Abstract
Stem cells can divide indefinitely and maintain their capability to differentiate into many cell types, the key features of self-renewal and pluripotency, which explains their importance to regenerative medicine. Embryonic stem cells (ESCs), the most highly pluripotent of all stem cells, have the added potential of tumorigenesis. This is thought to be driven in part through a shared gene expression program regulated by the transcription factor, Myc. Myc, first characterized as a potent oncogene, is shown to maintain pluripotency and self-renewal in mouse ESCs. Myc regulation of pluripotency and self-renewal is evident by its role in the generation of induced pluripotent stem (iPS) cells. Myc is thought to regulate target gene expression both locally through classical mechanisms, and globally through euchromatin remodeling. In this way, Myc can affect gene expression on a large enough scale to reprogram differentiated cells into iPS cells. Miz-1, a transcription factor named for its interaction with Myc, is thought to form a co-repressor complex with Myc, silencing Miz-1 target genes including those associated with differentiation and proliferation. Miz-1 contains BTB/POZ and 13 C2H2 zinc fingers and is thought to bind initiator sequences (INR) in the core promoters of target genes thereby modulating their expression. Still, relatively little is known about the function of Miz-1 as a transcriptional regulator and recent epigenetics analysis in hESCs suggest Miz-1 binds alternative sequences, not associated with the INR of target gene promoters. Using a Miz-1 maltose binding protein (MBP) fusion protein tag system, this study implemented an in vitro, high-throughput DNA binding assay and Multiple em for Motif Elicitation (MEME) analysis to identify putative Miz-1 DNA biding motifs de novo. The consensus motifs, ATCGAT and GATTACCGA were then confirmed by electrophoretic mobility shift analysis (EMSA) and further bioinformatics analysis revealed motif occurrences in functionally relevant gene ontology clusters including: transcription regulation, growth, chromatin, and developmental genes. MBP pull-down mass spectrometry analysis also identified interesting Miz-1 protein cofactors from hESC nuclear extracts that are associated with reported Miz-1 functions. Miz-1 DNA and protein interactions highlighted in this study confirm its role as a master transcriptional regulator, cofactor and antagonist of Myc in hESCs. Though, the findings also underline the importance of further characterization of pluripotency and self-renewal in hESCs so that potential therapies may be safe and effective.