Computational Methods for Assessing Chromatin Hierarchy

The hierarchical organization of chromatin is known to associate with diverse cellular functions; however, the precise mechanisms and the 3D structure remain to be determined. With recent advances in high-throughput next generation sequencing (NGS) techniques, genome-wide profiling of chromatin structures is made possible. Here, we provide a comprehensive overview of NGS-based methods for profiling “higher-order” and “primary-order” chromatin structures from both experimental and computational aspects. Experimental requirements and considerations specific for each method were highlighted. For computational analysis, we summarized a common analysis strategy for both levels of chromatin assessment, focusing on the characteristic computing steps and the tools. The recently developed single-cell level techniques based on Hi-C and ATAC-seq present great potential to reveal cell-to-cell variability in chromosome architecture. A brief discussion on these methods in terms of experimental and data analysis features is included. We also touch upon the biological relevance of chromatin organization and how the combination with other techniques uncovers the underlying mechanisms. We conclude with a summary and our prospects on necessary improvements of currently available methods in order to advance understanding of chromatin hierarchy. Our review brings together the analyses of both higher- and primary-order chromatin structures, and serves as a roadmap when choosing appropriate experimental and computational methods for assessing chromatin hierarchy. Keywords: 3D genome, Chromatin accessibility, Chromosome conformation capture, 3C-technologies, Hi-C, ATAC-se

Dynamics of the Methylome and Transcriptome during the Regeneration of Rice

Oryza sativa indica (cv. IR64) and Oryza sativa japonica (cv. TNG67) vary in their regeneration efficiency. Such variation may occur in response to cultural environments that induce somaclonal variation. Somaclonal variations may arise from epigenetic factors, such as DNA methylation. We hypothesized that somaclonal variation may be associated with the differential regeneration efficiency between IR64 and TNG67 through changes in DNA methylation. We generated the stage-associated methylome and transcriptome profiles of the embryo, induced calli, sub-cultured calli, and regenerated calli (including both successful and failed regeneration) of IR64 and TNG67. We found that stage-associated changes are evident by the increase in the cytosine methylation of all contexts upon induction and decline upon regeneration. These changes in the methylome are largely random, but a few regions are consistently targeted at the later stages of culture. The expression profiles showed a dominant tissue-specific difference between the embryo and the calli. A prominent cultivar-associated divide in the global methylation pattern was observed, and a subset of cultivar-associated differentially methylated regions also showed stage-associated changes, implying a close association between differential methylation and the regeneration programs of these two rice cultivars. Based on these findings, we speculate that the differential epigenetic regulation of stress response and developmental pathways may be coupled with genetic differences, ultimately leading to differential regeneration efficiency. The present study elucidates the impact of tissue culture on callus formation and delineates the impact of stage and cultivar to determine the dynamics of the methylome and transcriptome in culture