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  • Repression of CENP-A assembly in metaphase requires HJURP phosphorylation and inhibition by M18BP1. The Journal of cell biology Flores Servin, J. C., Brown, R. R., Straight, A. F. 2023; 222 (6)

    Abstract

    Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant CENP-A. CENP-A nucleosome assembly is uncoupled from replication and occurs in G1, but how cells control this timing is incompletely understood. The formation of CENP-A nucleosomes in vertebrates requires CENP-C and the Mis18 complex which recruit the CENP-A chaperone HJURP to centromeres. Using a cell-free system for centromere assembly in X. laevis egg extracts, we discover two activities that inhibit CENP-A assembly in metaphase. HJURP phosphorylation prevents the interaction between HJURP and CENP-C in metaphase, blocking the delivery of soluble CENP-A to centromeres. Non-phosphorylatable mutants of HJURP constitutively bind CENP-C in metaphase but are not sufficient for new CENP-A assembly. We find that the M18BP1.S subunit of the Mis18 complex also binds to CENP-C to competitively inhibit HJURP's access to centromeres. Removal of these two inhibitory activities causes CENP-A assembly in metaphase.

    View details for DOI 10.1083/jcb.202110124

    View details for PubMedID 37141119

  • DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nature methods Altemose, N., Maslan, A., Smith, O. K., Sundararajan, K., Brown, R. R., Mishra, R., Detweiler, A. M., Neff, N., Miga, K. H., Straight, A. F., Streets, A. 2022

    Abstract

    Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed directed methylation with long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein's binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies. We optimized and benchmarked DiMeLo-seq by mapping chromatin-binding proteins and histone modifications across the human genome. Furthermore, we identified where centromere protein A localizes within highly repetitive regions that were unmappable with short sequencing reads, and we estimated the density of centromere protein A molecules along single chromatin fibers. DiMeLo-seq is a versatile method that provides multimodal, genome-wide information for investigating protein-DNA interactions.

    View details for DOI 10.1038/s41592-022-01475-6

    View details for PubMedID 35396487