Chromatin High-order Structure Variation Of Zebrafish ZF4 Cells During Cold Acclimation

Most specious of fish belong to poikilotherms,thus cold stress has significant impact on the growth and reproduction of fish and the mechanism of cold acclimation of fish has always been a research hotspot.Environmental temperature changing instantly and unpredictable,traditional genetics can’t explain these cold adaptation traits emerging in a short time well,which requires epigenetics research.Fish,a eukaryotic organism,its chromatin high-order structure is complex and highly dynamic.Organisms can adapt to the environment quickly and gain survival advantages by regulating genes to produce advantageous traits in a short time according to the variation of environmental pressure.Chromatin accessible region and chromatin looping are main components of chromatin high-order structure: highly compressed chromatin is usually inactive,while accessible chromatin is usually active;Chromatin can also form chromatin looping to make long-range regulation,such as the interaction between far away enhancers and promoters.Traditional methods of chromatin interaction research suffer from the shortcomings of low throughput and resolution.The development of sequencing technology has brought up new chromatin conformation capture technology.Hi-C technology is high-lighted because it can detect the interaction of all sites in one experiment.However,Hi-C relies on adjacent ligation to find target fragments,and there is random ligation in it,so it’s prone to noise,moreover,Hi-C requires extremely high sequencing depth.To eliminate the drawbacks of Hi-C technology,NIH’s research team has developed the Trac-looping(transposase-mediated analysis of chromatin looping)technology,which was published on Nature Methods in August 2018.This method utilizing the “cut and paste” mechanism of transposase Tn5,using transposase complex to insert biotin labeled transposon(complementary sequence of sequencing primer)to the chromatin accessible region,making random DNA recombination to the adjacent DNA in the chromatin accessible region.After selecting positive fragments by biotin purification,intramolecular ligation in extremely low concentration solution,rolling amplification and sequencing PCR,chromatin accessible region and interaction site were analyzed by high throughput sequencing.Trac-looping technology makes biotin purification before adjacent ligation,reducing random ligation from the source,thus ensures the reliability of adjacent ligation,reduces noise and requirement of sequencing depth,while improves resolution.In this study,as preparatory work for Trac-looping,hyperactive transposase Tn5 is expressed and purified by toxic proteins protocol,and its activity is tested by gradient test with ZF4 cells genomic DNA under standard reaction conditions.Using the innovative Trac-looping technique to detect zebrafish ZF4 cells’ chromatin high-order structure variation after 30 days of cold acclimation: get the precise chromatin interaction maps of zebrafish ZF4 cells before and after cold acclimation;locate DNA sites with different chromatin accessibility;make genome annotation of the chromatin accessibility variant region;predict the Enhancer-Promoter interaction relating to cold acclimation based on Trac-looping,H3K4me3 and H3K27 ac data.