EnChIP Identification Of Fate Determinants Of The Mammary Epithelial Cells

Background and purposeThe mammary gland is a complex exocrine gland of mammals that completes the developmental process after birth.The mammary ducts are composed of the inner luminal cells and the outer myoepithelial cells.Many transcription factors are required for each stage of mammary gland development.The research on mammary gland development and mammary stem cells has made great progress,and the differentiation pattern of mammary epithelial cells has become a new focus of research.Yet little is known about the fate of individual cell types.Breast tumors from different cells of origin show distinct pathological features and prognostic outcome.Therefore,studying the fate of various cells can not only explain the differentiation pattern of mammary epithelial cells,but also provide novel strategies for the treatment and prevention of breast cancer.The fate of cells is ultimately determined by transcription factors.The enChIP technology is an effective method to study the interaction between promoters and transcription factors.We use engineered DNA-binding molecule-mediated chromatin immunoprecipitation(enChIP)combined with downstream proteomics analysis to identify proteins that interact with chromatin.Our goal is to find transcription factors that can regulate the fate of mammary epithelial cells.This research will provide new knowledge of the regulatory network guiding the differentiation of mammary epithelial cells and strategies for treating breast cancer.Methods and resultsIn this study,we used the enChIP technology to identify mammary cell fate determinants.We designed guide RNAs targeting the promoters of krt5 and krt8 genes,markers of myoepithelial cells and luminal epithelial cells respectively.Chromatin immunoprecipitation was used to test the specificity of guide RNAs and found they indeed targeted to krt5 promoters or krt8 promoters.We identified a series of factors(including Stk38,NF-κB pathway protein,Prdm1,Mga,etc.)related to the fate of mammary epithelial cells by chip-mass spectrometry.We further verified the enrichment and expression of Stk38,Rel A,Prdm1,and Mga in mammary epithelial cells based on Ch IP-q PCR and immunofluorescence of paraffin sections of human mammary tissues.We use RT-PCR experiments,immunofluorescence staining experiments,and Nano-Glo Dual-Luciferase experiments to explore the regulatory effects of these factors on the mammary epithelial cell lineage specification.The results showed that Stk38 and Rel A promoted the expression of CK5 and inhibited the expression of CK8;however,Prdm1 and Mga promoted the expression of CK8 and inhibited the expression of CK5.ConclusionIn this study,we found Stk38 and Rel A promote the phenotypes of myoepithelial cells and inhibit the phenotypes of luminal cells;Prdm1 promote the phenotypes of luminal cells and inhibit the phenotypes of myoepithelial cells.We found Mga is mainly expressed in luminal cells of the mammary gland and promotes the phenotype of luminal cells and inhibits the phenotype of myoepithelial cells.