The interplay of transcription regulation and chromatin organization is essential for normal tissue development and function, and maintenance of cellular differentiation. Loss of the normal differentiated state is a hallmark of many malignancies. A better understanding of all the mechanisms that regulate and maintain the differentiation state of a tissue will enable the development of strategies to maintain, enhance and induce differentiation to improve cancer outcomes and functional-differentiation-dependent outcomes such as milk production in the mammary gland. Transcription factors binding to regulatory elements and regulating chromatin states whilst enabling transcription initiation of target genes is one of the main processes underlying differentiation- dependent (tissue- and developmental stage specific) gene transcription. Recent genome wide studies have indicated that developmental gene expression is also regulated through transcription elongation control following promoter-proximal pausing. However, there are still gaps in our understanding of which cell-specific transcription factors interact with the elongation complex to regulate cell differentiation and function in vivo. We have shown that Singleminded-2s (Sim2s; expressed from Sim2), a member of the bHLH/PAS family of transcription factors, plays a major role in expression of differentiation-associated genes in the mammary gland, not as a classical DNA-binding transcription factor, but by interacting directly with the transcriptional elongation complex. Based on these exciting new results, we hypothesize that a subset of differentiation- dependent genes are regulated through enhancement of elongation in conjunction with changes in chromatin organization, mediated in the mammary gland by Sim2s. To address this hypothesis we propose two Specific Aims.
In Aim 1, we will couple Global Run On (GRO)-seq with RNA-seq and ChIP-seq analysis for Sim2s binding and histone modifications associated with active gene expression during mammary gland development using WT and Sim2 mammary gland knockout and over-expressing mice.
In Aim 2, we will define the physical basis for, and functional outcomes of interactions between Sim2s and P-TEFb components involved in transcription factor recruitment, elongation control and chromatin alterations associated with time- dependent hormone-dependent activation of the Csn2 gene. In addition, we will also determine the effect of over and under expression of elongation factors on hormone-induced Csn2 expression in WT and Sim2s over and under-expressing HC11 cells. These studies will better define mechanisms by which differentiation is regulated through selective gene expression and give new insight into the regulation of differentiation- dependent gene transcription in the mammary gland and functionally differentiated tissues in general.
Despite the importance of transcription regulation of gene expression in normal development and function of tissues, few studies have addressed the specific regulation of differentiation-dependent genes normally expressed in functionally differentiated cells. A better understanding of transcriptional regulation of differentiation and te factors involved will give new insight into the regulation of differentiation-dependent gene transcription in the mammary gland and functionally differentiated tissues in general. This is expected to have application in malignancies or prevention of malignant transformation as well as in human lactation, dairy research and production.