Kinetochore protein KNL1 links kinetochore-microtubule attachment and checkpoint signaling during mitosis, The

2014 Summer.Mitosis is the phase of the cell cycle in which replicated chromosomes physically separate, resulting in the formation of two genetically identical daughter cells. This process is not only essential for the development of a single fertilized cell into a multicellular organism, but also for replacement of damaged and dying cells during the span life of an organism. The distribution of chromosomes during mitotic cell division requires accurate yet dynamic attachment between the plus-ends of spindle microtubules (MTs) and kinetochores, which are protein structures assembled at the centromeric region of replicated chromatids. The tightly regulated connection between kinetochores and MTs allows for chromosome congression to the metaphase plate and subsequent separation of the replicated chromosomes during anaphase. Not surprisingly, the inability of cells to resolve erroneous kinetochore-MT attachments results in missegregation of chromosomes, which is linked to uncontrolled cell proliferation and cancer. Thus, proper kinetochore-MT attachment during cell division is essential for the maintenance of genetic integrity. Despite a growing understanding of the identity of proteins that compose the kinetochore and the processes for which they are required, the precise functions of many kinetochore proteins are still unknown. KNL1, a large kinetochore scaffolding protein, contributes to several signaling pathways coordinated by the kinetochore. Yet, how KNL1 recruits its various binding partners to the kinetochore, and whether KNL1 directly or indirectly modulates protein function during mitosis are unresolved questions. In this dissertation, I examine the function of KNL1 in the regulation of kinetochore-MT attachment and determine the regions of KNL1 required for the accumulation of an array of kinetochore proteins. By loss of function analyses using a set of KNL1 mutants, combined with functional assays in cells, I demonstrate that the KNL1 N-terminus is essential for Aurora B kinase activity at kinetochores and for correct kinetochore-MT dynamics. Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore-microtubule (MT) turnover and preventing premature stabilization of kinetochore-MT attachments. Therefore, KNL1 is required for correct Aurora B-mediated kinetochore-MT attachment regulation during mitosis. I provide evidence that the KNL1 N-terminus influences Aurora B activity by mediating the activity of Bub1 kinase, a kinetochore protein required for the spindle assembly checkpoint (SAC). The SAC mediates amplification of an inhibitory signal to prevent mitotic exit until all chromosomes are correctly attached to MTs. Although the SAC is known to be tightly coupled to kinetochore-MT attachment, how such coupling occurs at the kinetochore is a major unanswered question. The finding that KNL1 mediates Aurora B activity through Bub1 establishes KNL1 as a key integrator of multiple signaling pathways at the kinetochore. Finally, I determine the regions of KNL1 required for the accumulation of several kinetochore proteins, providing a broad view and better understanding of kinetochore organization inside the cell. Overall, results from these studies establish KNL1 as a central organizer of kinetochore architecture and function, and demonstrate the direct influence of this scaffolding protein on kinetochore-mediated regulatory processes during mitosis

Epigenetic patterns in a complete human genome

The completion of a telomere-to-telomere human reference genome, T2T-CHM13, has resolved complex regions of the genome, including repetitive and homologous regions. Here, we present a high-resolution epigenetic study of previously unresolved sequences, representing entire acrocentric chromosome short arms, gene family expansions, and a diverse collection of repeat classes. This resource precisely maps CpG methylation (32.28 million CpGs), DNA accessibility, and short-read datasets (166,058 previously unresolved chromatin immunoprecipitation sequencing peaks) to provide evidence of activity across previously unidentified or corrected genes and reveals clinically relevant paralog-specific regulation. Probing CpG methylation across human centromeres from six diverse individuals generated an estimate of variability in kinetochore localization. This analysis provides a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation

Epigenetic patterns in a complete human genome.

The completion of a telomere-to-telomere human reference genome, T2T-CHM13, has resolved complex regions of the genome, including repetitive and homologous regions. Here, we present a high-resolution epigenetic study of previously unresolved sequences, representing entire acrocentric chromosome short arms, gene family expansions, and a diverse collection of repeat classes. This resource precisely maps CpG methylation (32.28 million CpGs), DNA accessibility, and short-read datasets (166,058 previously unresolved chromatin immunoprecipitation sequencing peaks) to provide evidence of activity across previously unidentified or corrected genes and reveals clinically relevant paralog-specific regulation. Probing CpG methylation across human centromeres from six diverse individuals generated an estimate of variability in kinetochore localization. This analysis provides a framework with which to investigate the most elusive regions of the human genome, granting insights into epigenetic regulation

Complete genomic and epigenetic maps of human centromeres

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions

Complete genomic and epigenetic maps of human centromeres.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions

The complete sequence of a human genome.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies

The complete sequence of a human genome.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies