Supplementary Information accompanies the paper on Nature’s website (http://www.nature.com/nature). Acknowledgements We thank members of the Mellman/Warren laboratory for general support and advice, in particular J. Seemann and L. Pelletier. We also thank J. Kagan, A. Neild and C. Roy for confocal microscopy assistance, L. Zheng and A. Bothwell for help with the retroviral system, and T. Hughes for providing the EGFP complementary DNA. We thank O. Bloom, J. Unternaehrer and J. Chow for critical reading of the manuscript, and Olympus for providing the TIR-FM microscope. We also thank the Ludwig Institute for Cancer Research and the NIH for their support of our work. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to I.M. (e-mail: ira.mellman@yale.edu). .............................................................. A chromatin remodelling complex that loads cohesin onto human chromosomes Mohamed-Ali Hakimi*, Daniel A. Bochar*, John A. Schmiesing†, Yuanshu Dong*, Orr G. Barak*, David W. Speicher*, Kyoko Yokomori† & Ramin Shiekhattar* * The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA † University of California, Irvine, Department of Biological Chemistry, 240D, Med Sci I, Irvine, California 92697-1700, USA ............................................................................................................................................................................. Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions 1 . The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation 2–10 . The machineries that provide this accessibility are termed chromatin remodelling factors 11 . Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohe- sin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modi- fication of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin. We have previously shown that human SNF2h resides in two distinct complexes in HeLa nuclear extract. These are a complex of relative molecular mass (M r ) 670,000 (670K), WCRF/hACF, that eluted in the 1 M KCl fraction of the phosphocellulose (P11) chromatography, and a larger complex of M r 1,500–2,000K eluting in the 0.5-M KCl fraction of P11 (ref. 12). Moreover, immunopre- cipitation of SNF2h from the 0.5-M or 1-M KCl eluate of P11 demonstrated the specific association of WCRF180/hACF1 only with the SNF2h in the 1-M P11 fraction (Fig. 1a). To determine the components of the larger SNF2h-containing complex found in the 0.5-M P11 fraction, we purified SNF2h following the scheme in Fig. 1b. Silver stain analysis of the last chromatographic step revealed the association of SNF2h with a complex of approximately 15 polypep- tides (Fig. 1c). Western blot analysis of the Superose 6 column fractions indicated that SNF2h eluted with a broad profile encom- passing the relative molecular masses of 2,000K to 670K (Fig. 1c). Interestingly, although the two-subunit WCRF/hACF complex could not be detected in the initial steps of the purification, the 670 K SNF2h-containing complex was shown eluting in fractions (33–36) of Superose 6 (Fig. 1c, see the western blot for WCRF180). A combination of ion trap mass spectrometry and western blot analysis identified the other components of the M r 2,000K complex as subunits of the NuRD chromatin remodelling complex 13–15 , and the polypeptides involved in the sister chromatin cohesion. All four subunits of the core-cohesin complex—SMC1, SMC3, SA1/SA2 and hRAD21—were identified by mass spectrometry. Western blot analysis confirmed the coelution of hRAD21 and components of the NuRD complex with SNF2h in fractions 27–30 (Fig. 1c). These findings indicate that a fraction of SNF2h displays a chromato- graphic profile consistent with being a component of a complex that also contains cohesin and the subunits of the NuRD complex. To rigorously demonstrate that the cohesin-containing SNF2h Figure 1 Isolation of an SNF2h complex containing cohesin. a, HeLa nuclear extract was fractionated by chromatography and the fractions shown by an arrow were used for affinity-purification followed by western blotting as described 24 . b, Purification scheme. HeLa nuclear extract was fractionated by chromatography as described in Methods. c, Silver staining and western blot analysis of Superose 6 fractions (15 ml). The proteins analysed are indicated to the left of the figure. d, Western blot analysis of BioScale CHT5-I column fractions using antibodies shown on the right of the figure. Fractions 18–20 were pooled for analysis on a subsequent Mono Q column. e, Western blot analysis of Superose 6 column fractions using antibodies against proteins to the right of the figure. Throughout this purification fractions containing WCRF were excluded. letters to nature NATURE | VOL 418 | 29 AUGUST 2002 | www.nature.com/nature 994 © 2002 Nature Publishing Group