Protein moonlighting is a phenomenon in which a single polypeptide chain can perform a number of different unrelated functions. Here we present our analysis of moonlighting in the case of selected DNA repair proteins which include G:T mismatch-specific thymine DNA glycosylase (TDG), methyl-CpG-binding domain protein 4 (MBD4), apurinic/apyrimidinic endonuclease 1 (APE1), AlkB homologs, poly (ADP-ribose) polymerase 1 (PARP-1) and single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1). Most of their additional functions are not accidental and clear patterns are emerging. Participation in RNA metabolism is not surprising as bases occurring in RNA are the same or very similar to those in DNA. Other common additional function involves regulation of transcription. This is not unexpected as these proteins bind to specific DNA regions for DNA repair, hence they can also be recruited to regulate transcription. Participation in demethylation and replication of DNA appears logical as well. Some of the multifunctional DNA repair proteins play major roles in many diseases, including cancer. However, their moonlighting might prove a major difficulty in the development of new therapies because it will not be trivial to target a single protein function without affecting its other functions that are not related to the disease.
The Drosophila grainyhead (grh) and vertebrate Grainyhead-like (Grhl) transcription factors are among the most critical genes for epithelial development, maintenance and homeostasis, and are remarkably well conserved from fungi to humans. Mutations affecting grh/Grhl function lead to a myriad of developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member GRHL3 have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans, particularly the genetic disorder Van Der Woude Syndrome (VWS), as well as spina bifida, whereas mutations in mammalian Grhl2 lead to exencephaly and facial clefting. As transcription factors, Grhl proteins bind to and activate (or repress) a substantial number of target genes that regulate and drive a cascade of transcriptional networks. A multitude of large-scale datasets have been generated to explore the grh/Grhl-dependent transcriptome, following ablation or mis-regulation of grh/Grhl-function. Here, we have performed a meta-analysis of all 41 currently published grh and Grhl RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by grh/Grhl genes across disparate biological contexts. Moreover, we have also cross-referenced our results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct grh/Grhl targets, based on genomic occupancy by grh/Grhl genes. Lastly, to interrogate the predictive strength of our approach, we experimentally validated the expression of the top 10 candidate grhl target genes in epithelial development, in a zebrafish model lacking grhl3, and found that orthologues of seven of these (cldn23, ppl, prom2, ocln, slc6a19, aldh1a3, and sod3) were significantly down-regulated at 48 hours post-fertilisation. Therefore, our study provides a strong predictive resource for the identification of putative grh/grhl effector target genes.
The Grainyhead-like 1 (GRHL1) transcription factor is tissue-specific and is very highly expressed in the kidney. In humans the GRHL1 gene is located at the chromosomal position 2p25. A locus conferring increased susceptibility to essential hypertension has been mapped to 2p25 in two independent studies, but the causative gene has never been identified. Furthermore, a statistically significant association has been found between a polymorphism in the GRHL1 gene and heart rate regulation. The aim of our study was to investigate the physiological consequences of Grhl1 loss in a mouse model and ascertain whether Grhl1 may be involved in the regulation of blood pressure and heart rate.In our research we employed the Grhl1 "knock-out" mouse strain. We analyzed renal gene expression, blood pressure and heart rate in the Grhl1-null mice in comparison with their "wild-type" littermate controls. Most important results: The expression of many genes is altered in the Grhl1(-/-) kidneys. Some of these genes have previously been linked to blood pressure regulation. Despite this, the Grhl1-null mice have normal blood pressure and interestingly, increased heart rate.Our work did not discover any new evidence to suggest any involvement of Grhl1 in blood pressure regulation. However, we determined that the loss of Grhl1 influences the regulation of heart rate in a mouse model.
The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.
The involvement of Grainyhead-like (GRHL) transcription factors in various cancers is well documented. However, little is known about their role in clear cell renal cell carcinoma (ccRCC). We discovered that the expression of two of these factors-GRHL1 and GRHL2-are downregulated in ccRCC samples, and their expression is correlated with the expression of VHL gene. This suggests a functional link between the GRHL transcription factors and one of the best known tumor suppressors. Although the GRHL genes are not mutated in ccRCC, some of the single nucleotide polymorphisms in these genes may indicate an increased risk of ccRCC development and/or may allow to assess patients' prognoses and predict their responses to various forms of therapy. Silencing of GRHL2 expression in non-tumorigenic kidney cell line results in increased cell proliferation, increased resistance to apoptosis, as well as changes in the levels of selected proteins involved in the pathogenesis of ccRCC. These changes support the potential role for GRHL2 as a suppressor of ccRCC.
The Grainyhead-like (GRHL) family of transcription factors has three mammalian members, which are currently termed Grainyhead-like 1 (GRHL1), Grainyhead-like 2 (GRHL2), and Grainyhead-like 3 (GRHL3). These factors adopt a DNA-binding immunoglobulin fold homologous to the DNA-binding domain of key tumor suppressor p53. Their patterns of expression are tissue and developmentally specific. Earlier studies of the GRHL proteins focused on their functions in mammalian development. In recent years, these factors have been linked to many different types of cancer: squamous cell carcinoma of the skin, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, neuroblastoma, prostate cancer, and cervical cancer. The roles of GRHL proteins in these various types of cancer are complex, and in some cases appear to be contradictory: they can serve to promote cancer development, or they may act as tumor suppressors, depending on the particular GRHL protein involved and on the cancer type. The reasons for obvious discrepancies in results from different studies remain unclear. At the molecular level, the GRHL transcription factors regulate the expression of genes whose products are involved in cellular proliferation, differentiation, adhesion, and polarity. We herein review the roles of GRHL proteins in cancer development, and we critically examine relevant molecular mechanisms, which were proposed by different authors. We also discuss the significance of recent discoveries implicating the involvement of GRHL transcription factors in cancer and highlight potential future applications of this knowledge in cancer treatment.
The GRHL2 gene, encoding the Grainyhead-like 2 transcription factor, is essential for various biological processes. While GRHL2 has a complex role in cancer biology, its genetic variants have been also implicated in different forms of hearing loss (HL), including autosomal dominant non-syndromic hearing loss (DFNA28). Here, we report a novel c.1061C>T, p.(Ala354Val) mutation within the DNA binding domain (DBD) of GRHL2 that was identified in a three-generation HL family using a targeted multi-gene panel covering 237 HL-related genes. Unlike the previously reported DFNA28-causing variants that result in protein truncation, the impact of the p.(Ala354Val) missense change cannot be attributed to GRHL2 transcript level or composition, but to an alteration in protein function. Molecular dynamics simulations revealed destabilization of the p.(Ala354Val) mutant GRHL2 dimer interface and an altered DNA binding dynamics, leading to chaotic interaction patterns despite increased binding affinity to DNA. Functional assays demonstrated that the p.(Ala354Val) mutation and other DFNA28-related mutations in the DBD lead to loss of GRHL2 transcriptional transactivation activity, while the p.(Arg537Profs*11) mutation in the dimerization domain results in a gain-of-function effect. The findings indicate that both GRHL2 haploinsufficiency and gain-of-function contribute to HL and underscore the complex regulatory role of GRHL2 in maintaining proper function of the auditory system. Our study emphasizes the need to consider structural and functional aspects of gene variants to better understand their pathogenic potential. As GRHL2 is involved in a multitude of cellular processes, the data gathered here can be also applicable to other conditions.
The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG. Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes.
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