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.

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.

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.

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.

The crucial role of GATA-1 in CCR3 gene transcription: modulated balance by multiple GATA elements in the CCR3 regulatory region.

GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.

Serpent, suppressor of hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis.

The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, we characterized a PSC-active transcriptional enhancer that drives hh expression in supportive niche cells. Our findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance.

The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis.

Hematopoiesis is coordinated by a complex regulatory network of transcription factors and among them PU.1 (Spi1, Sfpi1) represents a key molecule. This review summarizes the indispensable requirement of PU.1 during hematopoietic cell fate decisions and how the function of PU.1 can be modulated by protein-protein interactions with additional factors. The mutual negative regulation between PU.1 and GATA-1 is detailed within the context of normal and leukemogenic hematopoiesis and the concept of 'differentiation therapy' to restore normal cellular differentiation of leukemic cells is discussed.

A cis-regulatory signature in ascidians and flies, independent of transcription factor binding sites.

BACKGROUND: Transcription initiation is controlled by cis-regulatory modules. Although these modules are usually made of clusters of short transcription factor binding sites, a small minority of such clusters in the genome have cis-regulatory activity. This paradox is currently unsolved. RESULTS: To identify what discriminates active from inactive clusters, we focused our attention on short topologically unconstrained clusters of two ETS and two GATA binding sites, similar to the early neural enhancer of Ciona intestinalis Otx. We first computationally identified 55 such clusters, conserved between the two Ciona genomes. In vivo assay of the activity of 19 hits identified three novel early neural enhancers, all located next to genes coexpressed with Otx. Optimization of ETS and GATA binding sites was not always sufficient to confer activity to inactive clusters. Rather, a dinucleotide sequence code associated to nucleosome depletion showed a robust correlation with enhancer potential. Identification of a large collection of Ciona regulatory regions revealed that predicted nucleosome depletion constitutes a general signature of Ciona enhancers, which is conserved between orthologous loci in the two Ciona genomes and which partitions conserved noncoding sequences into a major nucleosome-bound fraction and a minor nucleosome-free fraction with higher cis-regulatory potential. We also found this signature in a large fraction of short Drosophila cis-regulatory modules. CONCLUSION: This study indicates that a sequence-based dinucleotide signature, previously associated with nucleosome depletion and independent of transcription factor binding sites, contributes to the definition of a local cis-regulatory potential in two metazoa, Ciona intestinalis and Drosophila melanogaster.

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3.

Trps1, the gene mutated in human Tricho-Rhino-Phalangeal syndrome, represents an atypical member of the GATA-family of transcription factors. Here we show that Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3 in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types.

Distinct functions of Wnt/beta-catenin signaling in KV development and cardiac asymmetry.

The Wnt/beta-catenin pathway exhibits distinct and developmental stage-specific roles during cardiogenesis. However, little is known about the molecular mechanisms of Wnt/beta-catenin signaling in the establishment of cardiac left-right (LR) asymmetry. Using zebrafish as an animal model, we show here that Wnt/beta-catenin signaling is differentially required in cardiac LR patterning. At an early stage, during asymmetric signal generation, Wnt/beta-catenin signaling is necessary for Kupffer's vesicle development and for the regulation of both heart and visceral laterality. At a later stage, during asymmetric signal propagation, excessive Wnt/beta-catenin signaling inhibits the transmission of asymmetric cues from the lateral plate mesoderm (LPM) to the cardiac field but not to the developing gut; as such, it only regulates heart laterality. Molecular analysis identifies Gata4 as the downstream target of Wnt/beta-catenin signaling in the cardiac field that responds to the Wnt/beta-catenin signaling and regulates the competence of the heart field to express left-sided genes. In summary, our results reveal a previously unexpected role of Wnt-Gata4 signaling in the control of asymmetric signal propagation from the LPM to the cardiac field.

Transcription of steroidogenic acute regulatory protein in the rodent ovary and placenta: alternative modes of cyclic adenosine 3', 5'-monophosphate dependent and independent regulation.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Regulation of P450c17 expression in the early embryo depends on GATA factors.

The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the early mouse embryo. We show that P450c17 is expressed in differentiated mouse parietal and visceral endoderm lineages, in cultured mouse F9 embryonic carcinoma stem cells, in mouse embryonic stem cells, and in cultured mouse P19 stem cells. Bases -110 to -55 (which contain an SF-1 site and two potential GATA sites) of the rat cyp17 gene confer promoter activity in F9 cells. Overexpression of SF-1 has no effect, whereas overexpression of GATA4 in F9 cells increases transcription from -110/-55 fused to a reporter and increases endogenous P450c17 mRNA. Chromatin immunoprecipitation assays show that GATA4 binds to -215/+55 of mouse cyp17. Stimulating F9 cells with retinoic acid and cAMP differentiates them into visceral and parietal endoderm. Commensurate with cell differentiation, quantitative PCR showed increased GATA4 and GATA6 mRNAs, temporally followed by increased P450c17 mRNA. Small interfering RNA inhibition of GATA4 or GATA6 in undifferentiated or differentiated F9 cells diminished endogenous cyp17 expression. Thus, P450c17 is expressed in mouse embryonic stem cells, its expression increases upon differentiation to an early embryonic endoderm lineage, and GATA4/6 are responsible for activation of P450c17 gene expression at this early stage of embryonic development.

Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.

Trps1 deficiency enlarges the proliferative zone of growth plate cartilage by upregulation of Pthrp.

We have reported that elongation of the columnar proliferative zone of long bone growth plates in Trps1-/- mice during the late fetal stage in the previous study [1]. Since expression of Trps1 protein was found to overlap with that of mRNAs for Indian hedgehog (Ihh), PTH/PTHrP receptor (PPR), and PTHrP, we hypothesized that Trps1 may inhibit the hypertrophic differentiation of chondrocytes by interacting with the Ihh/PTHrP feedback loop. To investigate whether Trps1 has a role in this Ihh/PTHrP feedback loop, we compared the growth plates of Trps1-/- mice and wild-type (Trps1+/+) mice. Immunohistochemistry showed that Trps1 protein was strongly expressed in the periarticular and prehypertrophic zones of the fetal growth plate in wild-type mice on embryonic day 18.5 (E18.5). On the other hand, Ihh, PPR, and PTHrP mRNAs were predominantly expressed in the prehypertrophic zone at this stage of development. While expression of Ihh and PPR by prehypertrophic chondrocytes was unaffected in the growth plates of Trps1-/- mice, the range of PTHrP expression was expanded toward the proliferating zone in these mice. Quantitative real-time PCR analysis demonstrated upregulation of PTHrP in the epiphyseal growth plates of Trps1-/- mice. Furthermore, promoter analysis combined with the chromatin immunoprecipitation (ChIP) assay demonstrated that direct binding of Trps1 to the PTHrP promoter suppressed the transcription of PTHrP. Finally, organ culture of E14.5 tibiae in the absence or the presence of Pthrp revealed that the proliferative zone of the tibial growth plate was elongated by culture with Pthrp compared to that of control tibiae. Taken together, these data provide the first genetic evidence that lack of Trps1 leads to overexpression of PTHrP, and that Trps1 is required to maintain the normal organization of chondrocytes in the growth plate.

Gene induction following wounding of wild-type versus macrophage-deficient Drosophila embryos.

By using a microarray screen to compare gene responses after sterile laser wounding of wild-type and 'macrophageless' serpent mutant Drosophila embryos, we show the wound-induced programmes that are independent of a pathogenic response and distinguish which of the genes are macrophage dependent. The evolutionarily conserved nature of this response is highlighted by our finding that one such new inflammation-associated gene, growth arrest and DNA damage-inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild-type and serpent mutant embryos also shows a portfolio of 'macrophage-specific' genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound- and macrophage-related genes, our data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a 'pathogen response', which might prime the organism for what is likely to be an increased risk of infection.

Role of the GATA family of transcription factors in endocrine development, function, and disease.

The WGATAR motif is a common nucleotide sequence found in the transcriptional regulatory regions of numerous genes. In vertebrates, these motifs are bound by one of six factors (GATA1 to GATA6) that constitute the GATA family of transcriptional regulatory proteins. Although originally considered for their roles in hematopoietic cells and the heart, GATA factors are now known to be expressed in a wide variety of tissues where they act as critical regulators of cell-specific gene expression. This includes multiple endocrine organs such as the pituitary, pancreas, adrenals, and especially the gonads. Insights into the functional roles played by GATA factors in adult organ systems have been hampered by the early embryonic lethality associated with the different Gata-null mice. This is now being overcome with the generation of tissue-specific knockout models and other knockdown strategies. These approaches, together with the increasing number of human GATA-related pathologies have greatly broadened the scope of GATA-dependent genes and, importantly, have shown that GATA action is not necessarily limited to early development. This has been particularly evident in endocrine organs where GATA factors appear to contribute to the transcription of multiple hormone-encoding genes. This review provides an overview of the GATA family of transcription factors as they relate to endocrine function and disease.