DNMT3B Characterization in a Human Model of Pluripotency: Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) differentiation relies on a plethora of factors that regulate gene expression as lineage-specific transcription factors, but also epigenetics modifications, as DNA methylation. In particular, long noncoding RNAs (lncRNAs) are known to mediate the interaction between DNA and proteins in the nucleus. Here we wanted to study the interplay between lncRNAs and DNA methylation, focusing in particular on the de novo DNA methylase DNMT3B. We first investigated the self- renewal ability and the pluripotency state of an hESC line, showing that H9 hES cells are in a primed pluripotency state. Once having assessed the expression levels of DNMT3B in human embryonic stem cells and in different human colorectal cancer cell lines, we sought to exploit DNMT3B recruitment mechanism by isolating DNMT3B-interacting lncRNAs via RNA immunoprecipitation (RIP) in native conditions. To improve the efficiency of DNMT3B isolation, we also exploited the biotin-avidin interaction producing an avitag-DNMT3B construct which is biotinylated inside the cells and set up a Bio-RIP. Therefore, here we suggest and investigate a mechanism by which lncRNAs tether the DNMT3B enzyme to specific genomic loci in human embryonic stem cells.