Advancing Epigenetics Towards Systems Biology

Methyltransferase-based single-promoter analysis assay (Prot 38)

Tina B. Miranda, Einav Gal-Yam, Gerda Egger, Joy Lin, Shinwu Jeong, and Peter A.

Introduction

Nucleosomal positioning [reviewed in (1-3)] plays a pivotal role in the regulation of transcriptional initiation. Transcriptional co-activator complexes interact with nucleosomes (4) to induce nucleosomal rearrangements. Nucleosomes often have to unfold completely (5) or be disassembled (6) at the transcription start site, to allow for transcriptional initiation (7, 8).

Most of the studies done on the nucleosomal rearrangements of histones use conventional footprinting techniques, which rely on nuclease digestion and primer extension. However, promoters are molecular ‘modules’, which are controlled as individual entities. When analyzed by conventional methodologies this modularity is destroyed. Our lab has modified a previously described footprinting strategy (9,10), which now allows us to study the chromatin structure of individual molecules. MSPA (methylation-sensitive promoter analysis) allows for the study of unmethylated CpG islands by treatment of nuclei with the CpG-specific DNA methyltransferase SssI (M.Sssi), followed by genomic bisulfite sequencing of individual progeny DNA molecules (11-13). This gives single molecule resolution over the promoter and allows for the physical linkage between binding sites on individual promoter molecules to be maintained.

Our lab has successfully used this method to study the difference in nucleosomal positioning in the p16 promoters in two human cell lines (11), to identify transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress (12), and to study the changes in nucleosome occupancy silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells (13).

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Tina B. Miranda, Einav Gal-Yam, Gerda Egger, Joy Lin, Shinwu Jeong, and Peter A.

University of Southern California - Norris Comprehensive Cancer Center - 1441 Eastlake Ave
Los Angeles, CA 90033, USA

Tina B. Miranda, Einav Gal-Yam, Gerda Egger, Joy Lin, Shinwu Jeong, and Peter A