Czajkowsky, D. M. | bioRxiv Science

bioRxiv2017

Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks

Protein, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, we developed a cell lysate microarray on kilo-conditions (CLICK) from 4,837 yeast knockout (YKO) strains and 322 temperature-sensitive mutant strains to explore the impact of the genome-wide interruption on certain protein. Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3 which covers most of the known regulators in Saccharomyces cerevisiae. We also noted that several group of proteins that are linked to negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.

systems biology↗

bioRxiv2017

Sub-kb resolution Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells

Topologically associating domains (TADs) are fundamental elements of the 3D structure of the eukaryotic genome. However, while the structural importance of the insulator protein CTCF together with cohesin at TAD borders in mammalian cells is well established, the absence of such co-localization at most TAD borders in recent Hi-C studies of D. melanogaster is enigmatic, raising the possibility that these TAD border elements are not generally conserved among metazoans. Using in situ Hi-C with sub-kb resolution, we show that the genome of D. melanogaster is almost completely partitioned into more than 4,000 TADs (median size, 13 kb), nearly 7-fold more than previously identified. The overwhelming majority of these TADs are demarcated by pairs of Drosophila specific insulator proteins, BEAF-32/CP190 or BEAF-32/Chromator, indicating that these proteins may play an analogous role in Drosophila as that of the CTCF/cohesin pair in mammals. Moreover, we find that previously identified TADs enriched for inactive chromatin are predominantly assembled from the higher-level interactions between smaller TADs. In contrast, the contiguous small TADs in regions previously thought to be unstructured \"inter-TADs\" are organized in an open configuration with far fewer TAD-TAD interactions. Such structures can also be identified in some \"inter-TAD\" regions of the mammalian genome, suggesting that larger assemblages of small self-associating TADs separated by a \"burst\" of contiguous small, weakly associating TADs may be a conserved, basic characteristic of the higher order folding of the metazoan genome.

genomics↗