Cell-lineage-specific chromosomal instability in condensin II mutant mice
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Woodward2016.docx (18.17Mb)
Date
29/11/2016Author
Woodward, Jessica Christina
Metadata
Abstract
In order to equally segregate their genetic material into daughter cells during mitosis,
it is essential that chromosomes undergo major restructuring to facilitate compaction.
However, the process of transforming diffuse, entangled interphase chromatin into
discrete, highly organised chromosomal structures is extremely complex, and
currently not completely understood.
The complexes involved in chromatin compaction and sister chromatid decatenation
in preparation for mitosis include condensins I and II. Mutations in condensin subunits
have been identified in human tumours, reflecting the importance of accurate cell
division in the prevention of aneuploidy and tumour formation. Most mutations
described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic
Mutations in Cancer) are missense, and therefore likely to only partially affect
condensin function. Most functional genetic studies of condensin, however, have used
loss of function systems, which typically cause severe chromosome segregation
defects and cell death.
Mice carrying global hypomorphic mutations within the kleisin subunit of the
condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is
therefore a good system for understanding how condensin dysfunction can influence
tumourigenesis. However, little is known about which cellular processes are affected
in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse
model to study the consequences of the condensin II deficiency on cell cycle regulation
in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile,
with no obvious abnormalities other than the thymus, which is drastically reduced in
size. Previous studies reported greater than a hundred-fold reduction in the number of
CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously
expressed protein which functions in a fundamental cellular process would result in
such a cell-type specific block in development. To achieve this, I investigated the
possibility that condensin II is involved in interphase processes as well as in mitosis.
In addition, I studied the aspects of T cell development that may make this lineage
particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary
investigation into the biochemical properties of the condensin complexes.
During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific
phenotype arises due to abnormal cell division. However, I was unable to find any
evidence to support the hypothesis that the phenotype is a consequence of abnormal
interphase processes. Upon systematic analysis of several stages of hematopoietic
differentiation, I found that at a specific stage of T cell development, the mutation
results in an increased proportion of cells with abnormal ploidy, followed by a drastic
reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency
of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic
precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers
in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest
that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells
from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary
investigation into the biochemical properties of the condensin complex revealed
apparent imbalances in the expression of condensin subunits in T, B and erythroid
cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not
exclude the possibility that condensin subunits might be forming heavier-weight
complexes with non-SMC proteins. Further work must be carried out to determine
whether this sedimentation pattern is unique to T cells.