CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention Elena M. Pugachevaa,1, Naoki Kubob, Dmitri Loukinova, Md Tajmula, Sungyun Kangc, Alexander L. Kovalchuka, Alexander V. Strunnikovd, Gabriel E. Zentnerc,e, Bing Renb,f,g, and Victor V. Lobanenkova,1 aMolecular Pathology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bLudwig Institute for Cancer Research, University of California San Diego, La Jolla, CA 92093; cDepartment of Biology, Indiana University, Bloomington, IN 47405; dMolecular Epigenetics Laboratory, Guangzhou Institutes of Biomedicine and Health, Science Park, 510530 Guangzhou, China; eIndiana University Melvin and Bren Simon Cancer Center, Indiana University–Purdue University, Indianapolis, IN 46202; fDepartment of Cellular and Molecular Medicine, Center for Epigenomics, University of California San Diego School of Medicine, La Jolla, CA 92093-0653; and gMoores Cancer Center and Institute of Genomic Medicine, University of California San Diego School of Medicine, La Jolla, CA 92093-0653 Edited by Douglas Koshland, University of California, Berkeley, CA, and approved December 18, 2019 (received for review July 9, 2019) The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin unclear. Second, it is unclear how cohesin is retained at CTCF complex function together to shape chromatin architecture in mam- binding sites. Does CTCF directly recruit cohesin to chromatin malian cells, but the molecular details of this process remain unclear. via protein–protein interactions, or act to constrain cohesin Here, we demonstrate that a 79-aa region within the CTCF N terminus movement via other means during loop extrusion? is essential for cohesin positioning at CTCF binding sites and chromatin Here, we sought to clarify the relationship between CTCF and loop formation. However, the N terminus of CTCF fused to artificial cohesin during formation of chromatin loops. First, we show that zinc fingers was not sufficient to redirect cohesin to non-CTCF binding more than 95% of CTCF sites are also bound by cohesin in mul- sites, indicating a lack of an autonomously functioning domain in CTCF tiple cell lines, implying a nearly universal role of CTCF in the responsible for cohesin positioning. BORIS (CTCFL), a germline-specific retention of cohesin onto chromatin independent of the sequence paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding or function of the CTCF binding site. Second, we demonstrate that sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence the N terminus of CTCF is essential but not sufficient for cohesin that, besides the N terminus of CTCF, the first two CTCF zinc fingers, retention at CTCF sites and chromatin loop formation. We further and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to con- delineate a minimal 79-aa segment within the CTCF N terminus vert BORIS into CTCF with respect to cohesin positioning, thus pro- responsible for cohesin retention. Third, we also show that BORIS, viding additional molecular details of the ability of CTCF to retain a paralog of CTCF, is not able to retain cohesin. Using chimeric cohesin. Taken together, our data provide insight into the process proteins constructed between CTCF and BORIS, we found that by which DNA-bound CTCF constrains cohesin movement to shape the first two ZF domains of CTCF are also involved in cohesin spatiotemporal genome organization. localization to CTCF binding sites, and together with the N terminus of CTCF, can confer the ability to anchor cohesin to BORIS. CTCF | cohesin | BORIS | 3D genome organization Taken together, our data provide insights into the mechanism by he 3D structures of chromosomes in vertebrate cells play a Significance Tcritical role in nearly all nuclear processes, including tran- scriptional control of gene expression, replication of DNA, re- The DNA-binding protein CCCTC-binding factor (CTCF) and the pair of DNA damage, and splicing of messenger RNA (1–4). A cohesin complex function together to establish chromatin loops key player in chromatin organization is the CCCTC-binding factor and regulate gene expression in mammalian cells. It has been (CTCF), an 11-zinc finger (11ZF) protein (5, 6) that regulates proposed that the cohesin complex moving bidirectionally along formation of topologically associating domains and long-range DNA extrudes the chromatin fiber and generates chromatin loops chromatin loops in interphase nuclei (7–9). CTCF works together when it pauses at CTCF binding sites. To date, the mechanisms by with cohesin, a multipolypeptide complex with an evolutionarily which cohesin localizes at CTCF binding sites remain unclear. In conserved role in sister chromatid cohesion during mitosis, to the present study we define two short segments within the CTCF shape chromatin architecture (10–13). This process has been protein that are essential for localization of cohesin complexes at proposed to involve the loading of cohesin onto chromatin by CTCF binding sites. Based on our data, we propose that the N- factors such as NIPBL followed by ATP-dependent bidirectional terminus of CTCF and 3D geometry of the CTCF–DNA complex act movement of the cohesin complex and extrusion of the chromatin as a roadblock constraining cohesin movement and establishing fiber (14–16). According to this model, bidirectional movement of long-range chromatin loops. the cohesin complex along the chromatin fiber can be stalled at CTCF sites, especially at a pair of sites in convergent orientation, Author contributions: E.M.P., B.R., and V.V.L. designed research; E.M.P., N.K., M.T., and resulting in the appearance of topologically associating domains S.K. performed research; E.M.P., N.K., D.L., A.L.K., A.V.S., G.E.Z., and B.R. analyzed data; and E.M.P., G.E.Z., B.R., and V.V.L. wrote the paper. and chromatin loops in a cell population (15, 17). While this model is supported by a substantial amount of experimental evi- The authors declare no competing interest. dence (17–19), important questions remain. First, while it is well This article is a PNAS Direct Submission. known that CTCF and cohesin are frequent partners in the for- This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). mation of large-scale chromatin loops, genome-wide mapping of Data deposition: The data reported in this paper have been deposited in the Gene Ex- both factors has suggested that there are substantial fractions of pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. cohesin binding sites not occupied by CTCF (cohesin non-CTCF GSE136122 and GSE137216). – [CNC]) in various mammalian cell lines (20 22). Based on these 1To whom correspondence may be addressed. Email: [email protected] or findings, it has been suggested that there is a significant CTCF- [email protected]. independent role for cohesin in shaping genome architecture (21, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ 23, 24). However, the proportion of CNC sites varies between cell doi:10.1073/pnas.1911708117/-/DCSupplemental. types, and their implication for chromatin loop formation is largely First published January 14, 2020. 2020–2031 | PNAS | January 28, 2020 | vol. 117 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1911708117 Downloaded by guest on October 1, 2021 which the CTCF–DNA complex stalls cohesin movement to es- RAD21 or bound by CTCF but not RAD21 or vice versa if the tablish chromatin loops in mammalian cells. difference in tag density between the two factors was >threefold. The majority of sites was occupied by both CTCF and RAD21 Results (92.2%) (Fig. 1C), with only a minority of sites (4.8%) displaying CTCF and NIBPL Are Sufficient to Explain the Genomic Distribution of significant RAD21 ChIP-seq signal with low levels of CTCF Cohesin. While CTCF has been proposed to be the major factor binding, or CTCF binding with little RAD21 ChIP-seq signal required for cohesin anchoring on chromatin, there are con- (3%) (Fig. 1 B and C). Similar observations were made in three flicting reports as to the extent of overlap in their genomic dis- additional cell lines, namely human hepatocellular carcinoma tributions (10, 21, 22, 25, 26) (Fig. 1A). To resolve this issue, we cells (HepG2), mouse embryonic stem cells (mESCs), and mouse performed chromatin immunoprecipitation-sequencing (ChIP- B cell lymphoma cells (CH12), with cooccupancy of CTCF and seq) analysis of CTCF and cohesin binding in several cancer RAD21 at a majority of sites (93.6 to 95.4%) observed in each cell lines. In the MCF7 human breast cancer cell line, where a case, and relatively low numbers of sites bound by CTCF but not large percentage of CTCF-independent cohesin binding sites RAD21 and vice versa (Fig. 1D). Between cell types, CTCF and were previously reported (22), we observed robust correspon- cohesin (CAC) sites were the most reproducible, followed by dence between CTCF and RAD21 (a subunit of cohesin) binding CTCF sites without RAD21 enrichment, while there was very at the single-locus (Fig. 1B) and genome-wide scales, with 90.7% little overlap of CNC (SI Appendix, Fig. S1D). of RAD21 peaks overlapping CTCF binding sites (SI Appendix, We next set out to identify factors bound to CNC sites. Con- Fig. S1A). While a computational method identified ∼25,000 sistent with reports that the NIPBL protein promotes the loading more CTCF binding sites than RAD21 binding sites, an enrich- of cohesin onto chromatin (27, 28), the CNC sites are enriched ment of RAD21 ChIP-seq signal was readily visible at these for NIPBL (Fig. 1E) in MCF7, HepG2, mESC, and CH12 cells, additional CTCF sites (SI Appendix, Fig. S1 B and C).