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.

It remains unclear why acute depletion of CTCF (CCCTC-binding factor) and cohesin only marginally affects expression of most genes despite substantially perturbing three-dimensional (3D) genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling in mouse embryonic stem cells. We find that enhancer–promoter (E–P) interactions are largely insensitive to acute (3-h) depletion of CTCF, cohesin or WAPL. YY1 has been proposed as a structural regulator of E–P loops, but acute YY1 depletion also had minimal effects on E–P loops, transcription and 3D genome folding. Strikingly, live-cell, single-molecule imaging revealed that cohesin depletion reduced transcription factor (TF) binding to chromatin. Thus, although CTCF, cohesin, WAPL or YY1 is not required for the short-term maintenance of most E–P interactions and gene expression, our results suggest that cohesin may facilitate TFs to search for and bind their targets more efficiently.

Chromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. 20Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into "microTADs" with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCF/Cohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. 25Intriguingly, we detect "two-start" zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation. 30ONE SENTENCE SUMMARY Transcriptional regulatory elements shape 3D genome architecture of microTADs. MAIN TEXT 35Chromatin packages the eukaryotic genome via a hierarchical series of folding steps ranging from nucleosomes to chromosome territories (1). Structural analysis of chromosome folding has been revolutionized by the Chromosome Conformation Capture (3C) family of techniques, which uses proximity ligation of cross-linked genomic loci in vivo to estimate contact frequencies (2). Interphase chromosome structures such as compartments (3), topologically-40 associating domains (TADs) (4, 5), and CTCF/cohesin chromatin loops (6) have been characterized using 3C-based methods. Chromosome compartments correspond to large-scale active and inactive chromatin segments and appear as a plaid-like pattern in Hi-C contact maps at the megabase scale (3). At the intermediate scale of tens to hundreds of kilobases, topologically associating domains (TADs) spatially organize the mammalian genome into 45 continuous self-interacting domains. TADs are defined as local domains in which genomic loci contact each other more frequently within the domain than with loci outside. TADs appear as square boxes along the diagonal of 3D contact maps (4, 5). Mounting evidence suggests that CTCF and cohesin likely mediate TAD formation via a loop extrusion mechanism (7, 8) wherein the cohesin ring complex entraps chromatin loci and extrudes chromatin until blocked by CTCF 50 or other proteins. Stabilization of cohesin at CTCF sites result in sharp corner peaks in contact maps and are also referred to as CTCF/cohesin loops or loop domains. Various studies have