Mapping Higher-order Chromatin Structure Of Mouse Embryonic Fibroblast Cells By GST-MNase-Seq

Mammalian genomes encode genetic information in their linear sequence,but appropriate expression of their genes requires chromosomes to fold into complex three-dimensional structures.Chromatin is composed of nucleosome.Thus,Chromatin compactness is the key feature of chromatin that reflects its accessibility to transcription machinery.Tightly packed closed chromatin is considered a hallmark of gene silencing,and chromatin opening precedes lineage-specific gene expression thus providing an excellent indicator of cell fate commitment.The measurement of chromatin accessibility is a powerful approach to analyze the higher-order chromatin structure and its dynamics.To quantatitively measure the dynamic changes of chromatin accessibility in the mouse embryonic fibroblast cells,we develop a modified MNase-Seq protocol,in which the mouse embryonic fibroblast cells are partially digested by an engineered micrococcal nuclease tagged with a GST tag instead of commercial MNase in this study.We implemented a modified MNase-seq approach to quantitatively measure the local chromatin compaction and accessibility across the whole genome of mouse embryonic fibroblasts by altering MNase digestion conditions.Our results demonstrate the MNase accessible signal enrichment of active genes decreased with prolonging digestion time.Our results also demonstrate that MNase accessibility evaluates the opening chromatin state quantified,as well as open promoter genes defined by MNase-seq have a higher expression frequency than those compact promoter genes which has not any MNase accessible signals.Using this novel GST-MNase-Seq method,in combination with bioinformatics,three different types of accessible chromatin regions,named active and poised open chromatin regions and closed chromatin regions,are observed in the mouse embryonic fibroblast cells studied in this thesis.Interestingly,our results demonstrate that the active open chromatin regions obtained by this method are mainly included in the accessible chromatin regions observed previously by DNase-Seq analyses,however,the poised open chromatin regions are previously uncharacterized.We suapect that the genes,whose promoter regions are in poised open chromatin states,are crucial for cell fate determination during development.