Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth.

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a 'multi hit' effect may be achieved through the aberrant activation of just a single gene.

Quantitating transcription factor redundancy: The relative roles of the ELT-2 and ELT-7 GATA factors in the C. elegans endoderm.

The two GATA transcription factors ELT-2 and ELT-7 function in the differentiation of the C. elegans intestine. ELT-2 loss causes lethality. ELT-7 loss causes no obvious phenotype but enhances the elt-2(-) intestinal phenotype. Thus, ELT-2 and ELT-7 appear partially redundant, with ELT-2 being more influential. To investigate the different regulatory roles of ELT-2 and ELT-7, we compared the transcriptional profiles of pure populations of wild-type, elt-2(-), elt-7(-), and elt-7(-); elt-2(-) double mutant L1-stage larvae. Consistent with the mutant phenotypes, loss of ELT-2 had a>25 fold greater influence on the number of significantly altered transcripts compared to the loss of ELT-7; nonetheless, the levels of numerous transcripts changed upon loss of ELT-7 in the elt-2(-) background. The quantitative responses of individual genes revealed a more complicated behaviour than simple redundancy/partial redundancy. In particular, genes expressed only in the intestine showed three distinguishable classes of response in the different mutant backgrounds. One class of genes responded as if ELT-2 is the major transcriptional activator and ELT-7 provides variable compensatory input. For a second class, transcript levels increased upon loss of ELT-2 but decreased upon further loss of ELT-7, suggesting that ELT-7 actually overcompensates for the loss of ELT-2. For a third class, transcript levels also increased upon loss of ELT-2 but remained elevated upon further loss of ELT-7, suggesting overcompensation by some other intestinal transcription factor(s). In spite of its minor loss-of-function phenotype and its limited sequence similarity to ELT-2, ELT-7 expressed under control of the elt-2 promoter is able to rescue elt-2(-) lethality. Indeed, appropriately expressed ELT-7, like appropriately expressed ELT-2, is able to replace all other core GATA factors in the C. elegans endodermal pathway. Overall, this study focuses attention on the quantitative intricacies behind apparent redundancy or partial redundancy of two related transcription factors.

Effect on gene expression of three allelic variants in GATA motifs of ABO, RHD, and RHCE regulatory elements.

BACKGROUND: Only a few genetic variants have been reported in regulatory elements of blood group genes. Most of them affect GATA motifs, binding sites for the GATA-1 transcription factor. STUDY DESIGN AND METHODS: Samples from two patients and one donor with unusual or discrepant serology for ABO, RhD, and RhCE antigens were analyzed by DNA sequencing. Analyzed regions included the coding sequence and portions of regulatory elements. The effect of some variants on gene expression was evaluated in reporter gene assays. RESULTS: Three new alleles were identified. Their key variants are located in the ABO Intron 1 enhancer, the RHD proximal promoter, and the RHCE proximal promoter. IVS1 + 5859A was found in an African American patient with a group O forward type and a group B reverse type. 5'UTR-115C was the only RHD variant sequence found in a mixed-race black and Caucasian prenatal patient showing mixed-field agglutination with anti-D. 5'UTR-83T was found in several black donors and patients in the context of the genetically related RHCE*ceBI and RHCE*ceSM alleles. Reporter assays of promoter constructs including 5'UTR-115C or 5'UTR-83T showed a significant reduction in RH gene expression. CONCLUSION: Three new alleles in the ABO, RHD, and RHCE genes consist of single-nucleotide changes within GATA motifs, emphasizing the key role of GATA transcription factors in the expression of blood group genes.

E2F and GATA switches turn off WD repeat domain 77 expression in differentiating cells.

WDR77 (WD repeat domain 77) is expressed during earlier lung development when cells are rapidly proliferating, but is absent from adult lung. It is re-activated during lung tumorigenesis and is essential for lung cancer cell proliferation. Signalling pathways/molecules that control WDR77 gene expression are unknown. Promoter mapping, gel shift assay and ChIP revealed that the WDR77 promoter contains bona fide response elements for E2F and GATA transcriptional factors as demonstrated in prostate cancer, lung cancer and erythroid cells, as well as in mouse lung tissues. The WDR77 promoter is transactivated by E2F1, E2F3, GATA1 and GATA6, but suppressed by E2F6, GATA1 and GATA3 in prostate cancer PC3 cells. WDR77 expression is associated with E2F1, E2F3, GATA2 and GATA6 occupancy on the WDR77 gene, whereas, in contrast, E2F6, GATA1 and GATA3 occupancy is associated with the loss of WDR77 expression during erythroid maturation and lung development. More importantly, the loss of WDR77 expression that results from E2F and GATA switches is required for cellular differentiation of erythroid and lung epithelial cells. In contrast, lung cancer cells avoid post-mitotic differentiation by sustaining WDR77 expression. Altogether, the present study provides a novel molecular mechanism by which WDR77 is regulated during erythroid and lung development and lung tumorigenesis.

New expectations in the treatment of anemia in chronic kidney disease. / Nuevas expectativas en el tratamiento de la anemia en la enfermedad renal crónica.

The new drugs developed for the treatment of anemia in chronic kidney disease patients, together with their mechanisms of action are reviewed. At present, many of them are already in advanced stages of clinical trials and is expected to be incorporated into the therapeutic arsenal in the coming years. The potential benefits and possible limitations are also described.

GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut.

Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.

Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts.

When iron load exceeds that needed by fission and filamentous yeasts, iron-regulatory GATA-type transcription factors repress genes encoding iron acquisition systems. In contrast, under iron starvation, optimization of cellular iron utilization is coordinated by a specialized regulatory subunit of the CCAAT-binding factor that fosters repression of genes encoding iron-using proteins. Despite these findings, there is still limited knowledge concerning the mechanisms by which these iron-responsive regulators respond to high- or low-iron availability. To provide a framework for understanding common and distinct properties of iron-dependent transcriptional regulators, a repertoire of their functional domains in different fungal species is presented here. In addition, discovery of interacting partners of these iron-responsive factors contributes to provide additional insight into their properties.

High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2.

HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber (Cucumis sativus L.).

The shoot apical meristem (SAM) is essential for continuous organogenesis in higher plants, while the leaf is the primary source organ and the leaf shape directly affects the efficiency of photosynthesis. HANABA TARANU (HAN) encodes a GATA3-type transcription factor that functions in floral organ development, SAM organization, and embryo development in Arabidopsis, but is involved in suppressing bract outgrowth and promoting branching in grass species. Here the function of the HAN homologue CsHAN1 was characterized in cucumber, an important vegetable with great agricultural and economic value. CsHAN1 is predominantly expressed at the junction of the SAM and the stem, and can partially rescue the han-2 floral organ phenotype in Arabidopsis. Overexpression and RNAi of CsHAN1 transgenic cucumber resulted in retarded growth early after embryogenesis and produced highly lobed leaves. Further, it was found that CsHAN1 may regulate SAM development through regulating the WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) pathways, and mediate leaf development through a complicated gene regulatory network in cucumber.

GATA-dependent transcriptional and epigenetic control of cardiac lineage specification and differentiation.

Heart progenitor cells differentiate into various cell types including pacemaker and working cardiomyocytes. Cell-type specific gene expression is achieved by combinatorial interactions between tissue-specific transcription factors (TFs), co-factors, and chromatin remodelers and DNA binding elements in regulatory regions. Dysfunction of these transcriptional networks may result in congenital heart defects. Functional analysis of the regulatory DNA sequences has contributed substantially to the identification of the transcriptional network components and combinatorial interactions regulating the tissue-specific gene programs. GATA TFs have been identified as central players in these networks. In particular, GATA binding elements have emerged as a platform to recruit broadly active histone modification enzymes and cell-type-specific co-factors to drive cell-type-specific gene programs. Here, we discuss the role of GATA factors in cell fate decisions and differentiation in the developing heart.

Signaling molecules, transcription growth factors and other regulators revealed from in-vivo and in-vitro models for the regulation of cardiac development.

Several in-vivo heart developmental models have been applied to decipher the cardiac developmental patterning encompassing early, dorsal, cardiac and visceral mesoderm as well as various transcription factors such as Gata, Hand, Tin, Dpp, Pnr. The expression of cardiac specific transcription factors, such as Gata4, Tbx5, Tbx20, Tbx2, Tbx3, Mef2c, Hey1 and Hand1 are of fundamental significance for the in-vivo cardiac development. Not only the transcription factors, but also the signaling molecules involved in cardiac development were conserved among various species. Enrichment of the bone morphogenic proteins (BMPs) in the anterior lateral plate mesoderm is essential for the initiation of myocardial differentiation and the cardiac developmental process. Moreover, the expression of a number of cardiac transcription factors and structural genes initiate cardiac differentiation in the medial mesoderm. Other signaling molecules such as TGF-beta, IGF-1/2 and the fibroblast growth factor (FGF) play a significant role in cardiac repair/regeneration, ventricular heart development and specification of early cardiac mesoderm, respectively. The role of the Wnt signaling in cardiac development is still controversial discussed, as in-vitro results differ dramatically in relation to the animal models. Embryonic stem cells (ESC) were utilized as an important in-vitro model for the elucidation of the cardiac developmental processes since they can be easily manipulated by numerous signaling molecules, growth factors, small molecules and genetic manipulation. Finally, in the present review the dynamic role of the long noncoding RNA and miRNAs in the regulation of cardiac development are summarized and discussed.

Developmental control of polycomb subunit composition by GATA factors mediates a switch to non-canonical functions.

Polycomb repressive complex 2 (PRC2) plays crucial roles in transcriptional regulation and stem cell development. However, the context-specific functions associated with alternative subunits remain largely unexplored. Here we show that the related enzymatic subunits EZH1 and EZH2 undergo an expression switch during blood cell development. An erythroid-specific enhancer mediates transcriptional activation of EZH1, and a switch from GATA2 to GATA1 controls the developmental EZH1/2 switch by differential association with EZH1 enhancers. We further examine the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and reveal the existence of an EZH1-SUZ12 subcomplex lacking EED. EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin, and positively regulate gene expression. Loss of EZH2 expression leads to repositioning of EZH1 to EZH2 targets. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical functions during blood stem cell specification.

Trps1 deficiency inhibits the morphogenesis of secondary hair follicles via decreased Noggin expression.

A representative phenotype of patients with tricho-rhino-phalangeal syndrome (TRPS) is sparse hair. To understand the developmental defects of these patient's hair follicles, we analyzed the development of hair follicles histologically and biochemically using Trps1 deficient (KO) mice. First, we compared the numbers of primary hair follicles in wild-type (WT) and KO embryos at different developmental stages. No differences were observed in the E14.5 skins of WT and KO mice. However, at later time points, KO fetal skin failed to properly develop secondary hair follicles, and the number of secondary hair follicles present in E18.5 KO skin was approximately half compared to that of WT skin. Sonic hedgehog expression was significantly decreased in E17.5 KO skin, whereas no changes were observed in Eda/Edar expression in E14.5 or E17.5 skins. In addition, Noggin expression was significantly decreased in E14.5 and E17.5 KO skin compared to WT skin. In parallel with the suppression of Noggin expression, BMP signaling was promoted in the epidermal cells of KO skins compared to WT skins as determined by immunohistochemistry for phosphorylated Smad1/5/8. The reduced number of secondary hair follicles was restored in skin graft cultures treated with a Noggin and BMP inhibitor. Furthermore, decreased cell proliferation, and increased apoptosis in KO skin was rescued by Noggin treatment. Taken together, we conclude that hair follicle development in Trps1 KO embryos is impaired directly or indirectly by decreased Noggin expression.

A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.

During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.

Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium.

The small intestinal epithelium develops from embryonic endoderm into a highly specialized layer of cells perfectly suited for the digestion and absorption of nutrients. The development, differentiation, and regeneration of the small intestinal epithelium require complex gene regulatory networks involving multiple context-specific transcription factors. The evolutionarily conserved GATA family of transcription factors, well known for its role in hematopoiesis, is essential for the development of endoderm during embryogenesis and the renewal of the differentiated epithelium in the mature gut. We review the role of GATA factors in the evolution and development of endoderm and summarize our current understanding of the function of GATA factors in the mature small intestine. We offer perspective on the application of epigenetics approaches to define the mechanisms underlying context-specific GATA gene regulation during intestinal development.

Effects of IL-17 on erythroid progenitors growth: involvement of MAPKs and GATA transcription factors.

Interleukin-17 is Th17 cell cytokine implicated in regulation of hematopoiesis and inflammation. Besides promoting granulopoiesis, we have previously shown that IL-17 also affects erythropoiesis stimulating the development of early erythroid progenitors, BFU-E, but suppressing, at least partly via p38 MAPK, the growth of late stage erythroid progenitors, CFU-E. The aim of the present study was to investigate the involvement of other MAPKs, JNK and ERK1/2, as well as GATA transcription factors, in IL-17-mediated effects on murine bone marrow erythroid progenitors. Data obtained by use of specific MAPKs inhibitors indicated that MEK1/2-ERK1/2 MAPK signaling mediates IL-17-induced CFU-E inhibition, as well as that JNK and/or MEK1/2-ERK1/2 MAPKs activation underlies IL-17-induced stimulation of BFU-E growth. Furthermore, Western blot analyses demonstrated no effect on early hematopoiesis transcription factor, GATA-2, and enhanced expression level of erythroid-specific factor GATA-1 in murine bone marrow cells after IL-17 stimulation, which in light of previous reports that GATA-1 overexpression inhibits erythroid differentiation, could be related to IL-17-mediated inhibition of CFU-E growth. Although, no contribution for p38, JNK and ERK MAPKs in IL-17-induced GATA-1 expression was shown, data obtained using specific inhibitors pointed to the role of JNK and MEK1/2-ERK1/2 in GATA-1 downregulation. Overall, obtained data gave an insight into the mechanisms by which IL-17 exerts its effects on erythropoiesis, implying the involvement of JNK and ERK MAPKs, as well as GATA-1, in IL-17-regulated growth of erythroid progentors.

DNA demethylation-dependent AR recruitment and GATA factors drive Rhox5 homeobox gene transcription in the epididymis.

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.

The function of TRPS1 in the development and differentiation of bone, kidney, and hair follicles.

TRPS1 is a gene involved in Tricho-rhino-phalangeal syndrome (TRPS), an autosomal dominant skeletal disorder. TRPS1 encodes a GATA-type transcription factor that has nine zinc-finger motifs. A variety of mutations in TRPS1 including deletions and insertions, have been found in patients with TRPS type I and III. The functions of each domain of TRPS1 have been clarified from study of these mutations. Further studies on the localization and the function of TRPS1 have been performed using TRPS1Δgt and Trps1-deficient mice, which allow examination of the development and differentiation of all tissues with Trps1 expression. These studies suggest that TRPS1 exhibits a variety of functions in cartilage, kidneys, and hair follicles. In the growth plate cartilage, TRPS1 regulates the differentiation, proliferation, and apoptosis of chondrocytes through interaction of several signaling molecules. In addition, TRPS1 has a function downstream of BMP7, which regulates the mesenchymal-epithelial transition when nephrons are formed in renal development. Furthermore, TRPS1 suppresses the epithelial-mesenchymal transition and renal fibrosis induced by unilateral ureteral obstruction by decreasing Arkadia expression. Finally, TRPS1 is expressed in the dermal papillae and the mesenchymal cells surrounding the hair pegs, and the loss of TRPS1 largely influences the development of hair follicles. The molecular mechanisms of the function of TRPS1 in cartilage, kidneys, and hair follicles are discussed in this review.

Promotion of avian endothelial cell differentiation by GATA transcription factors.

In the avian embryo, endothelial cells originate from several sources, including the lateral plate and somite mesoderm. In this study, we show that Gata transcription factors are expressed in the lateral plate and in vasculogenic regions of the avian somite and are able to promote a vascular endothelial fate when ectopically expressed in somite precursors. A fusion of GATA4 to the transcriptional activator VP16 promoted endothelium formation, indicating that GATA transcription factors promote vasculogenesis via activation of downstream targets, while a fusion of GATA4 to the transcriptional repressor engrailed repressed expression of Vascular Endothelial Growth Factor Receptor 2, a marker of endothelial precursors. These findings indicate a role for GATA transcription factors in the differentiation of the endothelium.