Chromatin structure and architecture. We are interested in the biophysical and structural properties of native chromatin fragments. Using the extensively studied chicken folate receptor and beta-globin gene loci, we have characterized the hydrodynamic and gross structural properties of two distinct chromatin fragments. One fragment is the constitutively condensed heterochromatin region spanning 15.5 Kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes. The other is the transcriptionally inactive beta-globin gene locus, containing 16.2 Kbp of DNA. Despite their different histone protein to nucleic acid ratio, we have shown that both fragments adopt extended rod like structures consistent with models proposed for the condensed 30 nm chromatin fiber. The biophysical tools developed in these studies have allowed us to further expand our investigation. Using the same erythroid precursor cell line (6C2), in which the beta-globin genes are inactive, we have further dissected this gene cluster into a series of five distinct chromatin fragments ranging from 2.1 to 8.0 Kbp in size. The dependence of the sedimentation coefficient with size for these fragments is consistent with the extended rod like properties observed for the larger chromatin fragments described above. We are currently developing protocols to compare beta-globin gene chromatin fragments obtained from 6C2 cells with those released from 10-day old and adult chicken erythrocytes. As the beta-globin genes are transcribed in 10-day old erythrocytes but inactive in adult erythrocytes, these studies will allow us to relate the structures of constitutive and facultative heterochromatin with those of transcriptionally active and inactive chromatin. We are also developing a high resolution chromosome capture conformation assay to characterize the spatial arrangement of the individual nucleosomes within chromatin. These important studies will allow for a better understanding and evaluation of the relation between chromatin structure and gene expression, as well as DNA replication, repair and recombination. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend the biochemical and structural investigations and provide important mechanistic information, as exemplified by recently published works on the endosomal sorting complexes required for transport (ESCRT complexes) carried out in collaboration with Dr. James H. Hurley. Upon activation many cell surface receptors are ubiquitinated and internalized into endosomes, from which they are either recycled or degraded in lysosomes. It is the ESCRT-0 complex, composed of Hrs and STAM, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. We showed that the human Hrs and STAM proteins assemble with high affinity to form an elongated and monodisperse 1:1 complex. This represents the functionally active ESCRT-0 complex as its hydrodynamic properties are identical to those for the endogenous complex isolated in vivo. This unexpected stoichiometry is an important result, as it is consistent with the possibility that as few as one copy of each of the ESCRT-0, I and II complexes could direct the formation of an intralumenal vesicle, the first step required for the internalization of ubiquitinated cell surface receptors (Ren at al. 2009). Cytokinesis, the division of the cytoplasm, is the final step in cell division. Prior to complete cell division, a structure known as the midbody provides the final tether between the two daughter cells. Cleavage of the plasma membrane at the midbody, or abscission, involves the midbody protein Cep55: Cep55 recruits the ESCRT-I complex and ALIX, which in turn recruit ESCRT-III. These complexes, required for normal midbody morphology are believed to have membrane scission activity. Using the Cep55 ESCRT- and ALIX-binding region (EABR) we show that Cep55 forms a dimer. This dimer forms a 1:1 complex with ALIX (797-809) and a 1:1 complex with TSG101 (154-166), the ESCRT-I subunit responsible for Cep55 binding. Importantly, ALIX and TSG101 compete for binding to Cep55. As both ALIX and ESCRT-1 are required for cytokinesis, we conclude that multiple Cep55 are required for proper abscission (Lee et al., 2008).
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