BACKGROUND

In eukaryotic genomes, about 10% of genes are arranged in a head-to-head (H2H) orientation, and the distance between the transcription start sites of each gene pair is closer than 1 kb. Two genes in an H2H pair are prone to co-express and co-function. There have been many studies on bidirectional promoters. However, the mechanism by which H2H genes are regulated at the transcriptional level still needs further clarification, especially with regard to the co-regulation of H2H pairs. In this study, we first used the Hi-C data of chromatin linkages to identify spatially interacting H2H pairs, and then integrated ChIP-seq data to compare H2H gene pairs with and without evidence of spatial interactions in terms of their binding transcription factors (TFs). Using ChIP-seq and DNase-seq data, histones and DNase associated with H2H pairs were identified. Furthermore, we looked into the connections between H2H genes in a human co-expression network.

RESULTS

We found that i) Similar to the behaviour of two genes within an H2H pair (intra-H2H pair), a gene pair involving two distinct H2H pairs (inter-H2H pair) which interact with each other spatially, share common transcription factors (TFs); ii) TFs of intra- and inter-H2H pairs are distributed differently. Factors such as HEY1, GABP, Sin3Ak-20, POL2, E2F6, and c-MYC are essential for the bidirectional transcription of intra-H2H pairs; while factors like CTCF, BDP1, GATA2, RAD21, and POL3 play important roles in coherently regulating inter-H2H pairs; iii) H2H gene blocks are enriched with hypersensitive DNase and modified histones, which participate in active transcriptions; and iv) H2H genes tend to be highly connected compared with non-H2H genes in the human co-expression network.

CONCLUSIONS

Our findings shed new light on the mechanism of the transcriptional regulation of H2H genes through their linear and spatial interactions. For intra-H2H gene pairs, transcription factors regulate their transcriptions through bidirectional promoters, whereas for inter-H2H gene pairs, transcription factors are likely to regulate their activities depending on the spatial interaction of H2H gene pairs. In this way, two distinctive groups of transcription factors mediate intra- and inter-H2H gene transcriptions respectively, resulting in a highly compact gene regulatory network.

The GATA2 gene codes for a master hematopoietic transcription factor that is essential for the proliferation and maintenance of hematopoietic stem and progenitor cells. Heterozygous germline mutations in GATA2 have been initially associated with several clinical entities that are now collectively defined as GATA2 deficiency. Despite pleiotropic clinical manifestations, the high propensity for the development of myelodysplastic syndromes (MDS) constitutes the most common clinical denominator of this major MDS predisposition syndrome. The immunological phenotypes can be variable and mostly include deficiency of monocytes and/or B cells. Thus far, nearly 380 GATA2-deficient patients had been reported, with a roughly estimated prevalence of myeloid neoplasia of at least 75%. The most common abnormal karyotypes associated with GATA2-related MDS are monosomy 7, der(1;7) and trisomy 8. The overall clinical penetrance seems to be nearly complete for this transcriptopathy disorder. The high-risk MDS subtypes and karyotypes, and the underlying immunodeficiency guide decision-making toward timely stem cell transplantation.

The down-regulation of DOC-2/DAB2 gene, which encodes a unique phosphoprotein modulating signal pathways elicited by exogenous stimuli, is often associated with several cancer types; however, the underlying mechanism is still unknown. Dramatically different expression levels of DOC-2/DAB2 mRNA and protein are observed among several human transitional cell carcinoma (TCC) cell lines, suggesting that transcriptional regulation may play a role in these cells. In this study, we have shown that the histone acetylation status associated with the 5' upstream regulatory sequence of DOC-2/DAB2 gene is one of the key determinants for its gene expression. In addition, GATA6 but not other GATA family members, such as GATA2 and GATA4, can specifically induce DOC-2/DAB2 promoter activity, although GATA transcription factors share a very similar DNA-binding sequence. We also show that increased histone acetylation and the presence of GATA6 have a synergistic effect on DOC-2/DAB2 promoter activity, which results in the elevation of DOC-2/DAB2 protein expression. Thus, we conclude that transcriptional regulation of DOC-2/DAB2 gene in human TCC is determined by histone acetylation and a specific transcription factor (i.e., GATA6), which underlie the reduced DOC-2/DAB2 protein expression in TCC cells.

Monoamine oxidase A (MAOA) plays important roles in the pathogenesis of several neurological and cardiovascular disorders. The mechanism of transcriptional regulation of MAOA under basal and pathological conditions, however, remains incompletely understood. Here, we report systematic identification and characterization of cis elements and transcription factors that govern the expression of MAOA gene. Extensive computational analysis of MAOA promoter, followed by 5'-promoter deletion/reporter assays, revealed that the -71/-40 bp domain was sufficient for its basal transcription. Gel-shift and chromatin immunoprecipitation assays provided evidence of interactions of the transcription factors GATA-binding protein 2 (GATA2), Sp1 and TATA-binding protein (TBP) with this proximal promoter region. Consistently, over-expression of GATA2, Sp1 and TBP augmented MAOA promoter activity in a coordinated manner. In corroboration, siRNA-mediated down-regulation of GATA2/Sp1/TBP repressed the endogenous MAOA expression as well as transfected MAOA promoter activity. Tumor necrosis factor-α and forskolin activated MAOA transcription that was reversed by Sp1 siRNA; in support, tumor necrosis factor-α- and forskolin-induced activities were enhanced by ectopic over-expression of Sp1. On the other hand, MAOA transcription was diminished upon exposure of neuroblasts or cardiac myoblasts to ischemia-like conditions because of reduced binding of GATA2/Sp1/TBP with MAOA promoter. In conclusion, this study revealed previously unknown roles of GATA2, Sp1 and TBP in modulating MAOA expression under basal as well as pathophysiological conditions such as inflammation and ischemia, thus providing new insights into the molecular basis of aberrant MAOA expression in neuronal/cardiovascular disease states. Dysregulation of monoamine oxidase A (MAOA) have been implicated in several behavioral and neuronal disease states. Here, we identified three crucial transcription factors (GATA2, Sp1 and TBP) that regulate MAOA gene expression in a coordinated manner. Aberrant MAOA expression under pathophysiological conditions including inflammation and ischemia is mediated by altered binding of GATA2/Sp1/TBP with MAOA proximal promoter. Thus, these findings provide new insights into pathogenesis of several common diseases. GATA2, GATA-binding protein 2; Sp1, specificity protein 1; TBP, TATA-binding protein.

Identification of transcription factors that directly convert pluripotent stem cells (PSCs) into endothelial and blood cells and advances in the chemical modifications of messenger RNA (mRNA) offer alternative nucleic acid-based transgene-free approach for scalable production of these cells for drug screening and therapeutic purposes. Here we evaluated the effect of 5' and 3' RNA untranslated regions (UTRs) on translational efficiency of chemically-modified synthetic mRNA (modRNA) in human PSCs and showed that an addition of 5'UTR indeed enhanced protein expression. With the optimized modRNAs expressing ETV2 or ETV2 and GATA2, we are able to produce VE-cadherin+ endothelial cells and CD34+CD43+ hematopoietic progenitors, respectively, from human PSCs as well as non-human primate (NHP) PSCs. Overall, our findings provide valuable information on the design of in vitro transcription templates being used in PSCs and its broad applicability for basic research, disease modeling, and regenerative medicine.

Endothelial cells (ECs) form the lining of lymph and blood vessels. Changes in tissue requirements or wounds may cause ECs to behave as tip or stalk cells. Alternatively, they may differentiate into mesenchymal cells (MCs). These processes are known as EC activation and endothelial-to-mesenchymal transition (EndMT), respectively. EndMT, Tip, and Stalk EC behaviors all require SNAI1, SNAI2, and Matrix metallopeptidase (MMP) function. However, only EndMT inhibits the expression of VE-cadherin, PECAM1, and VEGFR2, and also leads to EC detachment. Physiologically, EndMT is involved in heart valve development, while a defective EndMT regulation is involved in the physiopathology of cardiovascular malformations, congenital heart disease, systemic and organ fibrosis, pulmonary arterial hypertension, and atherosclerosis. Therefore, the control of EndMT has many promising potential applications in regenerative medicine. Despite the fact that many molecular components involved in EC activation and EndMT have been characterized, the system-level molecular mechanisms involved in this process have not been elucidated. Toward this end, hereby we present Boolean network model of the molecular involved in the regulation of EC activation and EndMT. The simulated dynamic behavior of our model reaches fixed and cyclic patterns of activation that correspond to the expected EC and MC cell types and behaviors, recovering most of the specific effects of simple gain and loss-of-function mutations as well as the conditions associated with the progression of several diseases. Therefore, our model constitutes a theoretical framework that can be used to generate hypotheses and guide experimental inquiry to comprehend the regulatory mechanisms behind EndMT. Our main findings include that both the extracellular microevironment and the pattern of molecular activity within the cell regulate EndMT. EndMT requires a lack of VEGFA and sufficient oxygen in the extracellular microenvironment as well as no FLI1 and GATA2 activity within the cell. Additionally Tip cells cannot undergo EndMT directly. Furthermore, the specific conditions that are sufficient to trigger EndMT depend on the specific pattern of molecular activation within the cell.

Among the gliomas, glioblastoma (GBM) is the highest grade and the most malignant glioma tumor. GATA2 is a hematopoietic factor that has been intensely studied in hematopoietic malignancies. Recently, the functions of GATA2 as an oncogene in other types of human cancer have been reported. However, no role for GATA2 in the development and progression of glioma has been reported to date. In the present study, we found that the expression level of GATA2 is upregulated in GBM and is correlated with GBM outcome. Ectopic expression of GATA2 or RNAi-mediated knockdown of GATA2 significantly enhanced or inhibited proliferation, migration and invasion of glioma cells. Moreover, we found that epidermal growth factor receptor and extracellular signal-regulated kinase, as upstream components of the signaling pathway, upregulate GATA2 expression; moreover, GATA2 promotes Elk-1 expression. Therefore, a genetic approach or pharmacological intervention targeting GATA2 could potentially serve as an effective strategy for treating glioma patients.

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box-GATA-ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

Vasculogenesis and hematopoiesis are co-localized in the embryonic body, but precise phenotypes of the cells contributing to these processes are not defined. The aim of this study was to characterize phenotypic profiles and location of putative vasculogenic and hematopoietic cellular progenitors in the embryonic mouse heart. Confocal microscopy, as well as ultrastructural and stereomicroscopic analyses, was performed on immunohistochemical whole-mount-stained or sectioned hearts at stages 11.5-14 dpc. A FASC analysis was conducted to quantify putative vasculogenic and hematopoietic cells. We found subepicardial blood islands in the form of foci of accumulation of cells belonging to erythroblastic and megakaryocytic lineages at various stages of maturation, exhibiting phenotypes: GATA2(+)/CD41(+), GATA2(-)/CD41(+), GATA2(+)/CD71(-), GATA2(-)/CD71(+), Fli1(+)/CD71(+), Fli1(-)/CD71(+), with a majority of cells expressing the Ter119 antigen, but none of them expressing Flk1. The subepicardium and the outflow tract endothelium were recognized to be the areas where progenitor cells were scattered or adjoining the endothelial cells. These progenitor cells were characterized as possessing the following antigens: CD45(+)/Fli1(+), CD41(+)/Flk1(+), Flk1(+)/Fli1(+). A FACS analysis demonstrated that the CD41/Flk1 double-positive population of cells constituted 2.68% of total cell population isolated from 12.5 dpc hearts. Vessels and tubules were positive for CD31, Flk1, Fli1, Tie2, including blood islands endothelia. The endocardial wall endothelia were found to function as an anchoring apparatus for megakaryocytes releasing platelets into the cardiac cavities. Phenotypic characteristics of vasculogenic (Flk1(+)/Fli1(+)) and hematopoietic (GATA2(+)/CD71(+), CD41(+)/GATA2(+)) progenitors, as well as the putative hemogenic endothelium (Flk1(+)/CD41(+)) in embryonic mouse hearts, have been presented. Cardiac blood islands, the subepicardium and endothelium of the outflow tract cushions have been defined as areas where these progenitor cells can be found.

During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.

Several natural compounds exhibit strong capacity for decreasing triglyceride accumulation, enhancing lipolysis and inducing apoptosis. The present study reports the anti-adipogenic effects of Silybum marianum (SL), Citrus aurantium (CA), Taraxacum officinale (TO), resveratrol (RE), Curcuma longa (CU), caffeine (CF), oleuropein (OL) and docosahexaenoic acid (DHA) in reducing differentiation and increasing lipolysis and apoptosis. Analyses were performed on human primary visceral pre-adipocytes after 10 (P10) and 20 (P20) days of treatment during differentiation and on mature adipocytes after 7 days of treatment (A7). The percentage of apoptosis induced by TO extract in P10 and P20 cells was significantly higher than that induced by all other compounds and in CTRL cells. Triglyceride accumulation was significantly lower in cells treated with DHA, CF, RE in comparison to cells treated with OL and in CTRL cells. Treatments with CF, DHA and OL significantly incremented lipolysis in P20 cells in comparison to other compounds and in CTRL cells. On the contrary, the treatment of A7 cells with OL, CA and TO compounds significantly increased cell lipolysis. The addition of CF in differentiating P20 pre-adipocytes significantly increased the expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT1, WNT3A, SFRP5, and DLK1. Genes involved in promoting adipogenesis such as CCND1, CEBPB and SREBF1 were significantly down-regulated by the treatment. The screening of bioactive compounds for anti-adipogenic effects showed that in differentiating cells TO extract was the most effective in inducing apoptosis and CF and DHA extracts were more efficient in inhibition of differentiation and in induction of cell lipolysis.

The zinc finger transcription factors GATA1 and GATA2 participate in mast cell development. Although the expression of these factors is regulated in a cell lineage-specific and differentiation stage-specific manner, their regulation during mast cell development has not been clarified. Here, we show that the GATA2 mRNA level was significantly increased while GATA1 was maintained at low levels during the differentiation of mast cells derived from mouse bone marrow (BMMCs). Unlike in erythroid cells, forced expression or small interfering RNA (siRNA)-mediated knockdown of GATA1 rarely affected GATA2 expression, and vice versa, in mast cells, indicating the absence of cross-regulation between Gata1 and Gata2 genes. Chromatin immunoprecipitation assays revealed that both GATA factors bound to most of the conserved GATA sites of Gata1 and Gata2 loci in BMMCs. However, the GATA1 hematopoietic enhancer (G1HE) of the Gata1 gene, which is essential for GATA1 expression in erythroid and megakaryocytic lineages, was bound only weakly by both GATA factors in BMMCs. Furthermore, transgenic-mouse reporter assays revealed that the G1HE is not essential for reporter expression in BMMCs and peritoneal mast cells. Collectively, these results demonstrate that the expression of GATA factors in mast cells is regulated in a manner quite distinct from that in erythroid cells.

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.

The atypical cadherin FAT4 has established roles in regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in CCBE1 and ADAMTS3, encoding a matrix protein and protease, respectively, that regulate activity of the key pro-lymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1 and ADAMTS3 mutations underlie Hennekam syndrome suggested that all three genes might function in a common pathway. We identified FAT4 as a target gene of GATA2, a key transcriptional regulator of lymphatic vascular development and in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.

OBJECTIVE

To study the molecular pharmacological basis of the YiSui ShenXu Granule, a complex prescription of the Chinese traditional medicine used to treat beta-thalassemia.

METHODS

Real-time quantitative PCR method had been applied to analyze the genes expression: gamma-globin, Ckit, EpoR, Spi, FKLF, GATA1 and GATA2 in K562 cell treated and untreated with this complex prescription and its each single herbal medicine.

RESULTS

The results showed that this complex prescription increased the gamma-globin, EpoR, Spi and FKLF expression and decreased the Ckit, GATA1 and GATA2 expression. And all single herbal medicines of this complex prescription could change some of those gene expressions, but not the same as the complex prescription. Even that, this study results indicated that the YiSui ShenXu Granule has its molecular pharmacological basis in treating beta-thalassemia.

BACKGROUND

The majority of cases with severe pulmonary alveolar proteinosis (PAP) are caused by auto-antibodies against GM-CSF. A multitude of genetic and exogenous causes are responsible for few other cases. Goal of this study was to determine the prevalence of GATA2 deficiency in children and adults with PAP and hematologic disorders.

METHODS

Of 21 patients with GM-CSF-autoantibody negative PAP, 13 had no other organ involvement and 8 had some form of hematologic disorder. The latter were sequenced for GATA2.

RESULTS

Age at start of PAP ranged from 0.3 to 64 years, 4 patients were children. In half of the subjects GATA2-sequence variations were found, two of which were considered disease causing. Those two patients had the typical phenotype of GATA2 deficiency, one of whom additionally showed a previously undescribed feature - a cholesterol pneumonia. Hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (MDS). A 4 year old child with MDS and DiGeorge Syndrome Type 2 was rescued with repetitive whole lung lavages and her PAP was cured with heterologous stem cell transplant.

CONCLUSIONS

In children and adults with severe GM-CSF negative PAP a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor GATA2. Treatment with whole lung lavages as well as stem cell transplant may be successful.

Hematopoietic development requires the transcription factor GATA-2, and GATA-2 mutations cause diverse pathologies, including leukemia. GATA-2-regulated enhancers increase Gata2 expression in hematopoietic stem/progenitor cells and control hematopoiesis. The +9.5-kb enhancer activates transcription in endothelium and hematopoietic stem cells (HSCs), and its deletion abrogates HSC generation. The -77-kb enhancer activates transcription in myeloid progenitors, and its deletion impairs differentiation. Since +9.5-/- embryos are HSC deficient, it was unclear whether the +9.5 functions in progenitors or if GATA-2 expression in progenitors solely requires -77. We further dissected the mechanisms using -77;+9.5 compound heterozygous (CH) mice. The embryonic lethal CH mutation depleted megakaryocyte-erythrocyte progenitors (MEPs). While the +9.5 suffices for HSC generation, the -77 and +9.5 must reside on one allele to induce MEPs. The -77 generated burst-forming unit-erythroid through the induction of GATA-1 and other GATA-2 targets. The enhancer circuits controlled signaling pathways that orchestrate a GATA factor-dependent blood development program.

Primary lymphedema is a rare inherited condition characterized by swelling of body tissues caused by accumulation of fluid, especially in the lower limbs. In many patients, primary lymphedema has been associated with variations in a number of genes involved in the development and maintenance of the lymphatic system. In this study, we performed a genetic screening in patients affected by primary lymphedema using a next generation sequencing (NGS) approach. With this technology, based on a custom-made oligonucleotide probe library, we were able to analyze simultaneously in each patient all the coding exons of 10 genes (FLT4, FOXC2, CCBE1, GJC2, MET, HGF, GATA2, SOX18, VEGFC, KIF11) associated with primary lymphedema. In the study population, composed of 45 familial and 71 sporadic cases, we identified the presence of rare variants with a potential pathogenic effect in 33% of subjects. Overall, we found a total of 36 different rare nucleotidic alterations, 30 of which had not been previously described. Among these, we identified 23 mutations that we considered most likely to be disease causing. Patients with an FLT4 or FOXC2 alteration accounted for the largest percentage of the sample, followed by MET, HGF, KIK11, GJC2 and GATA2. No alterations were identified in SOX18, VEGFC, and CCBE1 genes. In conclusion, we showed that NGS technology can be successfully applied to perform molecular screening of lymphedema-associated genes in large cohort of patients with a reasonable effort in terms of cost, work, and time.

TAL1 (SCL/TAL1, T-cell acute leukemia protein 1) is a transcription factor that is involved in the process of hematopoiesis and leukemogenesis. It participates in blood cell formation, forms mesoderm in early embryogenesis, and regulates hematopoiesis in adult organisms. TAL1 is essential in maintaining the multipotency of hematopoietic stem cells (HSC) and keeping them in quiescence (stage G0). TAL1 forms complexes with various transcription factors, regulating hematopoiesis (E2A/HEB, GATA1-3, LMO1-2, Ldb1, ETO2, RUNX1, ERG, FLI1). In these complexes, TAL1 regulates normal myeloid differentiation, controls the proliferation of erythroid progenitors, and determines the choice of the direction of HSC differentiation. The transcription factors TAL1, E2A, GATA1 (or GATA2), LMO2, and Ldb1 are the major components of the SCL complex. In addition to normal hematopoiesis, this complex may also be involved in the process of blood cell malignant transformation. Upregulation of C-KIT expression is one of the main roles played by the SCL complex. Today, TAL1 and its partners are considered promising therapeutic targets in the treatment of T-cell acute lymphoblastic leukemia.

Identification of effective culture conditions to maintain and possibly expand human HSPCs in vitro is an important goal. Recent advances highlight the efficacy of chemicals in maintaining and converting cell fates. We screened 186 chemicals and found that a combination of CHIR-99021, Forskolin and OAC1 (CFO) maintained human CD34-positive cells in vitro. Efficiency of the culture system was characterized using flow cytometry for CD34-positive cells, a colony-forming assay and xeno-transplants. We found that human CD34-positive cells treated with this combination had enhanced expression of human HSPC markers and increased haematopoietic re-populating ability in immune-deficient mice. Single-cell RNA-seq analyses showed that the in vitro cultured human CD34-positive cells were heterogeneous. We found that CFO supports maintenance of human CD34-positive cells by activating HOXA9, GATA2 and AKT-cAMP signaling pathway. These data have implications in therapies requiring maintenance and/or expansion of human HSPCs.

BACKGROUND

DNA methylation of gene promoters is associated with transcriptional inactivation. Changes in DNA methylation can lead to differences in gene expression levels and thereby influence disease development. We hypothesized that epigenetics underlies the pathogenesis of minimal change nephrotic syndrome (MCNS).

METHODS

Genome-wide DNA methylation changes between relapse and remission in monocytes (n = 6) and naive T helper cells (Th0s) (n = 4) isolated from patients with MCNS were investigated using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method. We confirmed the MIAMI results using bisulfite-pyrosequencing analysis. Expression analysis was performed using quantitative real-time PCR.

RESULTS

Three gene loci (GATA2, PBX4, and NYX) were significantly less methylated in Th0s during relapse than in remission, compared to none in monocytes. In addition, the distance distribution from the regression line of all probes in MIAMI was significantly different between monocytes and Th0s. The mRNA levels of the three genes in Th0s were not significantly different between relapse and remission.

CONCLUSIONS

Our results demonstrate that the change in DNA methylation patterns from remission to relapse in MCNS occurs predominantly in Th0s rather than in monocytes and suggest that epigenetic regulation in Th0s underlies the pathogenesis of MCNS.

Mutations of the GATA binding protein 2 (GATA2) gene in myeloid malignancies usually cluster in the zinc finger 1 (ZF1) and the ZF2 domains. Mutations in different locations of GATA2 may have distinct impact on clinico-biological features and outcomes in AML patients, but little is known in this aspect. In this study, we explored GATA2 mutations in 693 de novo non-M3 AML patients and identified 44 GATA2 mutations in 43 (6.2%) patients, including 31 in ZF1, 10 in ZF2, and three outside the two domains. Different from GATA2 ZF2 mutations, ZF1 mutations were closely associated with French-American-British (FAB) M1 subtype, CEBPA double mutations (CEBPAdouble-mut), but inversely correlated with FAB M4 subtype, NPM1 mutations, and FLT3-ITD. ZF1-mutated AML patients had a significantly longer overall survival (OS) than GATA2-wild patients and ZF2-mutated patients in total cohort as well as in those with intermediate-risk cytogenetics and normal karyotype. ZF1 mutations also predicted better disease-free survival and a trend of better OS in CEBPAdouble-mut patients. Sequential analysis showed GATA2 mutations could be acquired at relapse. In conclusion, GATA2 ZF1 mutations are associated with distinct clinico-biological features and predict better prognosis, different from ZF2 mutations, in AML patients.