ABSTRACT
The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.
Subject(s)
Craniofacial Abnormalities/genetics , DNA-Binding Proteins/physiology , Histone Methyltransferases/physiology , MDS1 and EVI1 Complex Locus Protein/physiology , Skull/embryology , Transcription Factors/physiology , Zebrafish Proteins/physiology , Animals , Chromatin/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Ear, Middle/abnormalities , Ear, Middle/embryology , Facial Bones/embryology , Female , Genes, Lethal , Histone Code/genetics , Histone Methyltransferases/deficiency , Histone Methyltransferases/genetics , Histones/metabolism , Jaw/embryology , MDS1 and EVI1 Complex Locus Protein/deficiency , MDS1 and EVI1 Complex Locus Protein/genetics , Male , Methylation , Mice, Inbred C57BL , Protein Processing, Post-Translational/genetics , Species Specificity , Transcription Factors/deficiency , Transcription Factors/genetics , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
Our previous studies demonstrated that INPP4B, a member of the PI3K/Akt signaling pathway, is overexpressed in a subset of AML patients and is associated with lower response to chemotherapy and shorter survival. INPP4B expression analysis in AML revealed a right skewed frequency distribution with 25% of patients expressing significantly higher levels than the majority. The 75% low/25% high cut-off revealed the prognostic power of INPP4B expression status in AML, which would not have been apparent with a standard median cut-off approach. Our identification of a clinically relevant non-median cut-off for INPP4B indicated a need for a generalizable non-median dichotomization approach to optimally study clinically relevant genes. To address this need, we developed Subgroup Identifier (SubID), a tool which examines the relationship between a continuous variable (e.g. gene expression), and a test parameter (e.g. CoxPH or Fisher's exact P values). In our study, Fisher's exact SubID was used to reveal EVI1 as a transcriptional regulator of INPP4B in AML; a finding which was validated in vitro. Next, we used CoxPH SubID to conduct a pan-cancer analysis of INPP4B's prognostic significance. Our analysis revealed that INPP4Blow is associated with shorter survival in kidney clear cell, liver hepatocellular, and bladder urothelial carcinomas. Conversely, INPP4Blow was shown to be associated with increased survival in pancreatic adenocarcinoma in three independent datasets. Overall, our study describes the development and application of a novel subgroup identification tool used to identify prognostically significant rare subgroups based upon gene expression, and for investigating the association between a gene with skewed frequency distribution and potentially important upstream and downstream genes that relate to the index gene.
