The transcription factor GATA2 regulates normal hematopoiesis, particularly in- stem cell maintenance and myeloid differentiation. Various heteroallelic GATA2 gene mutations are associated with a variety of hematological neoplasms, including myelodysplastic syndromes and leukemias. Here, we report that impaired GATA2 expression induces myelodysplastic and myeloproliferative neoplasm development in elderly animals, and this neoplasm resembles chronic myelomonocytic leukemia in humans. GATA2 hypomorphic mutant (G2^fGN/fGN ) mice that were generated by the germline insertion of a neocassette into the Gata2 gene locus avoided the early embryonic lethality observed in Gata2-null mice. However, adult G2^fGN/fGN mice suffered from exacerbated leukocytosis concomitant with progressive anemia and thrombocytopenia and eventually developed massive granulomonocytosis accompanied by trilineage dysplasia. The reconstitution activity of G2^fGN/fGN mouse stem cells was impaired. Furthermore, G2^fGN/fGN progenitors showed myeloid lineage-biased proliferation and differentiation. Myeloid progenitor accumulation started at a younger age in G2^fGN/fGN mice and appeared to worsen with age. G2^fGN/fGN mice showed increased expression of transcripts encoding cytokine receptors, such as macrophage colony-stimulating factor receptor and interleukin-6 receptor, in granulocyte-monocyte progenitors. This increased expression could be correlated with the hypersensitive granulomonocytic proliferation reaction when the mice were exposed to lipopolysaccharide. Taken together, these observations indicate that GATA2 hypomorphism leads to a hyperreactive defense response to infections, and this reaction is attributed to a unique intrinsic cell defect in the regulation of myeloid expansion that increases the risk of hematological neoplasm transformation.

Neutrophils and eosinophils, 2 prominent granulocytes, are commonly derived from myelocytic progenitors through successive stages in the bone marrow. Our previous genome-wide transcriptomic data unexpectedly showed that genes encoding a multitude of neutrophil primary granule proteins (NPGPs) were markedly downregulated during the end period of eosinophilic terminal differentiation when cord blood (CB) cluster of differentiation (CD) 34+ cells were induced to differentiate toward the eosinophil lineage during a 24-day culture period. Accordingly, this study aimed to examine whether NPGP genes were expressed on the way to eosinophil terminal differentiation stage and to compare their expression kinetics with that of genes encoding eosinophil-specific granule proteins (ESGPs). Transcripts of all NPGP genes examined, including proteinase 3, myeloperoxidase, cathepsin G (CTSG), and neutrophil elastase, reached a peak at day 12 and sharply declined thereafter, while transcript of ESGP genes including major basic protein 1 (MBP1) attained maximum expression at days 18 or 24. Growth factor independent 1 (GFI1) and CCAAT/enhancer-binding protein α (C/EBPA), transactivators for the NPGP genes, were expressed immediately before the NPGP genes, whereas expression of C/EBPA, GATA1, and GATA2 kinetically paralleled that of eosinophil granule protein genes. The expression kinetics of NPGPs and ESGPs were duplicated upon differentiation of the eosinophilic leukemia cell line (EoL-1) immature eosinophilic cells. Importantly, confocal image analysis showed that CTSG was strongly coexpressed with MBP1 in differentiating CB eosinophils at days 12 and 18 and became barely detectable at day 24 and beyond. Our results suggest for the first time the presence of an immature stage where eosinophils coexpress NPGPs and ESGPs before final maturation.

GATA2 is a pioneering transcription factor governing androgen receptor expression and signaling in prostate cells. To understand the prognostic potential of GATA2 assessment in prostate cancer, we analyzed nuclear GATA2 expression on an annotated tissue microarray with 12,427 prostate cancer samples. Normal prostate glands were negative to weakly positive. GATA2 staining was found in almost all prostate cancers (95%). Strong GATA2 staining was linked to advanced tumor stage, high classical and quantitative Gleason grade (p < 0.0001 each), positive nodal stage (p = 0.0116), and early biochemical recurrence (p < 0.0001). GATA2 was linked to ERG-fusion-type cancers, with strong GATA2 staining in 29% of ERG-negative and 53% of ERG-positive cancers (p < 0.0001). Separate calculations in 3854 cancers with and 4768 cancers without TMPRSS2:ERG fusion revealed that these associations with tumor phenotype and patient outcome were largely driven by the subset of ERG-negative tumors. GATA2 expression was further linked to androgen receptor expression: Only 8% of androgen receptor-negative, but 56% of strongly androgen receptor expressing cancers had strong GATA2 expression (p < 0.0001). In conclusion, the results of our study demonstrate that increasing GATA2 levels are linked to prostate cancer progression and aggressiveness. The prognostic value of GATA2 is remarkable in ERG-negative cancers. However, the upregulation of GATA2 in ERG-positive cancers makes it unsuitable as a prognostic marker in this patient subset.

Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.

The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that the TEAD4 mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes; CDX2, GATA2 and CCN2, in the TEAD4-knockdown (KD) blastocysts. These expression levels significantly decreased in the TEAD4 KD blastocysts compared with controls. Of these downregulated genes, the CCN2 expression level decreased the most. We further analyzed the expression levels of TE-expressed genes; CDX2, GATA2 and TEAD4 in the CCN2 KD blastocysts. Strikingly, the CCN2 KD blastocysts showed the downregulation of CDX2, GATA2 and TEAD4 Furthermore, the ratio of TE-to-ICM cell numbers in the CCN2 KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation of CCN2 expression thorough TEAD4 in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation of TEAD4 and CCN2 is required for TE development with appropriate gene expression in bovine blastocysts.

The first two differentiation events in the embryo result in three cell types - epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2 Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17, while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype.

Fusobacterium nucleatum has pathogenic effects on oral squamous cell carcinoma and colon cancer, while the effects of continuously altered gene expression in normal human cells, as induced by persistent exposure to F. nucleatum, remain unclear. In this study, a microarray Significant Profiles (maSigPro) analysis was used to obtain the transcriptome profile of gingiva-derived mesenchymal stem cells (GMSCs) stimulated by F. nucleatum for 3, 7, 14, and 21 day, and the results revealed 790 (nine clusters) differentially expressed genes (DEGs), which were significantly enriched in cell adherens junctions and cancer-related pathways. On the basis of a short time-series expression miner (STEM) analysis, all the expressed genes in the GMSCs were grouped into 50 clusters according to dynamic gene expression patterns, and the expression levels of three gene clusters in the F. nucleatum-treated GMSCs were significantly different than the predicted values. Among the 790 DEGs, 50 tumor-associated genes (TAGs; such as L3MBTL4, CD163, CCCND2, CADM1, BCL7A, and IGF1) and five core dynamic DEGs (PLCG2, CHI3L2, L3MBTL4, SH2D2A, and NLRP3) were identified during F. nucleatum stimulation. Results from a GeneMANIA database analysis showed that PLCG2, CHI3L2, SH2D2A, and NLRP3 and 20 other proteins formed a complex network of which 12 genes were enriched in cancer-related pathways. Based on the five core dynamic DEGs, the related microRNAs (miRNAs) and transcription factors (TFs) were obtained from public resources, and an integrated network composed of the related TFs, miRNAs, and mRNAs was constructed. The results indicated that these genes were regulated by several miRNAs, such as miR-372-3p, miR-603, and miR-495-3p, and several TFs, including CREB3, GATA2, and SOX4. Our study suggests that long-term stimulation by F. nucleatum may trigger the expression of cancer-related genes in normal gingiva-derived stem cells.

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin β3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

KEY TEACHING POINTS • We describe a 45-year-old woman with GATA2 deficiency associated with verrucae, lymphedema, immunodeficiency, and a history of infections and skin cancer. • GATA2 deficiency has variable clinical expressivity with differing presentations, including infection, hematopoietic abnormalities, immunodeficiency, lymphedema, and cancer. • Cutaneous manifestations include verruca vulgaris, soft tissue infections, lymphedema, and panniculitis. • Patients may have verrucae that can progress to squamous cell carcinomas; dermatologists therefore play an important role in managing these patients as members of a multidisciplinary team.

The GATA family of transcription factors consists of six proteins (GATA1-6) that control a variety of physiological and pathological processes. In particular, GATA2 and GATA3 are coexpressed in a number of tissues, including in the urogenital and sympathoadrenal systems, in which both factors participate in the developmental process and tissue maintenance. Furthermore, accumulating studies have demonstrated that GATA2 and GATA3 are involved in distinct types of inherited diseases as well as carcinogenesis in diverse tissues. This review summarizes our current knowledge of how GATA2 and GATA3 participate in the transcriptional regulatory circuitry during the development of the sympathoadrenal and urogenital systems, and how their dysregulation results in the carcinogenesis of neuroblastoma, renal urothelial, and gynecologic cancers.

Keywords: GATA2; GATA3; gynecologic cancer; neuroblastoma; sympathoadrenal; urogenital; urothelial cancer.

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron-restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron-restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE-mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real-time RT-PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet-1, 5-Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF-dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over-expression of the engineered NRSF (a dominant-negative mutant or a DNA-binding domain and activation domain fusion protein). MS-275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC-1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin-specific protease 7 deubiquitinase inhibitors, P-22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE-mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin-specific protease 7-mediated deubiquitination and stabilization of NRSF.

Natural killer cell deficiency (NKD) is a primary immunodeficiency where the main defect lies in CD56⁺CD3^- natural killer (NK) cells which mediate cytotoxicity against tumors. Most cases are observed in children and adolescents with recurrent viral infections and cancer. GATA2 and MCM4 mutations are found in NKD patients with cancer. However, the question remains unclear whether NKD increases the risk of cancer. Mutations in the second zinc finger of GATA2 cause both NKD and haematopoietic malignancies. MCM4 splice site mutations are found in NKD patients and they increase susceptibility to DNA instability during replication. IRF8, RTEL1, and FCGR3A mutations are associated with NKD but their associations with cancer are unknown. Based on the studies, it is hypothesized that genetic mutations alone are sufficient to cause cancer. However, a number of NKD patients developed oncogenic viral infections which progressed into cancer. Here, we review the evidence of genetic mutations responsible for both NKD and cancer to identify whether NKD contributes to development of cancer. The findings provide insights into the role of NK cells in the prevention of cancer and the significance of assessing NK cell functions in susceptible individuals.

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine-paracrine factor in this tissue. GNRH has been shown to increase pituitary Adcyap1 gene expression through the interaction of CREB and jun/fos with CRE/AP1 cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognate cis-elements which regulate Adcyap1 gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulate Adcyap1 promoter activity via a GATA cis-element located at position -191 in the rat Adcyap1 gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation of Adcyap1 gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

Mechanisms underlying differentiation of embryonic hematopoietic stem/progenitor cells (HSPCs) remain unclear. In mouse, intra-aortic clusters (IACs) form in the aorta-gonad-mesonephros region and acquire HSPC potential after 9.5 days postcoitum (dpc). In this study we demonstrate that Twist1 is highly expressed in c-Kit+CD31+CD34+ IACs, which are equivalent to embryonic HSPCs, compared with adult HSPCs. Progenitor activities of colony-forming unit (CFU) of granulocytes and macrophages, CFU of macrophages, burst-forming unit of erythroid, and B lymphopoiesis were impaired in IACs of Twist1-/- relative to wild-type embryos. Microarray analysis and real-time polymerase chain reaction showed downregulated expression of Myb and Gata2 transcripts in Twist1-/- IACs. Chromatin immunoprecipitation and promoter binding assays indicated that Twist1 directly binds the Myb and Gata2 promoters in 10.5-dpc IACs. We conclude that Twist1 is a novel transcriptional regulator of HSPC differentiation through direct binding to promoter regions of key regulators of the process.

P. aeruginosa is one of the top three causes of opportunistic human bacterial infections. The remarkable variability in the clinical outcomes of this infection is thought to be associated with genetic predisposition. However, the genes underlying host susceptibility to P. aeruginosa infection are still largely unknown.

As a step towards mapping these genes, we applied a genome wide linkage analysis approach to a mouse model. A large F2 intercross population, obtained by mating P. aeruginosa-resistant C3H/HeOuJ, and susceptible A/J mice, was used for quantitative trait locus (QTL) mapping. The F2 progenies were challenged with a P. aeruginosa clinical strain and monitored for the survival time up to 7 days post-infection, as a disease phenotype associated trait. Selected phenotypic extremes of the F2 distribution were genotyped with high-density single nucleotide polymorphic (SNP) markers, and subsequently QTL analysis was performed. A significant locus was mapped on chromosome 6 and was named P . aeruginosa infection resistance locus 1 (Pairl1). The most promising candidate genes, including Dok1, Tacr1, Cd207, Clec4f, Gp9, Gata2, Foxp1, are related to pathogen sensing, neutrophils and macrophages recruitment and inflammatory processes.

We propose a set of genes involved in the pathogenesis of P. aeruginosa infection that may be explored to complement human studies.

The critical functions of dendritic cells (DCs) in immunity and tolerance have been demonstrated in many animal models but their non-redundant roles in humans are more difficult to probe. Human primary immunodeficiency (PID), resulting from single gene mutations, may result in DC deficiency or dysfunction. This relatively recent recognition illuminates the in vivo role of human DCs and the pathophysiology of the associated clinical syndromes. In this review, the development and function of DCs as established in murine models and human in vitro systems, discussed. This forms the basis of predicting the effects of DC deficiency in vivo and understanding the consequences of specific mutations on DC development and function. DC deficiency syndromes are associated with heterozygous GATA2 mutation, bi-allelic and heterozygous IRF8 mutation and heterozygous IKZF1 mutation. The intricate involvement of DCs in the balance between immunity and tolerance is leading to increased recognition of their involvement in a number of other immunodeficiencies and autoimmune conditions. Owing to the precise control of transcription factor gene expression by super-enhancer elements, phenotypic anomalies are relatively commonly caused by heterozygous mutations.

Flavanol intake positively influences several cardiometabolic risk factors in humans. However, the specific molecular mechanisms of action of flavanols, in terms of gene regulation, in the cell types relevant to cardiometabolic disease have never been systematically addressed. On this basis, we conducted a systematic literature review and a comprehensive bioinformatic analysis of genes whose expression is affected by flavanols in cells defining cardiometabolic health: hepatocytes, adipocytes, endothelial cells, smooth muscle cells and immune cells. A systematic literature search was performed using the following pre-defined criteria: treatment with pure compounds and metabolites (no extracts) at low concentrations that are close to their plasma concentrations. Differentially expressed genes were analyzed using bioinformatics tools to identify gene ontologies, networks, cellular pathways and interactions, as well as transcriptional and post-transcriptional regulators. The systematic literature search identified 54 differentially expressed genes at the mRNA level in in vitro models of cardiometabolic disease exposed to flavanols and their metabolites. Global bioinformatic analysis revealed that these genes are predominantly involved in inflammation, leukocyte adhesion and transendothelial migration, and lipid metabolism. We observed that, although the investigated cells responded differentially to flavanol exposure, the involvement of anti-inflammatory responses is a common mechanism of flavanol action. We also identified potential transcriptional regulators of gene expression: transcriptional factors, such as GATA2, NFKB1, FOXC1 or PPARG, and post-transcriptional regulators: miRNAs, such as mir-335-5p, let-7b-5p, mir-26b-5p or mir-16-5p. In parallel, we analyzed the nutrigenomic effects of flavanols in intestinal cells and demonstrated their predominant involvement in the metabolism of circulating lipoproteins. In conclusion, the results of this systematic analysis of the nutrigenomic effects of flavanols provide a more comprehensive picture of their molecular mechanisms of action and will support the future setup of genetic studies to pave the way for individualized dietary recommendations.

Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations in NBEAL2, resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to GATA1 mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1-deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients, and Gata1-deficient mice, expressed almost no NBEAL2. The differentiation of GATA1 patient-derived CD34+ stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.

Here we update receptor proximal and distant signaling events of the mast cell high affinity IgE receptor (FcεRI) launching immediate type I hypersensitivity and an inflammatory cytokine-chemokine cascade. Different physiologic antigen concentrations, their affinity, and valency for the IgE ligand produce distinct intracellular signaling events with different outcomes. Investigating mast cell degranulation has revealed a complex molecular machinery that relays proximal signaling to cytoskeletal reorganization, granule transport and membrane fusion. Several new phosphorylation- and calcium-responsive effectors have been described. FcεRI signaling also promotes de novo gene transcription. Recent progress has identified enhancers at genes that are upregulated in mast cells after stimulation through FcεRI using next generation sequencing methods. Enhancers at genes that respond to antigenic stimulation in human mast cells revealed Ca²⁺-dependency. Stimulation-responsive super enhancers in mouse mast cells have also been identified. Mast cell lineage-determining transcription factor GATA2 primes these enhancers to respond to antigenic stimulation.

Cardiomyopathy (CMP) is a group of myocardial diseases that progressively impair cardiac function. The mechanisms underlying CMP development are poorly understood, but lifestyle factors are clearly implicated as risk factors. This study aimed to identify molecular biomarkers involved in inflammatory CMP development and progression using a systems biology approach. The authors analysed microarray gene expression datasets from CMP and tissues affected by risk factors including smoking, ageing factors, high body fat, clinical depression status, insulin resistance, high dietary red meat intake, chronic alcohol consumption, obesity, high-calorie diet and high-fat diet. The authors identified differentially expressed genes (DEGs) from each dataset and compared those from CMP and risk factor datasets to identify common DEGs. Gene set enrichment analyses identified metabolic and signalling pathways, including MAPK, RAS signalling and cardiomyopathy pathways. Protein-protein interaction (PPI) network analysis identified protein subnetworks and ten hub proteins (CDK2, ATM, CDT1, NCOR2, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E and HIST1H4L). Five transcription factors (FOXC1, GATA2, FOXL1, YY1, CREB1) and five miRNAs were also identified in CMP. Thus the authors' approach reveals candidate biomarkers that may enhance understanding of mechanisms underlying CMP and their link to risk factors. Such biomarkers may also be useful to develop new therapeutics for CMP.

PEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. While aberrant expression of PEAK1 has been linked to cancer progression, its normal physiological role in vertebrate biology is not known. Here we provide evidence that PEAK1 plays a central role in orchestrating new vessel formation in vertebrates. Deletion of the PEAK1 gene in zebrafish, mice, and human endothelial cells (ECs) induced severe defects in new blood vessel formation due to deficiencies in EC proliferation, survival, and migration. Gene transcriptional and proteomic analyses of PEAK1-deficient ECs revealed a significant loss of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression, as well as downstream signaling to its effectors, ERK, Akt, and Src kinase. PEAK1 regulates VEGFR2 expression by binding to and increasing the protein stability of the transcription factor GATA-binding protein 2 (GATA2), which controls VEGFR2 transcription. Importantly, PEAK1-GATA2-dependent VEGFR2 expression is mediated by EC adhesion to the ECM and is required for breast cancer-induced new vessel formation in mice. Also, elevated expression of PEAK1 and VEGFR2 mRNA are highly correlated in many human cancers including breast cancer. Together, our findings reveal a novel PEAK1-GATA2-VEGFR2 signaling axis that integrates cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer.

Background: Abnormal DNA methylation is observed as an early event in breast carcinogenesis. However, how such alterations arise is still poorly understood. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play key roles in various biological processes. Here, we integrate miRNA expression and DNA methylation at CpGs to study how miRNAs may affect the breast cancer methylome and how DNA methylation may regulate miRNA expression.

Methods: miRNA expression and DNA methylation data from two breast cancer cohorts, Oslo2 (n = 297) and The Cancer Genome Atlas (n = 439), were integrated through a correlation approach that we term miRNA-methylation Quantitative Trait Loci (mimQTL) analysis. Hierarchical clustering was used to identify clusters of miRNAs and CpGs that were further characterized through analysis of mRNA/protein expression, clinicopathological features, in silico deconvolution, chromatin state and accessibility, transcription factor binding, and long-range interaction data.

Results: Clustering of the significant mimQTLs identified distinct groups of miRNAs and CpGs that reflect important biological processes associated with breast cancer pathogenesis. Notably, two major miRNA clusters were related to immune or fibroblast infiltration, hence identifying miRNAs associated with cells of the tumor microenvironment, while another large cluster was related to estrogen receptor (ER) signaling. Studying the chromatin landscape surrounding CpGs associated with the estrogen signaling cluster, we found that miRNAs from this cluster are likely to be regulated through DNA methylation of enhancers bound by FOXA1, GATA2, and ER-alpha. Further, at the hub of the estrogen cluster, we identified hsa-miR-29c-5p as negatively correlated with the mRNA and protein expression of DNA methyltransferase DNMT3A, a key enzyme regulating DNA methylation. We found deregulation of hsa-miR-29c-5p already present in pre-invasive breast lesions and postulate that hsa-miR-29c-5p may trigger early event abnormal DNA methylation in ER-positive breast cancer.

Conclusions: We describe how miRNA expression and DNA methylation interact and associate with distinct breast cancer phenotypes.

Keywords: Breast cancer; Methylation; Omics integration; Systems biology; miRNA.