Centromore regions are uniquely marked by the conserved histone variant CENP-A, which replaces H3 in centromeric nucleosomes. Using high-resolution fluorescence microscopy and optical mapping, we have shown that centromeric chromatin (CEN chromatin) contains interspersed subdomains of CENP-A and H3 nucleosomes. Since CEN chromatin also contains H3, we are interested in determining if modifications of core histones functionally distinguish centromeres from other regions of the genome. Centromeres have historically been considered heterochromatic, however, surprisingly, we have found that CEN chromatin in humans and flies contains “euchromatic” modifications of H3, signifying an open or flexible chromatin conformation. These initial studies have led us to investigate how centromeric chromatin affects transcription and to identify structural or functional elements that separate hetererochromatin, euchromatin and centromeric chromatin.

Due to their large sizes and repetitive DNA content, centromere regions have been excluded from genome projects. However, being such fundamental functional elements that are epigenetically regulated, we want to understand their genomic organization and the relationship between underlying DNA sequence and chromatin and protein organization. In collaboration with a colleague at NHGRI, we are using chromatin immunoprecipitation-PCR (ChIP-PCR) and optical mapping strategies to generate epigenomic profiles that overlay histone modifications onto the centromeric-pericentromeric genomic assemblies from several human chromosomes. This information is being used to identify genomic features that structurally, and potentially functionally, separate the centromere from the rest of the genome.

Centromeres/kinetochores are assembled along a distinct pathway. CENP-A is among the first proteins to be recruited, initiating a protein cascade that results in kinetochore formation and assembly of other kinetochore proteins. It is normal for chromosomes to have only one centromere. However, chromosome rearrangements often occur during meiosis or mitotis that produce chromosomes that have two (or more) centromeres. Chromosomes with two centromeres are called dicentric and, in humans, they are found in 1 in 1000 individuals. Barbara McClintock, a famous cytogeneticist and Nobel prize winner, studied maize (corn) in the 1930s and 1940s and showed that dicentric chromosomes in maize are unstable and lead to chromosome breakage because centromeres of dicentrics segregate to opposite spindle poles in anaphase. However, in humans, dicentric chromosomes are quite stable. This is because one of the centromeres is shut off, stabilizing the dicentric chromosome so that it behaves like it only has a single centromere.

Centromere inactivation is denoted by loss of centromeric proteins and a cytological change in chromosome appearance. Beyond these observations, the underlying mechanism of inactivation is unclear. We are focused on using genetic and biochemical assays to dissect the process of centromere inactivation, to identify chromatin changes that coincide with this event, and to understand how chromosome structure and centromere distance affects this process.

Eukaryotic chromosomes are arranged as multiple chromatin domains or blocks, all with different functions, analogous to individual cars that comprise a train. A given functional block may participate in chromosome architecture, movement and stability, transcription, and/or DNA replication. We study, in both humans and flies, how individual blocks of chromatin are maintained and how they influence, or are influenced by, surrounding chromatin domains. In many cancers, the genome is profoundly rearranged through duplications, deletions, and translocations. Not only is genome instability a hallmark of cancer, but it is associated with birth defects, miscarriage and infertility. We want to understand how genome rearrangements affect chromosomal functions. One approach that we have taken is to use engineered chromosomes in humans and Drosophila to study how chromatin blocks shift genomic position when chromosomes are rearranged, and how this affects chromosome behavior and gene expression.

We also study how specific patient-derived chromosomal rearrangements occur. We focus on two common rearrangements in humans, Robertsonian translocations (ROB) and isodicentric chromosome X (dicX). We hypothesize that breakage-prone regions of the genome may have similar structural and epigenomic attributes. First, we are using high-resolution genome arrays to pinpoint breakpoint regions in patient samples. Second, we are comparing DNA and chromatin signatures at breakpoint regions of specific chromosomal rearrangements to normal chromosomes and to other fragile sites in the genome. Finally, to extend our studies beyond patient-derived rearrangements, we have created an assay to engineer these abnormalities in vitro. Such a system allows us to monitor the rearranged chromosomes instantly after formation, and to scrutinize the epigenetic events that stabilize them or, alternatively, remove them from the karyotype.