Our understanding of the pathogenic mechanisms for orofacial clefting (OFC) is limited by the fact that less than half of the heritable risk for this disorder has been assigned to specific genes. Towards identifying pathological sequence variants among the many irrelevant ones detected in exomes and whole genomes of patients with this disorder, an understanding of the gene regulatory networks (GRNs) that govern the development of relevant tissues, including the oral periderm, is essential. We propose a systems biology approach to analyzing the periderm GRN. Using this approach in the past enabled us to identify three novel OFC risk genes. We will utilize two model organisms, zebrafish and mouse, because the periderm differentiation GRN appears to be highly conserved. In zebrafish, the periderm differentiates very early in embryogenesis, greatly facilitating the execution and interpretation of genetic perturbation analyses. Mouse, on the other hand, has the advantage that its craniofacial anatomy is more similar to that of humans.
In Aim 1, we will determine the zebrafish periderm differentiation GRN using a state-of-the-art network inference algorithm, NetProphet 2. This tool carries out both a coexpression analysis and a differential expression analysis. Input data sets will include RNA-seq expression profiles we will generate from loss-of-function (LOF) embryos for 4 key transcription factors (TF) known to participate in this GRN. We will also identify the direct gene linkages of these key TFs in the periderm GRN. Finally, we will test a novel candidate member of the periderm GRN, Tead, by carrying out LOF tests in zebrafish, thereby exploiting the strength of this model system.
In Aim 2 we will deduce the murine oral periderm differentiation GRN, also using the NetProphet algorithm. Input datasets will include expression profiles of periderm isolated from the palate shelves of wild-type mouse embryos, and from heterozygous mutants of three key TFs: Irf6, Grhl3 and Tfap2a. For each of the mutant genotypes there is evidence of abnormal periderm differentiation. We will also identify murine periderm enhancer candidates by sorting GFP-positive and -negative cells from Krt17-gfp transgenic embryos, performing ATAC-seq on both populations, and H3K27Ac ChIP-seq on cells from palate shelves and the nasal cavity. As in Aim 1, we will also identify the direct gene linkages of the key TFs. We will train a machine learning algorithm on palate periderm enhancers, and use the resulting scoring function to prioritize OFC-associated SNPs near genes that are expressed in periderm for those that are likely to directly affect risk for OFC. Finally, we will perform allele- specific reporter assays on the top candidate SNPs from each of three loci. The expected outcome is a deeper understanding of the specific TFs and cis-regulatory elements that control differentiation of the periderm. This will have a broad impact because it will enable human geneticists to prioritize candidate risk variants that emerge from whole-exome and -genome sequencing analyses of OFC.
Orofacial is a common disease with a major impact on the affected individual, their family and society. Here, we will determine the gene regulatory network for periderm, an embryonic tissue that is critical for development of the palate. Our new knowledge will identify candidate genes and help identify pathogenic variants located in regulatory elements which will improve risk assessment and provide new targets for rational therapies.
|Eshete, M A; Liu, H; Li, M et al. (2018) Loss-of-Function GRHL3 Variants Detected in African Patients with Isolated Cleft Palate. J Dent Res 97:41-48|
|Duncan, Kaylia M; Mukherjee, Kusumika; Cornell, Robert A et al. (2017) Zebrafish models of orofacial clefts. Dev Dyn 246:897-914|
|Liu, Huan; Busch, Tamara; Eliason, Steven et al. (2017) Exome sequencing provides additional evidence for the involvement of ARHGAP29 in Mendelian orofacial clefting and extends the phenotypic spectrum to isolated cleft palate. Birth Defects Res 109:27-37|
|Seberg, Hannah E; Van Otterloo, Eric; Cornell, Robert A (2017) Beyond MITF: Multiple transcription factors directly regulate the cellular phenotype in melanocytes and melanoma. Pigment Cell Melanoma Res 30:454-466|
|Liu, Huan; Leslie, Elizabeth J; Carlson, Jenna C et al. (2017) Identification of common non-coding variants at 1p22 that are functional for non-syndromic orofacial clefting. Nat Commun 8:14759|
|Leslie, Elizabeth J; Liu, Huan; Carlson, Jenna C et al. (2016) A Genome-wide Association Study of Nonsyndromic Cleft Palate Identifies an Etiologic Missense Variant in GRHL3. Am J Hum Genet 98:744-54|
|Liu, Huan; Leslie, Elizabeth J; Jia, Zhonglin et al. (2016) Irf6 directly regulates Klf17 in zebrafish periderm and Klf4 in murine oral epithelium, and dominant-negative KLF4 variants are present in patients with cleft lip and palate. Hum Mol Genet 25:766-76|
|Erives, Albert J (2015) Genes conserved in bilaterians but jointly lost with Myc during nematode evolution are enriched in cell proliferation and cell migration functions. Dev Genes Evol 225:259-73|
|Leslie, Elizabeth J; Taub, Margaret A; Liu, Huan et al. (2015) Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci. Am J Hum Genet 96:397-411|
|Lidral, Andrew C; Liu, Huan; Bullard, Steven A et al. (2015) A single nucleotide polymorphism associated with isolated cleft lip and palate, thyroid cancer and hypothyroidism alters the activity of an oral epithelium and thyroid enhancer near FOXE1. Hum Mol Genet 24:3895-907|
Showing the most recent 10 out of 13 publications