Defining genome architecture at base-pair resolution

Abstract

Complex gene regulation is one of the key requirements for the evolution of higher eukaryotes. 1 In these organisms, many genes are regulated by enhancers that are 10 4 -10 6 base pairs (bp) distant from the promoter. Enhancer sequences usually contain multiple small transcription factor binding sites (typically ~10bp), and physical contact between the promoter and enhancer is thought to be required to modulate gene expression. 2 Current methods have extensively defined chromatin architecture at scales above 1 kb but until now it has not been possible to define physical contacts at the scale of the key proteins determining gene expression. Here we define the interactions between different classes of regulatory elements (enhancers, promoters and boundary elements) in unprecedented detail, using a novel chromosome conformation capture method (Micro Capture-C (MCC)), which allows physical contacts to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show, using base pair resolution plots of ligation junctions, that transcription factors generate a key component of the contacts between enhancers and promoters. Our data show that contacts from CTCF sites highly correlate with cooccupancy of cohesin and that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. We also find that promoters make the strongest contacts with both enhancers and CTCF sites and that while CTCF sites contact promoters strongly they only make weak contacts with enhancers. The highly punctate nature of the contacts is an unexpected finding because the current view is that physical contacts are constrained by much larger domains such as topological associated domains (TADs). 3 Our results support a model in which chromatin loop extrusion 4-6 is dependent on cohesin loading at active promoters and enhancers, explaining the formation of tissue-specific chromatin domains without changes in CTCF binding. The data suggest that a separate mechanism to loop extrusion underlies enhancer-/promoter contacts, which likely involves DNA binding proteins at enhancers and promoters. The unprecedented

Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF

“…The preparation of Micro-Capture-C (MCC) libraries was performed as previously described 36 . 10 million cells per biological replicate were washed once with PBS, dissociated with trypsin and resuspended in 10 ml of culture media.…”

“…We have recently developed a novel MNase-based approach called Micro-Capture-C (MCC), which is capable of generating base pair resolution 3C data in mammalian cells 36 . This technique produces interaction profiles for selected viewpoints, but does not give an overview of the complete 3D structure of regions of interest.…”

“…Tiled-MCC analysis was performed using the MCC pipeline 36 (https://github.com/jojdavies/Micro-Capture-C). Briefly, adapter sequences were removed using Trim Galore (Babraham Institute, v.0.3.1) and paired-end reads were reconstructed using FLASH 47 (v.1.2.11).…”

Transcriptional enhancers and their communication with gene promoters

“…Capture Hi-C (CHi-C) enriches Hi-C libraries (generated using biotin pulldown of proximity ligation junctions) [ 278 – 281 ], while Capture-C instead enriches 3C libraries obtained without biotin pulldown [ 282 – 284 ]. Most recently reported Micro-Capture-C (Micro-C coupled with sequence capture) enables the profiling of pairwise contacts involving selected regions of interest, at up to single-base pair resolution [ 285 ]. These enrichment methods have detected multitudes of enhancer–promoter loops in many human and mouse cell types [ 243 , 286 – 291 ].…”

Interrogating cell type-specific cooperation of transcriptional regulators in 3D chromatin

“…The combinatory application of multiple tools, not only PIQ in the present study, and the introduction of reproducibility assessment could mitigate such biases ( Gusmao et al., 2016 ; Karabacak Calviello et al., 2019 ). The recent Micro-Capture-C has also introduced single-base pair maps of ligation junctions for analyzing directional footprinting and protein-protein contacts at high resolution between viewpoints and interacting elements ( Hua et al., 2021 ), which would significantly facilitate the identification of fine-scale TR cooperation in 3D chromatin.…”