Lucia Carbone, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project will study the relationship between global hypomethylation, activation of Transposable Elements (TEs), and chromosomal rearrangements in cancer. TEs are DNA sequences that can move and insert randomly in the genome causing mutations, including large-scale chromosomal rearrangements. CpG methylation is the main mechanism to repress these elements and limit genomic damage. Both reduction of CpG-methylation (hypomethylation) and chromosomal rearrangements are common in cancer, although a causal relationship is unclear at this point. Using gibbons, which display an unusually high rate of evolutionary chromosome changes and therefore are an excellent model system for studying mechanisms of genomic structural variation, we have shown an association between undermethylated TEs (Alus) and chromosomal breakpoints. In this pilot, we will look for a similar association in cancer (somatic) chromosomal breakpoints and test different approaches based on next-generation sequencing on one Acute Myeloid Leukemia (AML) patient. We will generate short and long-range paired-end Illumina libraries from the blasts and normal cells of the patient in order to map the somatic rearrangements. Furthermore, we will measure CpG-methylation of TEs using whole-genome bisulfite sequencing. Custom algorithms and bioinformatics workflows will be generated to identify the epigenetic state of repetitive elements nearby breakpoints and study changes related to malignant transformation. As this grant was recently been funded, there is no progress to report at this point.

Centromeres are DNA regions that ensure the correct separation of chromosomes during a cell's reproduction. However, due to the highly repetitive nature of DNA present in centromeres, further studies in identifying the make-up of this portion of DNA have been limited. In response to the roadblocks caused by the limited amount of research in this area, this project aims to advance our knowledge of centromere biology and evolution. This research will support outreach activities focused on the biology of gibbons, a critically endangered group of primates that has a unique centromere structure that could inform future studies. As the main threats for these species are from human activities, a large part of the outreach effort for this work seeks to increase awareness of the precarious state of this species. Current conservation efforts, encompassing different venues, include informal seminars to the public and collaborations with primate centers and museums. In addition, this project will promote education through training of postdoctoral fellows, graduate students, undergraduate and local high school programs by providing experiential learning and exposure to modern technologies and training workshops.

While expansions of DNA within centromeres are known for many species, most centromeres are stable over evolutionary time and relatively uniform across all centromeres in one genome. Decoupling equilibration events across chromosomes from the initial seeding events specific to a subset of chromosomes has not been possible in most model systems. This project capitalizes on the recently discovered centromeric expansion of a selfish element, the LAVA retroelement, in a subset of chromosomes in one gibbon genus (Hoolock). This project will build upon foundational discoveries in gibbon centromeres, the newly released gibbon genome sequence and novel genomics approaches for studying complex, repeat-rich regions. This project will test the hypothesis that both LAVA and neighboring satellite DNA bind inner kinetochore proteins, implicating genetic conflict in the seeding and expansion at centromeres. Second, based on observations that the Hoolock centromere structure is similar to that found in marsupials and plants, this project will include analyses of transcripts from centromeric repeats to determine whether transcription from centromere retroelements is associated with young centromere restructuring or with subsequent stabilization. Finally, this research will incorporate emerging next-generation sequencing technologies to assemble Hoolock centromeres from single chromosomes. Collectively, these aims will determine the impact of the organization and function of repeats among newly seeded centromeres and stabilized centromeres within one karyotype.

This project is funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.

PROJECT SUMMARY Chromosomal rearrangements are a great source of inter- and intra-specific genetic variation and are major contributors to human disease. Although position of rearrangement breakpoints can now be mapped at high resolution, interpreting the evolutionary or clinical implications of these events remains challenging. Depending on where they occur, rearrangements can disrupt the organization of genomic functional compartments, known as topologically associating domains (TADs). Within TADs, nearby loci (i.e. promoters and enhancers) interact frequently with each other, while interactions with loci outside TADs are prevented by TAD boundaries. Disruption of TAD boundaries can result in ectopic genes regulation, aberrant phenotypes, and genetic disorders. The functional outcomes of chromosomal rearrangements, therefore, can only be fully understood when studied in the context of genome topology. To shed light on some of the evolutionary implications of genome reorganization, we recently studied the gibbon genome, which has experienced rapid and recent karyotype evolution with respect to human and the other apes. In the gibbon genome, we observed that TADs remained genetically and epigenetically intact (genomic false-shuffle), because evolutionary breakpoints overlapped almost exclusively with TAD boundaries. Comparison with human and other mammals shows that these TAD boundaries are evolutionary conserved, indicating that TAD boundary establishment predated, and may have even contributed to, occurrence of evolutionary breakage. Motivated by our preliminary findings in gibbon, we propose to use a broad comparative and functional approach to assay multiple species with naturally highly rearranged genomes across the Boreoeutheria tree, and characterize the genetic context, epigenetic state, and evolutionary conservation of their TAD boundaries. We will determine if the false shuffle is a recurring mechanism of genome evolution and we will identify chromatin states associated with evolutionary fragility, as these regions and states could be relevant to human disease (Aim 1). Additionally, we will determine the level of conservation for TAD boundaries across different clades. Overall, the combination of these annotations will be a valuable resource to aid the interpretation of clinically and/or evolutionarily relevant rearrangements. We will then use an evolutionary-motivated approach to delete a subset of highly conserved and clade-specific TAD boundaries using CRISPR/Cas9 in cell lines and mouse, to assess the functional consequences of their deletion on DNA interaction, chromatin state, and gene expression (Aim 2). Finally, by analyzing differential gene expression and chromatin conformation between closely related species with structurally different genomes, we will evaluate the extent to which chromosomal rearrangements can alter short- and long-range functional interaction and contribute to differential gene expression (Aim 3). Overall, this study will elucidate the epigenetics changes associated with evolutionary genome reorganization and will help elucidating mechanisms by which genome rearrangement can lead to pathology.