The transcriptional regulation of the gene encoding α-synuclein (SNCA) is thought to play a critical role in the pathogenesis of Parkinson's disease (PD), as common genetic variability in this gene is associated with an elevated risk of developing PD. However, the relevant mechanisms are still poorly understood. So far, only few proteins have been identified as transcription factors (TFs) of SNCA in cellular models. Here we show that two of these TFs bind to the DNA in human brain tissue: the zinc finger protein ZSCAN21 occupies a region within SNCA intron 1, as described before, while GATA2 occupies a specific region within intron 2, where we have identified a new binding site within the complex structure of the 5'-promoter region of SNCA. Electrophoretic mobility shift assays confirmed these binding sites. Genetic investigations revealed no polymorphisms or mutations within these sites. A better understanding of TF-DNA interactions within SNCA may allow to develop novel therapies designed to reduce α-synuclein levels.

BACKGROUND

Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up- or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSHβ) by liganded thyroid hormone receptor (TR).

RESULTS

In an effort to better understand the mechanism that drives TSHβ down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSHβ promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSHβ promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner.

CONCLUSIONS

Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.

In a recent issue of Cell, Kumar and colleagues uncovered a synthetic lethal interaction between oncogenic KRAS and the transcription factor GATA2 in non-small cell lung carcinoma. Pharmacological inhibition of GATA2-mediated pathways with bortezomib and fasudil results in dramatic tumor inhibition. These observations unveil new armamentaria to fight this deadly disease.

Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.

PDGF acts as an autocrine and paracrine factor in certain tumors through upregulation of the PDGF beta-receptor expression. In order to elucidate the control mechanism for the receptor expression, we have isolated an enhancer from two P1 clones that together contain a 102 kb NotI region covering the entire human PDGFRB gene. They were partially digested with TspI and cloned into the PDGFRB enhancer trap vector to make a library for identification of enhancers. The digested DNA containing enhancer was identified by expression of GFP when transfected in PDGF beta-receptor expressing cells. One of the enhancer clones was further examined by making several deletion mutants in a luciferase vector. This enhancer was most active in neuroblastoma cells, IMR32 and BE2, but less active in hemangioma and in smooth muscle cell lines. Chip assay revealed that SP1, AP2, and GATA2 bound the enhancer in BE2 cells. Their interaction occurred dependently of the cell cycle and synchronously with their binding to the promoter. Transfection of GATA2 alone or with Ets, which binds adjacent to GATA, resulted in differentiation of BE2 cells in parallel with increased PDGF beta-receptor expression. Furthermore, over-expression of the PDGF beta-receptor in BE2 cells induced neurite extension.

OBJECTIVE

This research was conducted to investigate the potential of mouse embryonic stem (ES) cells to differentiate in vitro into gonadotropes.

METHODS

Undifferentiated ES cells were maintained on mitomycin C-inactivated fibroblasts in the presence of leukemia inhibitory factor (LIF). By a 5-day hanging drop culture devoid of them, ES cells were induced to form multidifferentiated structures called embryoid bodies (EBs). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze gene expression of gonadotrope markers in EBs at different time points during the culture.

RESULTS

Homeo box gene expressed in ES cells (Hesx1), LIM homeobox protein 3 (Lhx3), paired like homeodomain factor 1 (Prop1), GATA binding protein 2 (GATA2), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) mRNAs were detected at day 6 EBs and maintained throughout the culture to day 56. FSHbeta and LHbeta proteins were expressed in EBs from day 6 onward. Immunofluorescent labeling of FSHbeta and LHbeta showed that specific staining was restricted to the cytoplasm of some differentiated EB cells. With the prolongation of EB culture, the number of positive cells increased significantly. Both monohormonal and bihormonal cells were present, mainly in clusters within EBs and sparsely distributed among the outermost cells surrounding the EBs.

CONCLUSIONS

These results indicate that mouse ES cells can give rise to mature gonadotrope-like cells in EBs. It also shows that EBs may serve as a novel model system to study the development and function of gonadotropes.

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. We show that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3, which are activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3-expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1 Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. Our study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation.

Androgens regulate prostate physiology, and exert their effects through the androgen receptor. We hypothesized that androgen deprivation needs additional transcription factors to orchestrate the changes taking place in the gland after castration and for the adaptation of the epithelial cells to the androgen-deprived environment, ultimately contributing to the origin of castration-resistant prostate cancer. This study was undertaken to identify transcription factors that regulate gene expression after androgen deprivation by castration (Cas). For the sake of comparison, we extended the analysis to the effects of administration of a high dose of 17β-estradiol (E2) and a combination of both (Cas+E2). We approached this by (i) identifying gene expression profiles and enrichment terms, and by searching for transcription factors in the derived regulatory pathways; and (ii) by determining the density of putative transcription factor binding sites in the proximal promoter of the 10 most up- or down-regulated genes in each experimental group in comparison to the controls Gapdh and Tbp7. Filtering and validation confirmed the expression and localized EVI1 (Mecom), NFY, ELK1, GATA2, MYBL1, MYBL2, and NFkB family members (NFkB1, NFkB2, REL, RELA and RELB) in the epithelial and/or stromal cells. These transcription factors represent major regulators of epithelial cell survival and immaturity as well as an adaptation of the gland as an immune barrier in the absence of functional stimulation by androgens. Elk1 was expressed in smooth muscle cells and was up-regulated after day 4. Evi1 and Nfy genes are expressed in both epithelium and stroma, but were apparently not affected by androgen deprivation.

Patients with GATA2 haploinsufficiency have a significant predisposition to developing cytopenias, unique infectious manifestations, and myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). We report a unique case of a patient who presented with B-cell acute lymphoblastic leukemia (B-ALL) and was subsequently diagnosed with monocytopenia and mycobacterium avium complex (MonoMAC) syndrome/GATA2 haploinsufficiency. The development of MDS/AML in patients with GATA2 haploinsufficiency is well described, however, the development of ALL has not been reported in the literature. ALL may be associated with GATA2 haploinsufficiency. Clinicians should be attuned to the features of the MonoMAC syndrome in patients with ALL that would prompt additional testing and alter treatment.

We identified two novel GATA2 mutations in acute myeloid leukemia (AML). One mutation (p.R308P-GATA2) was a R308P substitution within the zinc finger (ZF)-1 domain, and the other (p.A350_N351ins8-GATA2) was an eight-amino-acid insertion between A350 and N351 residues within the ZF-2 domain. p.R308P-GATA2 did not affect DNA-binding and transcriptional activities, while p.A350_N351ins8-GATA2 reduced them, and impaired G-CSF-induced granulocytic differentiation of 32D cells. Although p.A350_N351ins8-GATA2 did not show a dominant-negative effect over wild-type (Wt)-GATA2 by the reporter assay, it might be involved in the pathophysiology of AML by impairing myeloid differentiation because of little Wt-GATA2 expression in primary AML cells harboring the p.A350_N351ins8 mutation.

BACKGROUND

A 29-year old patient presented with granulomatous lung disease and a family history of myelodysplastic syndrome/acute myeloid leukemia. She appeared to be a carrier of a mutation in the transcription factor GATA2. The case adds to the recent described heterogeneous clinical manifestations and syndromes in which, against a background of hematologic disorders, GATA2 mutations have been demonstrated, such as the Monomac and Emberger syndromes. In patients with a granulomatous disease and a history of (familial) hematologic disorders, the occurence of GATA2 mutations should be considered, as to gain further insight in the occurrence of granulomatous disease in a possible distinct phenotype among GATA2 mutation carriers.

BACKGROUND

Epigenetic modifications likely control the fate of hematopoietic stem cells (HSCs). The chromatin-modifying agents (CMAs), 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), have previously been shown to expand HSCs from cord blood and marrow. Here we assessed whether CMA can also expand HSCs present in growth factor-mobilized human peripheral blood (MPB).

METHODS

5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines, and the cells were cultured for 9 days.

RESULTS

After culture, a 3.6 ± 0.5-fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5-fold expansion of primitive colony-forming unit (CFU)-mix, and a 2.2 ± 0.5-fold expansion of long-term cobblestone-area-forming cells (CAFCs) was observed in 5azaD/TSA-expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather, a reduction in CD34+CD90+ cells (0.7 ± 0.1-fold) and CAFCs (0.3 ± 0.1-fold) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA-expanded MPB cells in contrast to controls. 5azaD/TSA-expanded MPB cells retained in vivo hematopoietic engraftment capacity.

CONCLUSIONS

MPB CD34+ cells from donors can be expanded using 5azaD/TSA, and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSC numbers for patients who fail to mobilize.

The development and function of stem and progenitor cells that produce blood cells are vital in physiology. GATA-binding protein 2 (GATA2) mutations cause GATA-2 deficiency syndrome involving immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA-2 physiological activities necessitate that it be strictly regulated, and cell type-specific enhancers fulfill this role. The +9.5 intronic enhancer harbors multiple conserved cis-elements, and germline mutations of these cis-elements are pathogenic in humans. Since mechanisms underlying how GATA2 enhancer disease mutations impact hematopoiesis and pathology are unclear, we generated mouse models of the enhancer mutations. While a multi-motif mutant was embryonically lethal, a single-nucleotide Ets motif mutant was viable, and steady-state hematopoiesis was normal. However, the Ets motif mutation abrogated stem/progenitor cell regeneration following stress. These results reveal a new mechanism in human genetics, in which a disease predisposition mutation inactivates enhancer regenerative activity, while sparing developmental activity. Mutational sensitization to stress that instigates hematopoietic failure constitutes a paradigm for GATA-2 deficiency syndrome and other contexts of GATA-2-dependent pathogenesis.

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2^mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2^mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator.This article is highlighted in the In This Issue feature, p. 813.

The underlying mechanism of the Chemokine-C Receptor 7 (CCR7) that leads to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. CCR7 is considered crucial for migration and invasion and has been associated with the risk of miscarriage. However, the functional role of CCR7 in RSA is not fully understood. Our study found that CCR7 mRNA and protein abundance were significantly decreased in the villous from RSA patients compared with healthy controls. Knockdown of CCR7 caused a significant reduction of migration and invasion in JAR and JEG-3 cells. Meanwhile, CCR7 functioned as a positive upstream factor of the AKT pathway contributing to the expression of GATA2, promoting trophoblast migration, and invasion via MMP2. Notably, a decreased abundance of CCR7 was positively correlated with the phosphorylation of AKT and with an abundance of GATA2 and MMP2 in human villous specimens of RSA compared with the control group. CCL19, a ligand of CCR7, could promote trophoblast migration and invasion by activating the deregulation of the CCR7-mediated pathway in RSA. We are convinced that CCR7 and its downstream factors may be possible mechanisms for the pathogenesis of RSA.

Chromatin conformation plays a key role in regulating gene expression and controlling cell differentiation. However, the whole-genome chromatin conformation changes that occur during leukemia cell differentiation are poorly understood. Here, we characterized the changes in chromatin conformation, histone states, chromatin accessibility, and gene expression using an all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation model. The results showed that the boundaries of topological associated domains (TADs) were stable during differentiation; however, the chromatin conformations within several specific TADs were obviously changed. By combining H3K4me3, H3K27ac, and Hi-C signals, we annotated the differential gene-regulatory chromatin interactions upon ATRA induction. The gains and losses of the gene-regulatory chromatin interactions are significantly correlated with gene expression and chromatin accessibility. Finally, we found that the loss of GATA2 expression and DNA binding are crucial for the differentiation process, and changes in the chromatin structure around the GATA2 regulate its expression upon ATRA induction. This study provided both statistical insights and experimental details regarding the relationship between chromatin conformation changes and transcription regulation during leukemia cell differentiation, and the results suggested that the chromatin conformation is a new type of potential drug target for cancer therapy.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Varicose veins of lower extremities (VVs) are a common multifactorial vascular disease. Genetic factors underlying VVs development remain largely unknown. Here we report the first large-scale study of VVs performed on a freely available genetic data of 408,455 European-ancestry individuals. We identified the 12 reliably associated loci that explain 13% of the SNP-based heritability, and prioritized the most likely causal genes CASZ1, PIEZO1, PPP3R1, EBF1, STIM2, HFE, GATA2, NFATC2, and SOX9. VVs-associated variants within these loci exhibited pleiotropic effects on several phenotypes including blood pressure/hypertension and blood cell traits. Gene set enrichment analysis revealed gene categories related to abnormal vasculogenesis. Genetic correlation analysis confirmed known epidemiological associations between VVs and deep venous thrombosis, weight, rough labor, and standing job, and found a genetic overlap with multiple traits that have not been previously suspected to share common genetic background with VVs. These traits included educational attainment, fluid intelligence and prospective memory scores, walking pace (negative correlation with VVs), smoking, height, number of operations, pain, and gonarthrosis (positive correlation with VVs). Finally, Mendelian randomization analysis provided evidence for causal effects of plasma levels of MICB and CD209 proteins, and anthropometric traits such as waist and hip circumference, height, weight, and both fat and fat-free mass. Our results provide novel insight into both VVs genetics and etiology. The revealed genes and proteins can be considered as good candidates for follow-up functional studies and might be of interest as potential drug targets.

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-ethyl-N-nitrosourea (ENU). Overall, 3 of 16 ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss- or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzf1 However, the engineered U2af1(S34F) missense mutation reverted to WT in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2a^Δi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2a^Δi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.

Chromosomal rearrangements between 3q21 and 3q26 elicit high-risk acute myeloid leukemia (AML), which is often associated with elevated platelet and megakaryocyte (Mk) numbers. The 3q rearrangements reposition a GATA2 enhancer near the EVI1 (or MECOM) locus, which results in both EVI1 overexpression and GATA2 haploinsufficiency. However, the mechanisms explaining how the misexpression of these 2 genes individually contribute to leukemogenesis are unknown. To clarify the characteristics of differentiation defects caused by EVI1 and GATA2 misexpression and to identify the cellular origin of leukemic cells, we generated a system to monitor both inv(3) allele-driven EVI1 and Gata2 expression in 3q-rearranged AML model mice. A cell population in which both EVI1 and Gata2 were highly induced appeared in the bone marrows before the onset of frank leukemia. This population had acquired serial colony-forming potential. Because hematopoietic stem/progenitor cells (HSPCs) and Mks were enriched in this peculiar population, we analyzed the independent EVI1 and GATA2 contributions to HSPC and Mk. We found that inv(3)-driven EVI1 promotes accumulation of Mk-biased and myeloid-biased progenitors, Mks, and platelets, and that Gata2 heterozygous deletion enhanced Mk-lineage skewing of EVI1-expressing progenitors. Notably, inv(3)-directed EVI1 expression and Gata2 haploinsufficient expression cooperatively provoke a leukemia characterized by abundant Mks and platelets. These hematological features of the mouse model phenocopy those observed in human 3q AML. On the basis of these results, we conclude that inv(3)-driven EVI1 expression in HSPCs and Mks collaborates with Gata2 haploinsufficiency to provoke Mk-lineage skewing and leukemogenesis with excessive platelets, thus mimicking an important feature of human AML.