBC. Additionally, ssGSEA analysis revealed the connection between GATA3 and immune infiltration. And the effects of neoadjuvant chemotherapy and immunotherapy on GATA3 expression were also explored. Finally, clinical samples were used to detect the relationship between GATA3 expression and tumor infiltrating lymphocyte (TIL) levels. Our results demonstrated that GATA3 was significantly overexpressed in TPBC tissues compared to normal tissues (P < 0.05). A positive correlation between GATA3 mRNA and protein levels was observed (R = 0.55, P < 0.05). Notably, high GATA3 expression was associated with poor overall survival (HR = 1.24, 95% confidence interval (CI) 1.25-11.76, P < 0.05). GSEA indicated significant enrichment of immune-related gene sets in low GATA3 expression groups. Furthermore, pathologic complete response (pCR) patients exhibited significantly lower GATA3 expression compared to residual disease (RD) patients. Mutation analysis revealed higher PIK3CA and TP53 mutation rates in high GATA3 expression groups. Finally, clinical validation data showed that the degree of TILs was significantly higher in the low GATA3 expression group. In conclusion, this study suggests that high GATA3 expression may be associated with poor prognosis and may reduce immune infiltration in TPBC.

Modulations of the mTORC2-GATA3 axis by an isorhamnetin activated endosomal-lysosomal system of the J774.1 macrophage-like cell line.

The endosomal-lysosomal system represents a crucial degradation pathway for various extracellular substances, and its dysfunction is linked to cardiovascular and neurodegenerative diseases. This degradation process involves multiple steps: (1) the uptake of extracellular molecules, (2) transport of cargos to lysosomes, and (3) digestion by lysosomal enzymes. While cellular uptake and lysosomal function are reportedly regulated by the mTORC1-TFEB axis, the key regulatory signal for cargo transport remains unclear. Notably, our previous study discovered that isorhamnetin, a dietary flavonoid, enhances endosomal-lysosomal proteolysis in the J774.1 cell line independently of the mTORC1-TFEB axis. This finding suggests the involvement of another signal in the mechanism of isorhamnetin. This study analyzes the molecular mechanism of isorhamnetin using transcriptome analysis and reveals that the transcription factor GATA3 plays a critical role in enhanced endosomal-lysosomal degradation. Our data also demonstrate that mTORC2 regulates GATA3 nuclear translocation, and the mTORC2-GATA3 axis alters endosomal formation and maturation, facilitating the efficient transport of cargos to lysosomes. This study suggests that the mTORC2-GATA3 axis might be a novel target for the degradation of abnormal substances.

GATA2­miR­374a axis promotes vascular smooth muscle cells proliferation, migration via targeting circTADA2A/RORA axis.

Evidence has shown that microRNAs (miRNAs/miRs) play key roles in biological functions of vascular smooth muscle cells (VSMCs). However, the role of miR-374a in VSMCs remains to be elucidated. The present study aimed to explore the influence of miR-374a on VSMCs and its molecular mechanism. The expression level of miR-374a was measured by reverse transcription-quantitative (RT-q) PCR. MTT and Transwell assay were employed to assess the role of miR-374a in proliferation and migration of VSMCs. To order to determine miR-374a targets, a dual-luciferase reporter assay was conducted, which was further verified by rescue experiments. Chromatin Immunoprecipitation Assay and JASPAR databases were applied to explore the regulatory association between GATA binding protein 2 (GATA2) and miR-374a. Western blotting or RT-qPCR were employed to detect the protein expression levels of GATA2 or RAR-related orphan receptor A (RORA). The present study found that miR-374a was elevated in VSMCs following treatment with platelet-derived growth factor-BB (PDGF-BB) compared with that in control group. In addition, the results demonstrated that a higher expression of a miR-374a could promote proliferation and migration of VSMCs while miR-374a inhibitor suppressed the PDGF-BB-induced proliferation and migration of VSMCs in vitro. Furthermore, circTADA2A bound to miR-374a and then upregulated RORA expression, which resulted in inhibition in VSMCs proliferation and migration. On the other hand, the result indicated that GATA2 overexpression could augment the proliferation, migration of PDGF-bb-induced VSMCs, which could be rescued by miR-374a inhibitor. The findings suggested that the GATA2/circTADA2A-miR-374a axis promoted the proliferation and migration of VSMCs by targeting RORA, which were closely related to atherosclerosis (AS). Thus the results might offer a new therapeutic target for AS.

A Rare Coincidence of Three Inherited Diseases in a Family with Cardiomyopathy and Multiple Extracardiac Abnormalities.

A genetic diagnosis of primary cardiomyopathies can be a long-unmet need in patients with complex phenotypes. We investigated a three-generation family with cardiomyopathy and various extracardiac abnormalities that had long sought a precise diagnosis. The 41-year-old proband had hypertrophic cardiomyopathy (HCM), left ventricular noncompaction, myocardial fibrosis, arrhythmias, and a short stature. His sister showed HCM, myocardial hypertrabeculation and fibrosis, sensorineural deafness, and congenital genitourinary malformations. Their father had left ventricular hypertrophy (LVH). The proband's eldest daughter demonstrated developmental delay and seizures. We performed a clinical examination and whole-exome sequencing for all available family members. All patients with HCM/LVH shared a c.4411-2A>C variant in ALPK3, a recently known HCM-causative gene. Functional studies confirmed that this variant alters ALPK3 canonical splicing. Due to extracardiac symptoms in the female patients, we continued the search and found two additional single-gene disorders. The proband's sister had a p.Trp329Gly missense in GATA3, linked to hypoparathyroidism, sensorineural deafness, and renal dysplasia; his daughter had a p.Ser251del in WDR45, associated with beta-propeller protein-associated neurodegeneration. This unique case of three monogenic disorders in one family shows how a comprehensive approach with thorough phenotyping and extensive genetic testing of all symptomatic individuals provides precise diagnoses and appropriate follow-up, embodying the concept of personalized medicine. We also present the first example of a splicing functional study for ALPK3 and describe the genotype-phenotype correlations in cardiomyopathy.

The Variation in the Traits Ameliorated by Inhibitors of JAK1/2, TGF-ß, P-Selectin, and CXCR1/CXCR2 in the Gata1low Model Suggests That Myelofibrosis Should Be Treated by These Drugs in Combination.

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-ß (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-ß) signaling and TGF-ß trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-ß content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-ß or CXCL1).

The Assessment of CDX1, IHH, SHH, GATA4, FOXA2, FOXF1 in Congenital Intra-Abdominal Adhesions.

Congenital abdominal adhesions are a rare condition that can result in a small bowel obstruction at any age, more frequently in pediatric populations. The cause remains unknown, and the importance of aberrant congenital bands is related to the difficulty of diagnosis, and cases of death with late detection have been documented. This research examines the expression of Caudal Type Homeobox 1 (CDX1), Indian Hedgehog (IHH), Sonic Hedgehog (SHH), GATA Binding Protein 4 (GATA4), Forkhead Box A2 (FOXA2) and Forkhead Box F1 (FOXF1) gene expression in human abdominal congenital adhesion fibroblast and endothelium cells by chromogenic in situ hybridization, with the aim of elucidating their potential association with the etiology of congenital intra-abdominal adhesion band development. The potential genes' signals were examined using a semi-quantitative approach. Significant correlations were observed between the expression of CDX1 (p <.001) and SHH (p=0.032) genes in fibroblasts from congenital intra-abdominal adhesions compared to fibroblasts from control peritoneal tissue. Statistically significant very strong correlations were found between the CDX1 and IHH comparing endothelium and fibroblast cells in congenital abdominal adhesion bands. There was no statistically significant difference found in the distribution of IHH, FOXA2, GATA4, and FOXF1 between the fibroblasts and endothelium of the patients compared to the control group. The presence of notable distinctions and diverse associations suggests the potential involvement of numerous morpho-pathogenetic processes in the development of intraabdominal adhesions.

Mesonephric-type adenocarcinomas of the ovary: prevalence, diagnostic reproducibility, outcome, and value of PAX2.

Mesonephric-type (or -like) adenocarcinomas (MAs) of the ovary are an uncommon and aggressive histotype. They appear to arise through transdifferentiation from Müllerian lesions creating diagnostic challenges. Thus, we aimed to develop a histologic and immunohistochemical (IHC) approach to optimize the identification of MA over its histologic mimics, such as ovarian endometrioid carcinoma (EC). First, we screened 1,537 ovarian epithelial neoplasms with a four-marker IHC panel of GATA3, TTF1, ER, and PR followed by a morphological review of EC to identify MA in retrospective cohorts. Interobserver reproducibility for the distinction of MA versus EC was assessed in 66 cases initially without and subsequently with IHC information (four-marker panel). Expression of PAX2, CD10, and calretinin was evaluated separately, and survival analyses were performed. We identified 23 MAs from which 22 were among 385 cases initially reported as EC (5.7%) and 1 as clear cell carcinoma. The interobserver reproducibility increased from fair to substantial (κ = 0.376-0.727) with the integration of the four-marker IHC panel. PAX2 was the single most sensitive and specific marker to distinguish MA from EC and could be used as a first-line marker together with ER/PR and GATA3/TTF1. Patients with MA had significantly increased risk of earlier death from disease (hazard ratio = 3.08; 95% CI, 1.62-5.85; p < 0.0001) compared with patients with EC, when adjusted for age, stage, and p53 status. A diagnosis of MA has prognostic implications for stage I disease, and due to the subtlety of morphological features in some tumors, a low threshold for ancillary testing is recommended.

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N) terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1 (friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor (MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Prognostic significance of GATA2 in patients with MDS/AML: a systematic review and meta-analysis.

GATA2 deficiency syndrome is a heterogeneous disorder characterized by a high risk of developing myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML). We conducted a meta-analysis of the literature to explore the prognostic significance of GATA2 mutations in patients diagnosed with MDS/AML, as previous studies have yielded conflicting findings regarding the impact of GATA2 mutations on patient outcomes. We conducted a comprehensive literature search of databases such as PubMed, Embase, the Cochrane Library, and the Web of Science to obtain studies on the prognostic significance of GATA2 mutations in patients with MDS/AML that were published through January 2024. We extracted the hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS), disease-free survival (DFS), and event-free survival (EFS). The meta-analysis was conducted by choosing either a fixed-effect model or a random-effect model, depending on the variability observed among the studies. A total of 13 cohort studies were included in the final meta-analysis, including 2714 patients with MDS, of whom 644 had GATA2 mutations. The results revealed that GATA2 mutations had an adverse impact on OS (HR = 1.54, 95% CI = 1.08-2.18, P = 0.02) and EFS (HR = 1.32, 95% CI = 1.01-1.72, P = 0.04), but no significant effect on DFS (HR = 1.21, 95% CI = 0.89-1.64, P = 0.23). GATA2 mutations were associated with a significantly shorter OS in MDS patients (HR = 2.56, 95% CI = 1.42-4.06, P = 0.002) but not in AML patients (HR = 1.08, 95% CI = 0.92-1.26, P = 0.37). Our meta-analysis revealed that GATA2 mutations are associated with unfavourable outcomes in patients with MDS/AML. Furthermore, patients harbouring these mutations should be prioritized for aggressive therapeutic interventions.

Down syndrome-associated leukaemias: current evidence and challenges.

Children with Down syndrome (DS) are at increased risk of developing haematological malignancies, in particular acute megakaryoblastic leukaemia and acute lymphoblastic leukaemia. The microenvironment established by abnormal haematopoiesis driven by trisomy 21 is compounded by additional genetic and epigenetic changes that can drive leukaemogenesis in patients with DS. GATA-binding protein 1 (GATA1) somatic mutations are implicated in the development of transient abnormal myelopoiesis and the progression to myeloid leukaemia of DS (ML-DS) and provide a model of the multi-step process of leukaemogenesis in DS. This review summarises key genetic drivers for the development of leukaemia in patients with DS, the biology and treatment of ML-DS and DS-associated acute lymphoblastic leukaemia, late effects of treatments for DS-leukaemias and the focus for future targeted therapy.

Insights into the efficient degradation mechanism of extracellular proteases mediated by Purpureocillium lilacinum.

Protease secretion is crucial for degrading nematode cuticles using nematophagous fungus Purpureocillium lilacinum, but the secretion pattern of protease remains poorly understood. This study aimed to explore the degradation mechanism of proteases by investigating the characteristics of protease secretion under various carbon and nitrogen sources, and different carbon to nitrogen (C:N) ratios in P. lilacinum. The results showed that corn flour as a carbon source and yeast extract as a nitrogen source specifically induced protease secretion in P. lilacinum. P. lilacinum produced significant amounts of gelatinase and casein enzyme at C:N ratios of 10:1, 20:1, and 40:1, indicating that higher C:N ratios were more beneficial for secreting extracellular proteases. Proteomic analysis revealed 14 proteases, including 4 S8 serine endopeptidases and one M28 aminopeptidase. Among four S8 serine peptidases, Alp1 exhibited a high secretion level at C:N ratio less than 5:1, whereas PR1C, PR1D, and P32 displayed higher secretion levels at higher C:N ratios. In addition, the transcription levels of GATA transcription factors were investigated, revealing that Asd-4, A0A179G170, and A0A179HGL4 were more prevalent at a C:N ratio of 40:1. In contrast, the transcription levels of SREP, AreA, and NsdD were higher at lower C:N ratios. The putative regulatory profile of extracellular protease production in P. lilacinum, induced by different C:N ratios, was analyzed. The findings offered insights into the complexity of protease production and aided in the hydrolytic degradation of nematode cuticles.

A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching.

BACKGROUND: GATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered. CASE PRESENTATION: The proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband. CONCLUSIONS: NGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

The different faces of GATA2 deficiency: implications for therapy and surveillance.

GATA2 deficiency is one of the most common genetic predispositions to pediatric myelodysplastic syndrome (MDS) in children and adolescents. The wide spectrum of disease comprises, among others, hematological, immunological and pulmonary manifestations, as well as occasionally distinct organ anomalies. Due to the elevated risk of progression, nearly all individuals with GATA2-related MDS eventually undergo a hematopoietic stem cell transplantation (HSCT) at some point in their lives. Nevertheless, the optimal timing, method, and even the indication for HSCT in certain cases are still matter of debate and warrant further research. In this article, we report five patients with different hematological and immunological manifestations of GATA2 deficiency ranging from immunodeficiency and refractory cytopenia of childhood without chromosomal aberrations to relapsed MDS-related acute myeloid leukemia. We discuss the adopted strategies, including intensity of surveillance, indication and timing of HSCT, based on morphological, clinical and molecular markers, as well as individual patient needs. We conclude that a better characterization of the natural disease course, a better understanding of the prognostic significance of somatic aberrations and a thorough evaluation of patients´ perspectives and preferences are required to achieve a personalized approach aimed at improving the care of these patients.

miR-107 reverses the multidrug resistance of gastric cancer by targeting the CGA/EGFR/GATA2 positive feedback circuit.

Chemotherapy is still the main therapeutic strategy for gastric cancer (GC). However, most patients eventually acquire multidrug resistance (MDR). Hyperactivation of the EGFR signaling pathway contributes to MDR by promoting cancer cell proliferation and inhibiting apoptosis. We previously identified the secreted protein CGA as a novel ligand of EGFR and revealed a CGA/EGFR/GATA2 positive feedback circuit that confers MDR in GC. Herein, we outline a microRNA-based treatment approach for MDR reversal that targets both CGA and GATA2. We observed increased expression of CGA and GATA2 and increased activation of EGFR in GC samples. Bioinformatic analysis revealed that miR-107 could simultaneously target CGA and GATA2, and the low expression of miR-107 was correlated with poor prognosis in GC patients. The direct interactions between miR-107 and CGA or GATA2 were validated by luciferase reporter assays and Western blot analysis. Overexpression of miR-107 in MDR GC cells increased their susceptibility to chemotherapeutic agents, including fluorouracil, adriamycin, and vincristine, in vitro. Notably, intratumor injection of the miR-107 prodrug enhanced MDR xenograft sensitivity to chemotherapies in vivo. Molecularly, targeting CGA and GATA2 with miR-107 inhibited EGFR downstream signaling, as evidenced by the reduced phosphorylation of ERK and AKT. These results suggest that miR-107 may contribute to the development of a promising therapeutic approach for the treatment of MDR in GC.

Intricate MIB1-NOTCH-GATA6 Interactions in Cardiac Valvular and Septal Development.

Genome-wide association studies and experimental mouse models implicate the MIB1 and GATA6 genes in congenital heart disease (CHD). Their close physical proximity and conserved synteny suggest that these two genes might be involved in analogous cardiac developmental processes. Heterozygous Gata6 loss-of-function mutations alone or humanized Mib1 mutations in a NOTCH1-sensitized genetic background cause bicuspid aortic valve (BAV) and a membranous ventricular septal defect (VSD), consistent with MIB1 and NOTCH1 functioning in the same pathway. To determine if MIB1-NOTCH and GATA6 interact in valvular and septal development, we generated compound heterozygote mice carrying different Mib1 missense (Mib1K735R and Mib1V943F) or nonsense (Mib1R530X) mutations with the Gata6STOP/+ heterozygous null mutation. Combining Mib1R530X/+ or Mib1K735R/+ with Gata6STOP/+ does not affect Gata6STOP/+ single mutant phenotypes. In contrast, combining Mib1V943F/+ with Gata6STOP/+ decreases the incidence of BAV and VSD by 50%, suggesting a suppressive effect of Mib1V943F/+ on Gata6STOP/+. Transcriptomic and functional analyses revealed that while the EMT pathway term is depleted in the Gata6STOP/+ mutant, introducing the Mib1V943F variant robustly enriches this term, consistent with the Mib1V943F/+ phenotypic suppression of Gata6STOP/+. Interestingly, combined Notch1 and Gata6 insufficiency led to a nearly fully penetrant VSD but did not affect the BAV phenotype, underscoring the complex functional relationship between MIB1, NOTCH, and GATA6 in valvular and septal development.

A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal RHD Exons.

Introduction: With over 360 blood group antigens in systems recognized, there are antigens, such as RhD, which demonstrate a quantitative reduction in antigen expression due to nucleotide variants in the non-coding region of the gene that result in aberrant splicing or a regulatory mechanism. This study aimed to evaluate bioinformatically predicted GATA1-binding regulatory motifs in the RHD gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal RHD exons. Methods: Publicly available open chromatin region data were overlayed with GATA1 motif candidates in RHD. Genomic DNA from weak D, Del or D- samples with normal RHD exons (n = 13) was used to confirm RHD zygosity by quantitative PCR. Then, RHD promoter, intron 1, and intron 2 regions were amplified for Sanger sequencing to detect potential disruptions in the GATA1 motif candidates. Electrophoretic mobility shift assay (EMSA) was performed to assess GATA1-binding. Luciferase assays were used to assess transcriptional activity. Results: Bioinformatic analysis identified five of six GATA1 motif candidates in the promoter, intron 1 and intron 2 for investigation in the samples. Luciferase assays showed an enhancement in transcription for GATA1 motifs in intron 1 and for intron 2 only when the R 2 haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel RHD c.1-110A>C variant disrupted the GATA1 motif in the promoter which was supported by a lack of a GATA1 supershift in the EMSA and 73% transcriptional activity in the luciferase assay. Two samples were D+/D- chimeras. Conclusion: The bioinformatic predictions enabled the identification of a novel DEL allele, RHD c.1-110A>C, which disrupted the GATA1 motif in the proximal promoter. Although the majority of the samples investigated here remain unexplained, we provide GATA1 targets which may benefit future RHD regulatory investigations.

GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

Human trophectoderm becomes multi-layered by internalization at the polar region.

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Ventricular Septal Defects: Molecular Pathways and Animal Models.

Ventricular septation is a complex process which involves the major genes of cardiac development, acting on myocardial cells from first and second heart fields, and on mesenchymal cells from endocardial cushions. These genes, coding for transcription factors, interact with each other, and their differential expression conditions the severity of the phenotype. In this chapter, we will describe the formation of the ventricular septum in the normal heart, as well as the molecular mechanisms leading to the four main anatomic types of ventricular septal defects: outlet, inlet, muscular, and central perimembranous, resulting from failure of development of the different parts of the ventricular septum. Experiments on animal models, particularly transgenic mouse lines, have helped us to decipher the molecular determinants of ventricular septation. However, a precise description of the anatomic phenotypes found in these models is mandatory to achieve a better comprehension of the complex mechanisms responsible for the various types of VSDs.