Assembly and Function of Centromeric Chromatin
The faithful segregation of chromosomes through each round of cell division is imperative to guard the human genome against
errors in chromosome number that can lead to birth defects, cell death or cancer. Integral to this mitotic process is a
specialized chromatin domain known as the centromere, which is present on the chromosome throughout the cell cycle. The
centromere directs the assembly of the transient macromolecular kinetochore structure during mitosis, which in turn mediates
the interaction between the chromosome and spindle microtubules and regulates the progression of cells through mitosis in
response to unaligned chromosomes. Centromeric chromatin is assembled around a unique centromeric nucleosome containing
Centromere protein-A (CENP-A), in place of histone H3 within the canonical histone octamer (which includes histones H4,
H2A and H2B). We are interested in the mechanism by which the CENP-A containing nucleosome directs the assembly of the
centromere and contributes to mitotic progression. Using a proteomics approach, we have identified the CENP-A
nucleosome-associated complex (CENP-ANAC, an assemblage of six centromere proteins [CENPs]) as the most proximal component of
the human centromere. We are interested in determining how this complex distinguishes CENP-A nucleosomes from general
histone-H3-containing chromatin and ultimately how this structure is assembled to ensure proper chromosome segregation, as
well as beginning to understand the role this macromolecular complex may play during interphase.
We are also interested in the mechanism by which a region of the chromosome is specified as a centromere and how assembly of
this chromatin domain is accomplished. The location of the mammalian centromere on each chromosome is stably inherited through
each cell cycle. In most eukaryotes (with the exception of S. cerevisiae) the location of the centromere is not dictated by
the underlying DNA sequence, and therefore the mechanism of centromere identification is thought to be epigenetic, literally
meaning "on top of" the genetic code. Due to the epigenetic nature of the centromere, each round of DNA synthesis presents a
challenge for it's stable propagation, since replication of the chromosome requires that new CENP-A nucleosomes are assembled
in the proper location in order to maintain the epigenetic mark through successive rounds of cell division. Epigenetic
inheritance is often dependent on covalent histone modifications as well as the incorporation of histone variants into
chromatin. In the case of the centromere, the CENP-A histone variant plays a central role; however, histone modifications
may also be involved. We seek to understand how centromeric chromatin is assembled and how this process is restricted to
sites already determined to be active centromeres. While the protein complexes involved in bringing pre-nucleosomal forms of
canonical histone H3 to the DNA, as well as the factors required for their assembly into chromatin, are well known, the
analogous proteins have not been identified for CENP-A nucleosome assembly. We are using tandem-affinity-tagging in human
cell culture coupled with mass spectrometry to identify the proteins and histone modifications involved in centromere
assembly. In addition, we use in vitro assays as well as siRNA in mammalian cell culture to determine how these complexes
function to assemble the human centromere.