Wednesday, September 27, 2023
9/27/2023
PROJECT SUMMARY Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Our lab is interested in understanding how human centromeres are assembled, maintained and propagated across the cell cycle. Mammalian centromeres are determined not by DNA sequence but rather through epigenetic acquisition of a histone H3 variant called CENP-A. We have previously shown that most CENP-A-containing chromatin consist of homotypic octameric nucleosomes and that DNA replication functions not only to replicate DNA but also to correct errors in ectopic CENP-A deposition, leading to removal of ectopic CENP-A and restricting CENP-A to centromeres only. In parallel, the same DNA replication machinery is capable to precisely recycle CENP-A onto the daughter centromeres to re-assemble CENP-A onto the same centromeric sequences. The combined actions of the DNA replication machinery of CENP-A removal from the chromosome arms and CENP-A retention and recycling at the centromere results in the epigenetic maintenance of human centromeres at a single locus on each chromosome. Our vision for the next five years is to harness the Telomere- to-Telomere genome assembly, that contains the first description of fully assembled human centromeric genomic maps and DNA methylation data, to build the CENCODE, an epigenomic landscape of human centromeres. The CENCODE will be built using available and new ChIP-sequencing and Cut & Run datasets of CENP-A, CENP- C and CENP-T/W/S/X nucleosome-like complex at each cell cycle point, as well as available DNA methylation data, mapped to the centromeres within the Telomere-to-Telomere genome assembly to create a novel genomic and epigenomic map for human centromeres. We will determine the relationship between CENP-A binding and DNA methylation at neocentromeres and at ectopic sites of CENP-A deposition using long-read nanopore sequencing, and the functional importance of centromeric DNA methylation. This project will lay the foundation for future examination of centromeric epigenetic changes in aging and cancer. Next we will determine the positional stability of human centromeres and whether CENP-A is capable to specify centromere position precisely and stably across a single cell cycle and throughout cellular proliferation at base-pair resolution, through the use of patient derived cell lines that harbor a neocentromere. The contribution of overexpressed CENP-A and/or HJURP, both known to be elevated in several types of cancer, to human centromere drift and/or expansion will be determined. We will explore error corrections mechanisms of ectopically loaded centromeric proteins that aim to prevent failure of chromosome segregation as well as neocentromere formation. Finally, innovative single molecule approaches will be used to define histone compositions and combinations of epigenetic posttranslational modifications within single CENP-A-containing nucleosomes located across the genome: at repetitive human centromeres, at non-centromeric ectopic sites, and at neocentromeres.
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