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-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β 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-β 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-β or CXCL1).

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.

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.
METHODS: 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.

In previous work, we found that PC was differentially expressed in cows at different lactation stages. Thus, we deemed that PC may be a candidate gene affecting milk production traits in dairy cattle. In this study, we found the polymorphisms of PC by resequencing and verified their genetic associations with milk production traits by using an animal model in a cattle population. In total, we detected six single-nucleotide polymorphisms (SNPs) in PC. The single marker association analysis showed that all SNPs were significantly associated with the five milk production traits (p < 0.05). Additionally, we predicted that allele G of 29:g.44965658 in the 5\' regulatory region created binding sites for TF GATA1 and verified that this allele inhibited the transcriptional activity of PC by the dual-luciferase reporter assay. In conclusion, we proved that PC had a prominent genetic effect on milk production traits, and six SNPs with prominent genetic effects could be used as markers for genomic selection (GS) in dairy cattle, which is beneficial for accelerating the improvement in milk yield and quality in Chinese Holstein cows.

Transcriptional mechanisms establish and maintain complex genetic and protein networks to control cell state transitions. The hematopoietic transcription factor GATA1 is a master regulator of erythropoiesis and megakaryopoiesis, and human GATA1 genetic variants cause anemia and megakaryoblastic leukemia. Multiomic analyses revealed that GATA1 controls expression of transporters and metabolic enzymes that dictate intracellular levels of endogenous small molecules, including heme, metal ions, and sphingolipids. Besides its canonical function as a hemoglobin component, heme facilitates or antagonizes GATA1 function to regulate erythropoiesis via mechanisms dependent or independent of the heme-binding transcription factor BTB domain and CNC homology 1 (BACH1). GATA1 regulates the expression of genes encoding heme biosynthetic enzymes and BACH1. GATA1 maintains homeostasis of bioactive ceramides during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Disrupting ceramide homeostasis impairs critical cytokine signaling and is detrimental to erythroid cells. During erythroid maturation, GATA1 induces a zinc transporter switch that favors export versus import, thus dictating the intracellular zinc level, erythroblast survival, and differentiation. In aggregate, these studies support an emerging paradigm in which GATA factor-dependent transcriptional mechanisms control the intracellular levels of endogenous small molecules and small molecule-dependent feedback loops that serve as vital effectors of transcription factor activity, genome function, and cell state transitions.

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome is a distinct form of leukemia or preleukemia that mirrors the hematological features of acute megakaryoblastic leukemia. However, it typically resolves spontaneously in the early stages. TAM originates from fetal liver (FL) hematopoietic precursor cells and emerges due to somatic mutations in GATA1 in utero. In TAM, progenitor cells proliferate and differentiate into mature megakaryocytes and granulocytes. This process occurs both in vitro, aided by hematopoietic growth factors (HGFs) produced in the FL, and in vivo, particularly in specific anatomical sites like the FL and blood vessels. The FL\'s hematopoietic microenvironment plays a crucial role in TAM\'s pathogenesis and may contribute to its spontaneous regression. This review presents an overview of current knowledge regarding the unique features of TAM in relation to the FL hematopoietic microenvironment, focusing on the functions of HGFs and the pathological features of TAM.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP‑like cell line HEL western blotting, RT‑qPCR, lentivirus‑mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation‑specific (ETS) transcription factor friend leukemia integration factor 1 (Fli‑1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1‑mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation‑specific‑related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA‑mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.