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.

PC Gene Affects Milk Production Traits in Dairy Cattle.

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.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation.

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.

The hematopoietic microenvironment of the fetal liver and transient abnormal myelopoiesis associated with Down syndrome: A review.

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.

TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia.

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.

An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

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 MEP­like cell line HEL western blotting, RT­qPCR, lentivirus­mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation­specific (ETS) transcription factor friend leukemia integration factor 1 (Fli­1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1­mediated 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 transformation­specific­related 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 shRNA­mediated 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.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

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.

Epigenetic dissection of human blood group genes reveals regulatory elements and detailed characteristics of KEL and four other loci.

BACKGROUND: Blood typing is essential for safe transfusions and is performed serologically or genetically. Genotyping predominantly focuses on coding regions, but non-coding variants may affect gene regulation, as demonstrated in the ABO, FY and XG systems. To uncover regulatory loci, we expanded a recently developed bioinformatics pipeline for discovery of non-coding variants by including additional epigenetic datasets. METHODS: Multiple datasets including ChIP-seq with erythroid transcription factors (TFs), histone modifications (H3K27ac, H3K4me1), and chromatin accessibility (ATAC-seq) were analyzed. Candidate regulatory regions were investigated for activity (luciferase assays) and TF binding (electrophoretic mobility shift assay, EMSA, and mass spectrometry, MS). RESULTS: In total, 814 potential regulatory sites in 47 blood-group-related genes were identified where one or more erythroid TFs bound. Enhancer candidates in CR1, EMP3, ABCB6, and ABCC4 indicated by ATAC-seq, histone markers, and co-occupancy of 4 TFs (GATA1/KLF1/RUNX1/NFE2) were investigated but only CR1 and ABCC4 showed increased transcription. Co-occupancy of GATA1 and KLF1 was observed in the KEL promoter, previously reported to contain GATA1 and Sp1 sites. TF binding energy scores decreased when three naturally occurring variants were introduced into GATA1 and KLF1 motifs. Two of three GATA1 sites and the KLF1 site were confirmed functionally. EMSA and MS demonstrated increased GATA1 and KLF1 binding to the wild-type compared to variant motifs. DISCUSSION: This combined bioinformatics and experimental approach revealed multiple candidate regulatory regions and predicted TF co-occupancy sites. The KEL promoter was characterized in detail, indicating that two adjacent GATA1 and KLF1 motifs are most crucial for transcription.

[Mitochondrial metabolism and erythroid differentiation].

The transcription factor GATA-1 is essential for erythroid differentiation. Recently, FAM210B, which encodes a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1. To clarify the role of FAM210B, we depleted endogenous FAM210B in human iPS-derived erythroid progenitor (HiDEP-1) cells, and found that erythroid differentiation was more pronounced in the FAM210B depleted cells. Comprehensive metabolite analysis revealed a decline in mitochondrial function accompanied by increased lactate production, indicative of anaerobic glycolysis. Mass spectrometry revealed that FAM210B could interact with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. This review will discuss the potential association between mitochondrial metabolism and erythropoiesis.

Toll-interacting protein is activated by the transcription factor GATA1 and Sp1 to negatively regulate NF-κB and MAPK pathways in the Japanese eel (Anguilla japonica).

Toll-interacting protein (Tollip) serves as a crucial inhibitory factor in the modulation of Toll-like receptor (TLR)-mediated innate immunological responses. The structure and function of Tollip have been well documented in mammals, yet the information in teleost remained limited. This work employed in vitro overexpression and RNA interference in vivo and in vitro to comprehensively examine the regulatory effects of AjTollip on NF-κB and MAPK signaling pathways. The levels of p65, c-Fos, c-Jun, IL-1, IL-6, and TNF-α were dramatically reduced following overexpression of AjTollip, whereas knocking down AjTollip in vivo and in vitro enhanced those genes' expression. Protein molecular docking simulations showed AjTollip interacts with AjTLR2, AjIRAK4a, and AjIRAK4b. A better understanding of the transcriptional regulation of AjTollip is crucial to elucidating the role of Tollip in fish antibacterial response. Herein, we cloned and characterized a 2.2 kb AjTollip gene promoter sequence. The transcription factors GATA1 and Sp1 were determined to be associated with the activation of AjTollip expression by using promoter truncation and targeted mutagenesis techniques. Collectively, our results indicate that AjTollip suppresses the NF-κB and MAPK signaling pathways, leading to the decreased expression of the downstream inflammatory factors, and GATA1 and Sp1 play a vital role in regulating AjTollip expression.

Inherent genome instability underlies trisomy 21-associated myeloid malignancies.

Constitutional trisomy 21 (T21) is a state of aneuploidy associated with high incidence of childhood acute myeloid leukemia (AML). T21-associated AML is preceded by transient abnormal myelopoiesis (TAM), which is triggered by truncating mutations in GATA1 generating a short GATA1 isoform (GATA1s). T21-associated AML emerges due to secondary mutations in hematopoietic clones bearing GATA1s. Since aneuploidy generally impairs cellular fitness, the paradoxically elevated risk of myeloid malignancy in T21 is not fully understood. We hypothesized that individuals with T21 bear inherent genome instability in hematopoietic lineages that promotes leukemogenic mutations driving the genesis of TAM and AML. We found that individuals with T21 show increased chromosomal copy number variations (CNVs) compared to euploid individuals, suggesting that genome instability could be underlying predisposition to TAM and AML. Acquisition of GATA1s enforces myeloid skewing and maintenance of the hematopoietic progenitor state independently of T21; however, GATA1s in T21 hematopoietic progenitor cells (HPCs) further augments genome instability. Increased dosage of the chromosome 21 (chr21) gene DYRK1A impairs homology-directed DNA repair as a mechanism of elevated mutagenesis. These results posit a model wherein inherent genome instability in T21 drives myeloid malignancy in concert with GATA1s mutations.

ARHGAP18 is Upregulated by Transcription Factor GATA1 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma.

Rho GTPase activating protein 18 (ARHGAP18), a member of the RhoGAP gene family that increases GTP hydrolysis and inhibits RhoGTPase, was recently discovered to play a role in the development of breast cancer. However, its exact biological role in hepatocellular carcinoma (HCC) remains unclear. In our present study, we comprehensively assessed ARHGAP18 expression and its correlation with the prognostic value of cancer patients in databases. Cell proliferation and colony formation assays were employed to monitor cell growth. Luciferase reporter assay, Chromatin immunoprecipitation qPCR (ChIP-qPCR), immunofluorescence were performed for mechanism research. The expression of genes and proteins was detected by real-time PCR and western blotting. According to the findings of this research, ARHGAP18 protein levels are increased in HCC tissues compared to adjacent nontumor tissues, and ARHGAP18 overexpression is associated with poor survival. The results of a gain- and loss-of-function experiment with HCC cells in vitro demonstrated that ARHGAP18 stimulated cell proliferation, migration, and invasion. Mechanistically, we found that the transcription factor GATA binding protein 1 (GATA1) could bind to the ARHGAP18 promoter and facilitate ARHGAP18 expression. Further studies revealed that the effects of ARHGAP18 silencing on HCCLM3 and Bel-7402 cells were blocked by GATA1 overexpression. In conclusion, GATA1-mediated ARHGAP18 up-regulation plays an important role in HCC tumorigenesis and might be a potential therapeutic target for HCC.

Down syndrome and leukemia: An insight into the disease biology and current treatment options.

Children with Down syndrome (DS) have a 10- to 20-fold greater predisposition to develop acute leukemia compared to the general population, with a skew towards myeloid leukemia (ML-DS). While ML-DS is known to be a subtype with good outcome, patients who relapse face a dismal prognosis. Acute lymphocytic leukemia in DS (DS-ALL) is considered to have poor prognosis. The relapse rate is high in DS-ALL compared to their non-DS counterparts. We have a better understanding about the mutational spectrum of DS leukemia. Studies using animal, embryonic stem cell- and induced pluripotent stem cell-based models have shed light on the mechanism by which these mutations contribute to disease initiation and progression. In this review, we list the currently available treatment strategies for DS-leukemias along with their outcome with emphasis on challenges with chemotherapy-related toxicities in children with DS. We focus on the mechanisms of initiation and progression of leukemia in children with DS and highlight the novel molecular targets with greater success in preclinical trials that have the potential to progress to the clinic.

Transient erythroblastopenia due to a GATA1 variant in an infant female.

Diamond-Blackfan anemia (DBA) is a congenital anemia with erythroid cell aplasia. Most of the causative genes are ribosomal proteins. GATA1, a hematopoietic master transcription factor required for erythropoiesis, also causes DBA. GATA1 is located on Xp11.23; therefore, DBA develops only in males in an X-linked inheritance pattern. Here, we report a case of transient erythroblastopenia and moderate anemia in a female newborn infant with a de novo GATA1 variant. In this patient, increased methylation of the GATA1 wild-type allele was observed in erythroid cells. Skewed lyonization of GATA1 may cause mild transient erythroblastopenia in a female patient.

Aggregates of nonmuscular myosin IIA in erythrocytes associate with GATA1- and GFI1B-related thrombocytopenia.

BACKGROUND: The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES: To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS: We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS: We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 µm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION: Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

A novel GATA1 variant p.G229D causing the defect of procoagulant platelet formation.

INTRODUCTION: GATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. OBJECTIVE: To determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. METHODS: Comprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. RESULTS: The patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbß3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. CONCLUSIONS: The G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders.

Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination.

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process.

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

Synergistic roles of DYRK1A and GATA1 in trisomy 21 megakaryopoiesis.

Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.

HEXIM1 is an essential transcription regulator during human erythropoiesis.

ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.