A Role for Sim2s in Elongation Regulation of Milk Protein Gene Transcription in the Mammary Gland

Jessica Elswood, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, USA

J. Elswood, S. Pearson, M. Rijnkels, W. Porter
Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University

Identification of the mechanisms underlying the establishment and maintenance of functional differentiation is critical to our understanding of lactation. Genome wide analyses have recently demonstrated that regulation of differentiation occurs in large part by transcription elongation. Elongation proceeds after RNA Polymerase II (RNAPII) is released from its paused state through association with the positive transcriptional elongation factor (P-TEFb) containing catalytic enzyme, Cdk9. However, the mechanism by which regulation of these processes occur has yet to be elucidated in the mammary gland. We have previously shown that Singleminded-2s, (Sim2s) a member of the bHLH/PAS family of transcription factors, is required for establishing and maintaining mammary gland differentiation. Overexpression of Sim2s results in heightened expression of the milk protein genes beta-Casein (Csn2) and Wap. We have further demonstrated that conditional loss of Sim2s in mouse mammary epithelial cells results in decreased Csn2 gene expression. Building on chromatin immunopreciptitation (ChIP) and re-ChIP analyses documenting the association of Sim2s at the Csn2 promoter and interaction of the RNAPII complex with Sim2s respectively, we further analyzed the role of Sim2s in transcriptional regulation of differentiation-dependent gene expression. ChIP analysis of exon 7 at the Csn2 gene locus uncovered association of Sim2s, RNAPII, and Cdk9. Using mouse mammary epithelial cell lines (HC11 and CIT3) we found that inhibition of Cdk9 caused loss of milk protein gene expression. Additionally, we found an interaction between Cdk9 and Sim2s, which further indicates that Sim2s associates with transcription elongation machinery. This suggests Sim2s acts, not only as a traditional transcription factor, but also plays a role in enhancing transcription elongation of milk protein genes. Together these data demonstrate a novel paradigm of milk protein gene regulation in the mammary gland.

Download PDF

Leave a Reply

Meet Our Sponsors