Delivery of chromosomes, the basic units of inheritance, to each daughter cell during cell division is mediated by the centromere. Unlike typical genes, in metazoans this central genetic element is determined epigenetically rather than by DNA sequence. With support from this grant and using gene replacement in human cells, we have established that chromatin containing the centromere-specific histone H3 variant CENP-A is the epigenetic mark which acts through a two step mechanism to identify, maintain and propagate human or yeast centromere function indefinitely. In the first step, centromere position is replicated and maintained in a cell cycle-dependent manner by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into histone H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is directed by either CENP-A's amino- or carboxy-terminal tails which serve to recruit inner kinetochore proteins. We will now test and extend the two-step model of epigenetic centromere specification by exploiting the rapid, inducible, degron-mediated protein degradation approach whose utility we established in mammalian cells in the prior grant period. With this, we will determine the importance of CENP-A for maintenance of centromeric chromatin and determine the influence of CENP-B and/or chromatin modifications for 'de novo'CENP-A reloading onto centromeric chromatin. The mechanism(s) will be identified through which the CENP-A amino-terminal tail stabilizes CENP-B binding and nucleates kinetochore assembly. Further, the structure of CENP-A chromatin will be determined across the cell cycle using volume and size measurements with solid-state nanopores. Histone composition of CENP-A-containing chromatin, the lengths and DNA sequences bound by CENP-A or the CENP-T/W/S/X nucleosome-like complex at each cell cycle point will be determined by high-throughput DNA sequencing with and without TALEN-mediated inactivation of both CENP- B alleles. Finally, following our discovery that CENP-A and other components known for essential roles in centromere assembly are rapidly recruited to sites of DNA damage, we will assess the roles of CENP-A and its partners in DNA repair of DNA double strand breaks produced by an inducible/inactivatable, site specific CRISPR/Cas9 nuclease that we have generated. This will include determination of the mechanism of transient CENP-A recruitment to sites of DNA damage, the extent of chromatin remodeling following DNA damage, and dependence of overall repair on CENP-A.

Public Health Relevance

Centromeres direct faithful delivery of one copy of each chromosome to each new cell. Remarkably, unlike typical genes, this central genetic element is not determined by DNA sequence, but rather by a DNA-protein complex that knows how to direct its own replication at the same DNA location. Our goals here are to understand how centromeres function and the genetic mechanisms that may generate failure of normal chromosome delivery have broad medical implications. Among these, errors of chromosome segregation lead to infertility. Moreover, many human tumors have highly abnormal numbers of chromosomes (that is, they are aneuploid), with initial chromosomal loss participating in the early steps of the transformation cascade in inherited cancers caused by heterozygous mutation in tumor suppressor genes and the more widespread aneuploidy characteristic of advanced tumors thought to drive acquisition of malignant growth properties.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Deatherage, James F
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Ludwig Institute for Cancer Research Ltd
La Jolla
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