Meta-Analysis of Grainyhead-Like Dependent Transcriptional Networks: A Roadmap for Identifying Novel Conserved Genetic Pathways

Abstract

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.

Hyperactive WNT/CTNNB1 signaling induces a competing cell proliferation and epidermal differentiation response in the mouse mammary epithelium

“…Of note, transient overexpression of GRHL3, but not OVOL1 or KLF4, was sufficient to induce endogenous Rptn but not its neighboring gene, Tchh (Figure 4H, Supplementary File 3). This is in line with the fact that Rptn was previously suggested to be a conserved target gene for GRHL transcription factors in both mouse and human (Mathiyalagan et al, 2019).…”

Grainyhead-like (Grhl) Target Genes in Development and Cancer

“…Future studies should aim to characterise the involvement of targets implicated in one process (e.g., edn1 in craniofacial development) across multiple other processes, to determine which of these pathways are core regulatory mechanisms involved in the most basic steps of tissue organisation and maintenance. Through the ongoing evolution of “big-data” (single cell RNA-SEQ, spatial transcriptomics [ 162 ], ChIP-SEQ, bioinformatic meta-analyses [ 42 ] etc.) combined with an increased focus on identifying both upstream regulators and transcriptional co-factors of the Grhl family, the future of understanding the Gene Regulatory Networks (both critically conserved and tissue/organ/disease specific) is likely to be substantially improved in the coming years.…”

“…Although this TF family directly regulates multiple processes common to both development and cancer, what is becoming very clear is that the regulatory specificity of these genes is highly context-dependent [ 42 ]. A nice analysis of GRHL2-function in the context of the EMT has recently been published [ 43 ], and so we will not cover this role again here; however, clearly the role of Grhl-factors in maintaining epithelial integrity and stability, and preventing mesenchymal transition is essential.…”

GRHL2-controlled gene expression networks in luminal breast cancer

“…A meta-analysis combining all RNA-seq, micro-array, and ChIP-seq experiments, identified common candidate genes for regulation by GRH or GRHL1-3. The authors noticed a striking lack of correlation between findings in normal epithelia as compared to cancerous cells with CDH1 being identified as a target in normal epithelia but not cancer [ 78 ]. Likewise, the findings reported in our study represent candidate GRHL2-regulated genes and pathways in luminal breast cancer that partly overlap but are also distinct from GRHL2 regulation in normal epithelia and other cancer types.…”