Transcription Factor Grainyhead Research Articles

Temporal and spatial staging of lung alveolar regeneration is determined by the grainyhead transcription factor Tfcp2l1

Alveolar epithelial type 2 (AT2) cells harbor the facultative progenitor capacity in the lung alveolus to drive regeneration after lung injury. Using single-cell transcriptomics, software-guided segmentation of tissue damage, and invivo mouse lineage tracing, we identified the grainyhead transcription factor cellular promoter 2-like 1 (Tfcp2l1) as a regulator of this regenerative process. Tfcp2l1 loss in adult AT2 cells inhibits self-renewal and enhances AT2-AT1 differentiation during tissue regeneration. Conversely, Tfcp2l1 blunts the proliferative response to inflammatory signaling during the early acute injury phase. Tfcp2l1 temporally regulates AT2 self-renewal and differentiation in alveolar regions undergoing active regeneration. Single-cell transcriptomics and lineage tracing reveal that Tfcp2l1 regulates cell fate dynamics across the AT2-AT1 differentiation and restricts the inflammatory program in murine AT2 cells. Organoid modeling shows that Tfcp2l1 regulation of interleukin-1 (IL-1) receptor expression controlled these cell fate dynamics. These findings highlight the critical role Tfcp2l1 plays in balancing epithelial cell self-renewal and differentiation during alveolar regeneration.

Mild overexpression of a novel transcription factor Grainy head like-2 (Grhl2) elicits hybrid phenotype in human astrocytoma cells

The Grainy head like-2 (Grhl2) transcription factor plays a major role in embryonic and cancer development. The role of Grhl2 has been intensively studied in various cancers but not for brain cancer. Hence, in this study, we provide a preliminary understanding on the role of Grhl2 that regulate the transition of astrocytoma cells. The human A172 astrocytoma cell line, a mesenchymal cell characterized by mild overexpression of Grhl2 transcription factor, was used in this study. At first, the Grhl2 stably overexpressing A172 clones into three types i.e., Grhl2+ (mild), Grhl2++ (moderate) and Grhl2+++ (high) based on mRNA and protein expression levels of Grhl2 were characterized. Phenotypic characteristics of vector and Grhl2+ cells were observed using phase contrast microscopy. Quantitative PCR (qPCR), Western blot and immunofluorescence were used to detect the level of mesenchymal markers (N-cadherin/vimentin) and also epithelial markers (E-cadherin/ β- catenin) in vector and Grhl2+ cells. The migration and invasion characteristics of vector and Grhl2+ cells were determined by scratch assay and Boyden chamber assay. Further, the Grhl2+ cells were characterized to determine the effect of temozolomide chemotherapy drug which were widely used in treating brain cancer. As expected, in phase contrast image, we observed the mesenchymal characteristic of A172 cells becomes hybrid phenotype i.e., mixture of mesenchymal (spindle-like fibroblast morphology) and epithelial (cobblestone like appearance) cells upon Grhl2 mild expression (Grhl2+) when compared to vector cells. Further, we found that there was a significant upregulation of E-cadherin at both mRNA and protein levels in Grhl2+ cells when compared to vector cells. There was a significant upregulation of β-catenin, N-cadherin and vimentin at mRNA levels, but there was no significant upregulation at the protein levels in Grhl2+ cells compared to the vector cells. The migration and invasion were diminished in Grhl2+ cells when compared to the vector control cells. We observed that the Grhl2+ were sensitive to the temozolomide compared to the vector cells. This infers that the Grhl2+ cells were unable to attain complete transition of mesenchymal to epithelial state, and hence we categorized the Grhl2+ cells as hybrid phenotype. The results provide a better understanding of the largely unknown function of Grhl2 in human astrocytoma cells as tumor progression or suppression.

Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan

Aging is impacted by interventions across species, often converging on metabolic pathways. Transcription factors regulate longevity yet approaches for their pharmacological modulation to exert geroprotection remain sparse. We show that increased expression of the transcription factor Grainyhead 1 (GRH-1) promotes lifespan and pathogen resistance in Caenorhabditis elegans. A compound screen identifies FDA-approved drugs able to activate human GRHL1 and promote nematodal GRH-1-dependent longevity. GRHL1 activity is regulated by post-translational lysine methylation and the phosphoinositide (PI) 3-kinase C2A. Consistently, nematodal longevity following impairment of the PI 3-kinase or insulin/IGF-1 receptor requires grh-1. In BXD mice, Grhl1 expression is positively correlated with lifespan and insulin sensitivity. In humans, GRHL1 expression positively correlates with insulin receptor signaling and also with lifespan. Fasting blood glucose levels, including in individuals with type 2 diabetes, are negatively correlated with GRHL1 expression. Thereby, GRH-1/GRHL1 is identified as a pharmacologically malleable transcription factor impacting insulin signaling and lifespan.

GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.

The transcription factor grainy head (Grh) functions in the protection of the epithelium against the external environment by generating strongly adhesive layers, and this function is conserved in vertebrates and invertebrates. In Drosophila, the top model for holometabolous insects, Grh is necessary during embryonic development, epidermal differentiation, central nervous system specification and epithelial repair. However, the function of this gene in hemimetabolous insect epithelia remains unknown. To examine the function of Grh signaling in regulating epithelium development in Hemimetabola, we focused on the Blattella germanica epidermal layer using a gene knockdown strategy. The spatiotemporal expression pattern of BgGrh was detected, and knockdown of BgGrh and BgCad96ca, which provide positive feedback to BgGrh, caused severe defects in new epithelium development and impeded the molting process required to discard the old integument. Knockdown of the expression of BgGrh and BgCad96ca caused increased expression of chitin synthase gene (BgCHS1) and chitinase gene (BgCht5), the upregulations of which should be mediated by the higher level of hormone receptor 3 (BgHr3) gene. In conclusion, epithelium development is regulated by Grh signaling, which might represent a potential target for the control of urban pest cockroaches.

Establishment of chromatin accessibility by the conserved transcription factor Grainy head is developmentally regulated.

The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.

Epithelial Migration and Non-adhesive Periderm Are Required for Digit Separation during Mammalian Development.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

Structure and cis-regulatory analysis of a Drosophila grainyhead neuroblast enhancer.

Evolutionary analysis of cis-regulatory DNA reveals that enhancers consist of clusters of conserved sequence blocks (CSBs) that are made up of both unique and repeated sequence elements. This study seeks to address the basis for spatial and temporal regulation of neuroblas. A search for temporally restricted CNS NB enhancers identified one within the transcription factor grainyhead (grh) gene locus. The intronic enhancer, grh-15, contains two separable semi-autonomous activities, one that drives expression predominantly within the developing brain NBs and another in ventral cord NBs. To gain insight into the function of the CSBs constituting the brain-specific enhancer, we have systematically deleted each CSB and compared the activity of the altered enhancer to that of the full brain-specific enhancer. While our results indicate that information regulating enhancer activity is highly redundant, we have found that individual CSBs convey expression in subsets of larval lineages that are generated from either Type I or Type II NBs. These studies also highlight how evolutionary sequence conservation can be used as a guide the functional analysis of cis-regulatory DNA.

Combinatorial action of Grainyhead, Extradenticle and Notch in regulating Hox mediated apoptosis in Drosophila larval CNS.

Hox mediated neuroblast apoptosis is a prevalent way to pattern larval central nervous system (CNS) by different Hox genes, but the mechanism of this apoptosis is not understood. Our studies with Abdominal-A (Abd-A) mediated larval neuroblast (pNB) apoptosis suggests that AbdA, its cofactor Extradenticle (Exd), a helix-loop-helix transcription factor Grainyhead (Grh), and Notch signaling transcriptionally contribute to expression of RHG family of apoptotic genes. We find that Grh, AbdA, and Exd function together at multiple motifs on the apoptotic enhancer. In vivo mutagenesis of these motifs suggest that they are important for the maintenance of the activity of the enhancer rather than its initiation. We also find that Exd function is independent of its known partner homothorax in this apoptosis. We extend some of our findings to Deformed expressing region of sub-esophageal ganglia where pNBs undergo a similar Hox dependent apoptosis. We propose a mechanism where common players like Exd-Grh-Notch work with different Hox genes through region specific enhancers to pattern respective segments of larval central nervous system.

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease

Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. We obtained liver samples from 12 patients with ALD and cirrhosis and 9healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in livertissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with micegiven a control vector. Levels of hypoxia-inducible factor1 alpha (HIF1α) mRNA, a target of MIR122, were increased inliver tissues from patients and mice with ALD, compared withcontrols. Mice with hepatocyte-specific disruption of Hif1αdeveloped less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.

An intrinsic mechanism controls reactivation of neural stem cells by spindle matrix proteins

The switch between quiescence and proliferation is central for neurogenesis and its alteration is linked to neurodevelopmental disorders such as microcephaly. However, intrinsic mechanisms that reactivate Drosophila larval neural stem cells (NSCs) to exit from quiescence are not well established. Here we show that the spindle matrix complex containing Chromator (Chro) functions as a key intrinsic regulator of NSC reactivation downstream of extrinsic insulin/insulin-like growth factor signalling. Chro also prevents NSCs from re-entering quiescence at later stages. NSC-specific in vivo profiling has identified many downstream targets of Chro, including a temporal transcription factor Grainy head (Grh) and a neural stem cell quiescence-inducing factor Prospero (Pros). We show that spindle matrix proteins promote the expression of Grh and repress that of Pros in NSCs to govern their reactivation. Our data demonstrate that nuclear Chro critically regulates gene expression in NSCs at the transition from quiescence to proliferation.

Stable Binding of the Conserved Transcription Factor Grainy Head to its Target Genes Throughout Drosophila melanogaster Development.

It has been suggested that transcription factor binding is temporally dynamic, and that changes in binding determine transcriptional output. Nonetheless, this model is based on relatively few examples in which transcription factor binding has been assayed at multiple developmental stages. The essential transcription factor Grainy head (Grh) is conserved from fungi to humans, and controls epithelial development and barrier formation in numerous tissues. Drosophila melanogaster, which possess a single grainy head (grh) gene, provide an excellent system to study this conserved factor. To determine whether temporally distinct binding events allow Grh to control cell fate specification in different tissue types, we used a combination of ChIP-seq and RNA-seq to elucidate the gene regulatory network controlled by Grh during four stages of embryonic development (spanning stages 5-17) and in larval tissue. Contrary to expectations, we discovered that Grh remains bound to at least 1146 genomic loci over days of development. In contrast to this stable DNA occupancy, the subset of genes whose expression is regulated by Grh varies. Grh transitions from functioning primarily as a transcriptional repressor early in development to functioning predominantly as an activator later. Our data reveal that Grh binds to target genes well before the Grh-dependent transcriptional program commences, suggesting it sets the stage for subsequent recruitment of additional factors that execute stage-specific Grh functions.

A Grainyhead-Like 2/Ovo-Like 2 Pathway Regulates Renal Epithelial Barrier Function and Lumen Expansion.

Grainyhead transcription factors control epithelial barriers, tissue morphogenesis, and differentiation, but their role in the kidney is poorly understood. Here, we report that nephric duct, ureteric bud, and collecting duct epithelia express high levels of grainyhead-like homolog 2 (Grhl2) and that nephric duct lumen expansion is defective in Grhl2-deficient mice. In collecting duct epithelial cells, Grhl2 inactivation impaired epithelial barrier formation and inhibited lumen expansion. Molecular analyses showed that GRHL2 acts as a transcriptional activator and strongly associates with histone H3 lysine 4 trimethylation. Integrating genome-wide GRHL2 binding as well as H3 lysine 4 trimethylation chromatin immunoprecipitation sequencing and gene expression data allowed us to derive a high-confidence GRHL2 target set. GRHL2 transactivated a group of genes including Ovol2, encoding the ovo-like 2 zinc finger transcription factor, as well as E-cadherin, claudin 4 (Cldn4), and the small GTPase Rab25. Ovol2 induction alone was sufficient to bypass the requirement of Grhl2 for E-cadherin, Cldn4, and Rab25 expression. Re-expression of either Ovol2 or a combination of Cldn4 and Rab25 was sufficient to rescue lumen expansion and barrier formation in Grhl2-deficient collecting duct cells. Hence, we identified a Grhl2/Ovol2 network controlling Cldn4 and Rab25 expression that facilitates lumen expansion and barrier formation in subtypes of renal epithelia.

Putative orthologues of genetically identified Drosophila melanogaster chitin producing and organising genes in Apis mellifera

The cuticle is a versatile coat allowing insects to survive in a variety of ecological situations. The polysaccharide chitin is a major cuticle component defining cuticle function. The key players of chitin synthesis and organisation, including chitin synthase-1 (CS-1), the chitin deacetylases vermiform (Verm) and serpentine (Serp), the extracellular obstructor-A (Obst-A) protein, the membrane-bound Knickkopf (Knk) and retroactive (Rtv) proteins, the cytoplasmic mummy (Mmy) protein and the transcription factor grainyhead (Grh) have been characterised genetically in the fruit fly Drosophila melanogaster. Here, we report on the identification of putative orthologues of these factors in Apis mellifera and on their expression at different developmental stages. Overall, differences in gene organisation and expression pattern between these two species may compile different cuticle compositions that reflect their life style and ecology. Our data will serve to elucidate the honeybee-specific mechanisms of cuticle formation.

Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression

Until now the essential transcription factor that determines the epithelial phenotype of breast cancer has not been identified and its role in epithelial-to-mesenchymal transition (EMT) and tumor progression remain unclear. Here, by analyzing large expression profiles of human breast cancer cells, we found an extraordinary correlation between the expression of Grainyhead transcription factor Grhl2 and epithelial marker E-cadherin. Knockdown of Grhl2 expression by shRNA in human mammary epithelial cell MCF10A leads to down-regulation of E-cadherin and EMT. Grhl2 is down-regulated in disseminated cancer cells that have undergone EMT, and over-expression of Grhl2 is sufficient to induce epithelial gene expression. Large clinical datasets reveal that expression of Grhl2 is significantly associated with poor relapse free survival and increased risk of metastasis in breast cancer patients. In mouse models, over-expression of Grhl2 significantly promotes tumor growth and metastasis. Further testing of several Grhl2 regulated genes leads to the same conclusions that the tumorigenic and metastatic potentials of tumor cells are linked to epithelial phenotype but not mesenchymal phenotype. In conclusion, our findings indicate that Grhl2 plays an essential role in the determination of epithelial phenotype of breast cancers, EMT and tumor progression.

The Grainyhead transcription factor Grhl3/Get1 suppresses miR-21 expression and tumorigenesis in skin: modulation of the miR-21 target MSH2 by RNA-binding protein DND1

Epidermal differentiation and stratification, crucial for barrier formation, are regulated by a complex interplay of transcription factors, including the evolutionarily conserved Grainyhead-like 3 (Grhl3/Get1); Grhl3-deleted mice exhibit impaired epidermal differentiation and decreased expression of multiple differentiation genes. To test whether Grhl3 regulates epidermal genes indirectly by controlling the expression of specific microRNAs (miRs), we performed miR profiling and identified 11 miRs that are differentially regulated in Grhl3(-/-) skin, one of which is miR-21, previously shown to be upregulated in diseased skin, including in psoriasis and squamous cell skin cancer. We found that miR-21 is normally expressed in the post-mitotic suprabasal layers of the epidermis, overlapping with Grhl3. The miR-21 promoter is bound and repressed by Grhl3 indicating that these two factors are involved in a regulatory loop maintaining homeostasis in the epidermis. Although miR-21 overexpression in normal keratinocytes had mild effects on the expression of several known miR-21 targets, an enhanced downregulation of the miR-21 tumor-related targets, including MSH2, was observed in Ras-transformed keratinocytes. The increased sensitivity of transformed keratinocytes to miR-21's effects occurs in part through downregulation of the RNA-binding protein DND1 during the transformation process. Additionally, we observed increased tumorigenesis in mice subcutaneously injected with transformed keratinocytes lacking Grhl3. These findings indicate that decreased Grhl3 expression contributes to tumor progression and upregulation of the oncomir miR-21 in squamous cell carcinoma of the skin.

The transcription factor Grainy head and the steroid hormone ecdysone cooperate during differentiation of the skin of Drosophila melanogaster

The arthropod epidermis is an epithelium that deposits the apical cuticle, which is a stratified extracellular matrix (ECM) protecting the animal against pathogens, preventing dehydration and also serving as an exoskeleton. Differentiation of the cuticle conceivably implies coordinated production, secretion and localization of its components. The underlying molecular mechanisms are poorly explored. In this work, we show that the transcription factor Grainy head and the steroid hormone ecdysone drive the production of two partially overlapping sets of cuticle factors. Nevertheless, Grainy head is needed to modulate the expression of ecdysone signalling factors; the significance of this cross-talk is yet unclear. In addition, we found that ecdysone signalling negatively regulates its own impact. In conclusion, our findings suggest that at least two independently triggered pathways have evolved in parallel to cooperatively ensure the stereotypic implementation of the cuticle. As Grainy head is also essential for epithelial differentiation in vertebrates, we speculate that it acts to decode the ancient skin programme common to all animals. Full differentiation of the skin necessitates a second, complementing taxon-specific programme that requires its own decoder, which is represented by ecdysone in arthropods, whereas the vertebrate specific one remains to be identified.

The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.

The evolutionary diversification of LSF and Grainyhead transcription factors preceded the radiation of basal animal lineages

BackgroundThe transcription factors of the LSF/Grainyhead (GRH) family are characterized by the possession of a distinctive DNA-binding domain that bears no clear relationship to other known DNA-binding domains, with the possible exception of the p53 core domain. In triploblastic animals, the LSF and GRH subfamilies have diverged extensively with respect to their biological roles, general expression patterns, and mechanism of DNA binding. For example, Grainyhead (GRH) homologs are expressed primarily in the epidermis, and they appear to play an ancient role in maintaining the epidermal barrier. By contrast, LSF homologs are more widely expressed, and they regulate general cellular functions such as cell cycle progression and survival in addition to cell-lineage specific gene expression.ResultsTo illuminate the early evolution of this family and reconstruct the functional divergence of LSF and GRH, we compared homologs from 18 phylogenetically diverse taxa, including four basal animals (Nematostella vectensis, Vallicula multiformis, Trichoplax adhaerens, and Amphimedon queenslandica), a choanoflagellate (Monosiga brevicollis) and several fungi. Phylogenetic and bioinformatic analyses of these sequences indicate that (1) the LSF/GRH gene family originated prior to the animal-fungal divergence, and (2) the functional diversification of the LSF and GRH subfamilies occurred prior to the divergence between sponges and eumetazoans. Aspects of the domain architecture of LSF/GRH proteins are well conserved between fungi, choanoflagellates, and metazoans, though within the Metazoa, the LSF and GRH families are clearly distinct. We failed to identify a convincing LSF/GRH homolog in the sequenced genomes of the algae Volvox carteri and Chlamydomonas reinhardtii or the amoebozoan Dictyostelium purpureum. Interestingly, the ancestral GRH locus has become split into two separate loci in the sea anemone Nematostella, with one locus encoding a DNA binding domain and the other locus encoding the dimerization domain.ConclusionsIn metazoans, LSF and GRH proteins play a number of roles that are essential to achieving and maintaining multicellularity. It is now clear that this protein family already existed in the unicellular ancestor of animals, choanoflagellates, and fungi. However, the diversification of distinct LSF and GRH subfamilies appears to be a metazoan invention. Given the conserved role of GRH in maintaining epithelial integrity in vertebrates, insects, and nematodes, it is noteworthy that the evolutionary origin of Grh appears roughly coincident with the evolutionary origin of the epithelium.

Grainyhead like 3 – a newly identified TNFalpha regulated transcription factor – is regulated by the Src kinases & NO

One major independent risk factor of cardiovascular diseases (CAD) is aging. Therefore, it is important to understand vascular aging on a cellular level. It is known that TNFalpha is increased in serum of CAD patients, induces senescence and apoptosis of endothelial cells (EC) and decreases their migratory capacity. Recently, we discovered the transcription factor Grainyhead like 3 (GRHL3) as a TNFalpha regulated gene. GRHL3 -/- mice demonstrated defects in cell migration. Therefore, the aim of our study was to investigate the role of GRHL3 in EC migration and the underlying mechanisms. After verifying expression of GRHL3 in EC, GRHL3 was cloned and overexpressed in EC. GRHL3 induced migration to a similar degree as vascular endothelial growth factor (VEGF). To get insights in the underlying mechanisms, we analyzed a known VEGF-induced migration activator, endothelial nitric oxide synthase (eNOS). eNOS phosphorylation on S1179 was increased upon overexpression of GRHL3, suggesting that GRHL3 increased EC cell migration through a nitric oxide (NO) dependent pathway. To determine whether a potentional positive feedback loop of nitric oxide (NO) and GRHL3 exists, we next tested the effect of NO on endogenous GRHL3 expression by real time PCR. Incubation with the NO donor papanonoate dramatically increased GRHL3 mRNA expression (papanonoate: 3.75-fold +/- 1.34 of control). Furthermore, decreased migratory capacity of senescent EC concomitant with an increase in Src kinase activity has been shown. To analyze whether Src has an influence on GRHL3, we determined the nuclear levels of this promigratory transcription factor in mouse embryonic fibroblasts (MEF), deficient for Src, Fyn and Yes (SFY -/-). A higher concentration of GRHL3 protein was observed in nuclear extracts of SFY -/- MEF. To demonstrate that the Src kinase family is indeed a negative regulator of GRHL3 expression in EC, we treated the cells with the Src kinase family inhibitor PP2 and analyzed expression of GRHL3. Indeed, expression of GRHL3 was dramatically increased by PP2 (PP2: 4.50-fold +/- 1.72 of control). In conclusion, GRHL3 is an important inducer of EC migration mediated through activation of eNOS/NO. NO in turn induces GRHL3 expression, suggesting a positive feedback loop. In addition, the Src kinase family negatively regulates endogenous GRHL3.