APPLICATION OF CHIP-SEQ, RNA-SEQ AND CRISPR/CAS9 SYSTEM TO ANALYZE THE MECHANISM OF SOX9, A MAJOR TRANSCRIPTION FACTOR FOR CHONDROGENESIS

Abstract

In recent days, many kinds of technologies including ChIP-sequencing, RNA-sequencing and genome-editing using Crispr/Cas9 system have been applied to molecular biology, agricultural sciences and so on. Especially genome editing technology is though to be powerful tool for targeted mutageneeis in various organisms and this high mutagenesis efficiency have accelerated the use of this system in various kind of researches. Actually in agricultural field, this has been applied to generate a tomato, rice and so on, which have desired characteristics. Today I would like to show how we have used these new technologies to demonstrate the mechanism of SOX9, a transcription factor, which regulates chondrogenesis. In vertebrae, most of the skeleton is formed through endochondral ossification. Endochondral bone formation requires multiple steps. SOX9 (SRY (sex-determining region Y)-related HMG box containing protein 9), a transcription factor, has been shown to contribute to the overt steps of chondrogenesis as a master regulatory factor. Here I would like to show that many kinds of genes including ECM proteins, ECM modification enzymes, receptors and transportors have been proven to be targets of this transcription factor by use of RNA-seq and ChIP-seq. Also I would like to show this transcription factor regulates one of its most major target genes, Type II collagen α, Col2a1 through its binding to an enhancer in its intron 6 by use of Cripsr/Cas9 mediated mutagenesis.