title: HOXB5 binds to multi-species conserved sequence (MCS+9.7) of RET gene and regulates RET expression.
abstract: RET gene is crucial for the development of enteric nervous system, and dys-regulation of RET expression causes Hirschsprung disease. HOXB5 regulates RET transcription, and perturbations in transcriptional regulation by HOXB5 caused reduced RET expression and defective enteric nervous system development in mice. The mechanisms by which HOXB5 regulate RET transcription are unclear. Thus, unraveling the regulatory mechanisms of HOXB5 on RET transcription could lead to a better understanding of the etiology of Hirschsprung disease. In this study, we identified and confirmed HOXB5 binding to the multi-species conserved sequence (MCS+9.7) in the first intron of the RET gene. We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos. The deletion of HOXB5 binding site interfered with HOXB5 trans-activation. Furthermore, transfection of HOXB5 induced endogenous RET transcription, enhanced the co-precipitation of TATA-box binding protein with the transcription start site of RET, and induced histone H3K4 trimethylation in chromatin regions upstream and downstream of RET transcription start site. In conclusion, (i) HOXB5 physically interacted with MCS+9.7 and enhanced RET transcription, (ii) HOXB5 altered chromatin conformation and histone modification of RET locus, which could facilitate the formation of transcription complex, and enhance RET transcription, (iii) expression of RET was mediated by a complex regulatory network of transcription factors functioning in a synergistic, additive and/or independent manners. Hence, dys-regulation of RET expression by HOXB5 could result in insufficient RET expression and Hirschsprung disease.
authors.affiliation: Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region; Faculty of Medicine, Shenzhen University, Shenzhen, Guangdong Province, China.
authors.forename: Joe Jiang
authors.initials: JJ
authors.lastname: Zhu
authors.affiliation: Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region.
authors.forename: Mandy KaMan
authors.initials: MK
authors.lastname: Kam
authors.affiliation: Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region.
authors.forename: Maria-Mercè
authors.initials: MM
authors.lastname: Garcia-Barceló
authors.affiliation: Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region.
authors.forename: Paul Kwong Hang
authors.initials: PK
authors.lastname: Tam
authors.affiliation: Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region. Electronic address: vchlui@hku.hk.
authors.forename: Vincent Chi Hang
authors.initials: VC
authors.lastname: Lui
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002642:Chick Embryo; D005786:Gene Expression Regulation; D006627:Hirschsprung Disease / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007438:Introns; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D051096:Proto-Oncogene Proteins c-ret / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C489126:HOXB5 protein, human; D018398:Homeodomain Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human
keywords: Enhancer; HOXB5; Hirschsprung disease; RET; Transcription