The transcription factor Trp63 is a lineage-specific transcription factor that is highly expressed and plays an important role in epithelial-rich tissue. This is epitomized by the striking phenotype of the p63 knockout mice, which lack a mature stratified epidermis and exhibit a block in development of multiple ectodermal appendages such as mammary glands, hair and teeth. Remarkably, in perfect phenocopy of the mouse model, dominant mutations of the Trp63 gene are found in human disorders with various combinations of ectodermal dysplasia, orofacial clefting and limb malformations. Deciphering the underlying molecular mechanism by which p63 transcriptionally regulates developmental processes has proven to be quite challenging, in part, due to the existence of multiple p63 isoforms. However, recent studies have strongly suggested that ?Np63 proteins are the dominant isoforms expressed in epithelial cells where they can function as potent transcriptional regulators, dictate stem/progenitor cell renewal and orchestrate the balance between proliferation and differentiation. Hence, there is an emerging need for identifying specific molecular targets of ?Np63, particularly other developmentally regulated transcription factors, which act together with ?Np63 in mediating some of the biological functions of p63. However our knowledge of how ?Np63 fits into the global transcriptional circuitry of mammary glands is not well characterized. We have generated a novel allele of ?Np63-GFP mice, where the expression of GFP is driven by ?Np63-specific regulatory elements thus allowing prospective isolation of ?Np63-enriched myoepithelial/basal populations. Furthermore, we have developed and successfully utilized ChIP-grade antibodies directed specifically to the ?Np63 isoform. These valuable resources coupled with the powerful technology of next-generation sequencing will allow us to a) Perform ChIP using anti-?Np63 specific antibodies and deep sequencing (ChIP-seq) to identify relevant ?Np63 targets sites in mammary glands at high resolution and deep coverage, b) Analyze the results obtained from the global ?Np63-binding sites by comparing them against the existing p63-target database to identify key members of the ?Np63-driven transcriptional circuitry in myoepithelial cells that are likely to be relevant for the biological function of mammary glands. Given the essential role of p63 in mammary gland development and its close association with breast cancer, the identification of a well-defined ?Np63 regulatory program is a critical step in understanding the molecular mechanism of its function and in integrating ?Np63 with existing regulatory networks. Our studies to probe the in vivo ?Np63-regulated transcriptome in a specific and global fashion will provide novel insights into mechanisms of mammary gland development and differentiation that has not been possible before.

Public Health Relevance

The p53-family member, p63, is a critical regulator for controlling the balance between myoepithelial proliferation and differentiation and for masterminding cell lineage choices in mammary glands. Alterations in the biological activity of p63 are often found in human patients with developmental disorders and breast cancer. Thus it is important to understand the molecular details of how p63 regulates epithelial growth and what signaling pathways and specific target genes are regulated by this factor so as to develop better new therapies and cures.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
1R03HD073891-01A1
Application #
8516752
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Raiten, Daniel J
Project Start
2013-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$79,438
Indirect Cost
$29,438
Name
State University of New York at Buffalo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Sethi, Isha; Sinha, Satrajit; Buck, Michael J (2014) Role of chromatin and transcriptional co-regulators in mediating p63-genome interactions in keratinocytes. BMC Genomics 15:1042
Rizzo, Jason M; Sinha, Satrajit (2014) Analyzing the global chromatin structure of keratinocytes by MNase-seq. Methods Mol Biol 1195:49-59