In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish
the critical task of correct cell division. In some organisms, centromeres are
composed of a euchromatic central core region embedded in a stretch of
heterochromatin and the inheritance and maintenance of centromeres are controlled
by dynamic epigenetic phenomena. Although the size of centromeres differs between
organisms, its organization, and the placement of euchromatic and heterochromatic
regions is conserved from the fission yeast, Schizosaccharomyces pombe, to
humans, Homo sapiens. However, relatively little is known about centromeres in the
filamentous fungi from the Ascomycota, representing the largest group of fungi and
fungal pathogens. Further, studies from humans, flies, yeast and plants have shown
that the inheritance of centromeres is not strictly guided by centromeric DNA content,
which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to
align ans assemble the sequenced contigs of centromeric regions of higher
eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad)
analysis has helped to map the centromeres of the filamentous fungus Neurospora
crassa early on. The research presented in this dissertation used N. crassa as a
model to focus on characterizing different features of centromeres with an emphasis
on the centromere-specific histone H3 (CenH3) protein. Data included here represent
the first study on centromere-specific proteins in Neurospora, and demonstrate that
the central core of the centromeres are heterochromatic, showing enrichment of silent
histone marks, which is in contrast to the centromere arrangement in fission yeast.
The CenH3 protein, whose deposition on the genome licenses formation or
maintenance of centromeres, shows highly divergent N-terminal regions and a
conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of
CenH3 is also observed in the Ascomycota, which provides an opportunity for
functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with
CenH3 genes from other species within the Ascomycota. The results from this
experimental approach provide good measures for (1) determining the specific
regions of CenH3 required for the assembly of centromeres during meiotic and mitotic
cell divisions and (2) analyzing the resistance to changes in the organization of
centromeres in N. crassa.
The genetic analysis showed that the divergent N-terminal region is essential
for the proper assembly of centromeres, and that the conserved carboxy-terminus of
CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq
analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3-
GFP) from certain N. crassa centromeres does not result in obvious phenotypic
defects, e.g. diminished growth or evidence for aneuploidy. Further, the low
enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined
during meiosis, which results in irreversible and progressive decreases in enrichment.
It remains to be determined if this process is random as far as selection of
centromeres is concerned. Together the results presented here suggest that during
meiosis more stringent structural requirements for centromere assembly apply and
that these are dependent on CenH3, and that depletion of CenH3 from centromeres
does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae. / Graduation date: 2013
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/35915 |
| Date | 10 December 2012 |
| Creators | Phatale, Pallavi A. |
| Contributors | Freitag, Michael |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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