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molecular mechanism remains to be fully elucidated. Method: To investigate the role of HINTW in avian sex determination, a prokaryotic expression vector containing its key structural domain was constructed, and its in vitro expression was achieved. Pull-down assays were performed to capture interacting proteins from male and female gonadal tissues, followed by a silver staining analysis to compare interaction profiles between ovaries and testes. Mass spectrometry was utilized to identify differentially bound proteins. Additionally, functional characterization and co-immunoprecipitation (Co-IP) assays were conducted to validate the interaction between HINTW and its candidate binding partner. Result: A total of 1590 differentially bound proteins were identified between ovarian and testicular tissues. Functional analysis and Co-IP assays confirmed a specific interaction between HINTW and UBE2I in the ovary, suggesting that HINTW may facilitate female differentiation in chicken embryos through its interaction with UBE2I. Conclusions: This study provides novel insights into the molecular mechanisms underlying HINTW-mediated female differentiation in chickens and contributes to a deeper understanding of avian sex determination.<\/jats:p>","DOI":"10.3390\/genes16040366","type":"journal-article","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T13:48:20Z","timestamp":1742824100000},"page":"366","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Molecular Mechanism of HINTW (HINT Gene in W-Chromosome)-Mediated UBE2I (Ubiquitin Conjugating Enzyme E2 I) Interaction in Female Differentiation of Chicken Embryos"],"prefix":"10.3390","volume":"16","author":[{"given":"Changhua","family":"Sun","sequence":"first","affiliation":[{"name":"College of Biological and Chemical Engineering, Yangzhou Polytechnic College, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6973-5959","authenticated-orcid":false,"given":"Jiuzhou","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Animal & Avian Sciences, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6137-6242","authenticated-orcid":false,"given":"Malik Ahsan","family":"Ali","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China"},{"name":"College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Sun","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1829-1718","authenticated-orcid":false,"given":"Yingjie","family":"Niu","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qisheng","family":"Zuo","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Han","sequence":"additional","affiliation":[{"name":"Poultry Institute, Jiangsu Institute of Poultry Sciences\/Chinese Academy of Agricultural Sciences, Yangzhou 225003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5692-4577","authenticated-orcid":false,"given":"Bichun","family":"Li","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Department of Animal & Avian Sciences, University of Maryland, College Park, MD 20742, USA"},{"name":"Poultry Institute, Jiangsu Institute of Poultry Sciences\/Chinese Academy of Agricultural Sciences, Yangzhou 225003, China"},{"name":"College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2489-2173","authenticated-orcid":false,"given":"Kai","family":"Jin","sequence":"additional","affiliation":[{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"},{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China"},{"name":"College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3645","DOI":"10.1091\/mbc.11.10.3645","article-title":"Wpkci, encoding an altered form ofPKCI, is conserved widely on the avian W chromosome and expressed in early female embryos: Implication of its role in female sex determination","volume":"11","author":"Hori","year":"2000","journal-title":"Mol. 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