SUMMARY We describe a Hi-C based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically-associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, “gene looping” factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction.

Materials and MethodsMicro-C protocol for mammals was modified from the original protocol for yeast in (1, 2). The protocol was optimized for the input cell number from 1k to 5M and first applied to the mammalian system in (3). We first briefly summarize the critical steps and concepts in the Micro-C method, and then provide detailed step-by-step instructions. Micro-C experiment 1. Cell culture and crosslinkingHere, we performed a dual crosslinking protocol to fix protein-DNA and protein-protein interactions. In addition to formaldehyde, we used the non-cleavable and membrane-permeable protein-protein crosslinker DSG (disuccinimidyl glutarate, 7.7Å) or EGS (ethylene glycol bis(succinimidyl succinate), 16.1Å) to crosslink the primary amines between proximal proteins. The dual-crosslinking method significantly increases the signal-to-noise ratio of Micro-C data in yeast (2).In brief, 1k -5M cells were resuspended by trypsin and fixed by freshly made 1% formaldehyde at room temperature for 10 minutes. The crosslinking reaction was quenched by adding Tris buffer (pH = 7.5) to final 0.75 M at room temperature. Fixed cells were washed twice with 1X PBS and protein-protein interactions fixed by 3 mM DSG for 45 minutes at room temperature. The DSG solution was freshly made at a 300 mM concentration in DMSO and diluted to 3 mM in 1X PBS before use. The crosslinking reaction was quenched by 0.75 M Tris buffer and washed twice with 1X PBS. Crosslinked cells were snap-frozen in liquid nitrogen and stored at -80°C (pellets are stable for up to a year). Note that freshly made crosslinking solution is critical to producing high-reproducibility Micro-C data, and Tris buffer is a faster and stronger quenching agent than glycine.

Over the past decade, 3C-related methods, complemented by increasingly detailed microscopic views of the nucleus, have provided unprecedented insights into chromosome folding in vivo. Here, to overcome the resolution limits inherent to the majority of genome-wide chromosome architecture mapping studies, we extend a recently-developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human embryonic stem cells and fibroblasts. Micro-C maps robustly capture well-described features of mammalian chromosome folding including .

SummaryCovalent histone modifications are highly conserved and play multiple roles in eukaryotic transcription regulation. Here, we mapped 26 histone modifications genome-wide in exponentially growing yeast and during a dramatic transcriptional reprogramming—the response to diamide stress. We extend prior studies showing that steady-state histone modification patterns reflect genomic processes, especially transcription, and display limited combinatorial complexity. Interestingly, during the stress response we document a modest increase in the combinatorial complexity of histone modification space, resulting from roughly 3% of all nucleosomes transiently populating rare histone modification states. Most of these rare histone states result from differences in the kinetics of histone modification that transiently uncouple highly correlated marks, with slow histone methylation changes often lagging behind the more rapid acetylation changes. Explicit analysis of modification dynamics uncovers ordered sequences of events in gene activation and repression. Together, our results provide a comprehensive view of chromatin dynamics during a massive transcriptional upheaval.