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Hierarchical Looping of Zigzag Nucleosome Chains in Metaphase Chromosomes

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Hierarchical Looping of Zigzag Nucleosome Chains in Metaphase Chromosomes Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes Sergei A. Grigoryeva,1, Gavin Bascomb, Jenna M. Buckwaltera, Michael B. Schuberta, Christopher L. Woodcockc, and Tamar Schlickb,d,1 aDepartment of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033; bDepartment of Chemistry and Courant Institute of Mathematical Sciences, New York University, New York, NY 10012; cBiology Department, University of Massachusetts, Amherst, MA 01003; and dNYU-ECNU Center for Computational Chemistry, NYU Shanghai, Shanghai 200062, China Edited by Michael Levitt, Stanford University School of Medicine, Stanford, CA, and approved December 22, 2015 (received for review September 14, 2015) The architecture of higher-order chromatin in eukaryotic cell nuclei is However, evidence for 30-nm fibers in interphase nuclei of living largely unknown. Here, we use electron microscopy-assisted nucleo- cells has been controversial (reviewed in refs. 9 and 10). For exam- some interaction capture (EMANIC) cross-linking experiments in ple, whereas a distinct 30-nm fiber architecture is observed in ter- combination with mesoscale chromatin modeling of 96-nucleosome minally differentiated cells (11, 12), neither continuous nor periodic arrays to investigate the internal organization of condensed chroma- 30-nm fibers are observed in the nuclei of proliferating cells (13–15). tin in interphase cell nuclei and metaphase chromosomes at nucleo- However, zigzag features of the chromatin fibers are well supported somal resolution. The combined data suggest a novel hierarchical by nucleosome interaction mapping in vitro (16) and in vivo (15). looping model for chromatin higher-order folding, similar to rope For chromatin architecture within metaphase chromosomes, flaking used in mountain climbing and rappelling. Not only does such fluorescence studies of mitotic chromosome condensation in vivo packing help to avoid tangling and self-crossing, it also facilitates rope (17), cryo-EM observations of unfixed and unstained chromosomes unraveling. Hierarchical looping is characterized by an increased in situ (18), and small-angle X-ray scattering (19) show no struc- frequency of higher-order internucleosome contacts for metaphase tures resembling folded 30-nm fibers and instead suggest random chromosomes compared with chromatin fibers in vitro and interphase folding of soft polymers. Evidence is also accumulating that during chromatin, with preservation of a dominant two-start zigzag orga- chromosome condensation in mitosis, chromatin higher-order nization associated with the 30-nm fiber. Moreover, the strong de- structure is dramatically altered at the global level (20) by signifi- pendence of looping on linker histone concentration suggests a cant increase in looping (21). A random type of looping, however, hierarchical self-association mechanism of relaxed nucleosome zigzag cannot explain sharp chromosomal boundaries separating the chains rather than longitudinal compaction as seen in 30-nm fibers. translocated genomic regions in metaphase chromosomes (22) as Specifically, concentrations lower than one linker histone per nucle- well as formation of highly localized fibers of transgenic DNA, osome promote self-associations and formation of these looped up to 250 nm in diameter, detected by fluorescence imaging networks of zigzag fibers. The combined experimental and modeling in vivo (17). In contrast, a hierarchical or layered looping could evidence for condensed metaphase chromatin as hierarchical loops explain the above aspects of chromosome organization; in ad- and bundles of relaxed zigzag nucleosomal chains rather than dition, it could help reconcile the experiments in living cells with randomly coiled threads or straight and stiff helical fibers reconciles in vitro data and determine which aspects of the secondary structure aspects of other models for higher-order chromatin structure; it are retained in the metaphase chromosome and how these features constitutes not only an efficient storage form for the genomic correlate with the polymer melt model (18, 23). material, consistent with other genome-wide chromosome confor- Here we apply the EM-assisted nucleosome interaction cap- mation studies that emphasize looping, but also a convenient orga- ture (EMANIC) technique, which captures nearest-neighbor nization for local DNA unraveling and genome access. Significance chromatin higher-order structure | nucleosome | linker histone | mesoscale modeling | electron microscopy Though the two-start zigzag geometry of a nucleosome chain is dominant in chromatin fibers in vitro, it remains unknown he physical packaging of megabase pairs of genomic DNA whether it is preserved in living cells. Our combined nucleosome Tstored as the chromatin fiber in eukaryotic cell nuclei has been interaction capture technique and mesoscale modeling reveal one of the great challenges in biology (1). The limited resolution that the zigzag motif is persistent in interphase and metaphase and disparate levels that can be studied by both experimental and chromatin. For metaphase chromosomes, we observe a striking modeling studies of chromatin, which exhibits multiple spatial and increase in occurrence of long-range internucleosome interac- temporal scales par excellence, make it challenging to present an tions, inconsistent with hierarchical coiling or a liquid melt. In- integrated structural view, from nucleosomes to chromosomes (2). stead, the data point to a new mechanism for higher-order Because all fundamental template-directed processes of DNA folding, via hierarchical looping, similar to rope flaking used in depend on chromatin architecture, advances in our understanding mountaineering. This mechanism brings together distant DNA of chromatin higher-order organization are needed to help in- elements in a manner that can be controlled by linker histones terpret numerous regulatory events from DNA damage repair to and histone modifications. Implications for genomic accessibility epigenetic control. and epigenetic functions naturally arise. At the primary structural level, the DNA makes ∼1.7 left-super- helical turns around eight core histones to form a nucleosome core. Author contributions: S.A.G. and T.S. designed research; S.A.G., G.B., J.M.B., and M.B.S. performed research; S.A.G., C.L.W., and T.S. contributed new reagents/analytic tools; The nucleosome cores are connected by linker DNA to form nu- S.A.G., G.B., J.M.B., M.B.S., C.L.W., and T.S. analyzed data; and S.A.G., C.L.W., and T.S. cleosome arrays. An X-ray crystal structure of the nucleosome core wrote the paper. has been solved at atomic resolution (3), and a short, four-nucleo- The authors declare no conflict of interest. some array has also been solved (4). Next, at the secondary structural This article is a PNAS Direct Submission. level, the nucleosome arrays, aided by linker histones (H1 or H5), 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. ∼ form a compact chromatin fiber with a diameter of 30 nm and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. longitudinal compaction of 5–7 nucleosomes per 11 nm (5–8). 1073/pnas.1518280113/-/DCSupplemental. 1238–1243 | PNAS | February 2, 2016 | vol. 113 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1518280113 Downloaded by guest on September 23, 2021 chromatin cross-linking by formaldehyde in situ (25). To facili- tate direct imaging of internucleosomal interactions in this chromatin, we introduced a mild trypsin treatment to digest the linker histone with minimal effect on the core histones (Fig. 1B). This procedure opens nucleosome arrays for EM analysis (Fig. 1E), while the residual DNA-depleted protein husks sediment to the bottom (fraction P in intact chromatin in Fig. 1C). Fig. 2 shows typical EM images of cross-linked arrays. We record several thousand nucleosomes with nucleosome loop sizes up to 22 nucleosomes (Fig. S3 A–C; see also EMANIC dataset image files). EMANIC scoring of EM data from uncross-linked control cells and cells cross-linked with 0.3% and 1% formaldehyde reveal significant differences between the cross-linked HeLa and control arrays. Fig. 3A and Fig. S3 B and C show the relative abundance of cases where two consecutive nucleosomes are cross-linked (i ± 1), cases involving loops of one or more nucleosomes between the cross-linked pair Fig. 1. Cross-linking and unfolding of native nucleosome chains for EM (loops), cases where two nucleosomal arrays are cross-linked in trans analysis. (A) Scheme of the experimental procedure steps. Interactions ±1, (trans), and a low number of nonassigned or obscured contacts ±2, etc. result from intrafiber internucleosomal interactions; t (trans) results (N/A). Strikingly, between the control and 1% formaldehyde cross- from interfiber internucleosomal interactions. (B) SDS/PAGE of histones from linking, the extent of the i ± 1 interaction decreased approximately − HeLa oligonucleosomes cross-linked with formaldehyde in situ, then iso- threefold, P < 5 × 10 4, and combined loops increased ∼2.5-fold, − lated and treated with 0.5 μg/mL trypsin at +37 °C for the indicated pe- P < 5 × 10 8 (Fig. 3A), which suggests that most of the i ± 1in- riods of time. (C) Agarose gel electrophoresis of DNA from sucrose gradient teractions seen in uncross-linked control chromatin originate from fractionation of the intact and trypsin-treated chromatin isolated from formaldehyde-cross-linked
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