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Title: The functional analysis of heterochromatin protein 1 (HP1) in Xenopus laevis
Author: Kao, Cheng-Fu
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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In my study, I employed the South African clawed-toe frog, Xenopus laevis, as a vertebrate model system to address two questions. First, is HP1 protein necessary for Xenopus development? Second, What are the molecular partners for HP1? I identified two full-length cDNA clones encoding HP1 homologues from Xenopus, xHP1α and xHP1γ. Both of these clones contain a chromodomain (CD) and chromoshadow domain (CSD). Functional analysis demonstrates that xHP1α and xHP1γ can homo- and heteromerdise in vitro and this is dependent on the CSD. A chromatin pull-down assay localised a novel chromatin-binding domain to the hinge region of xHP1α, which is not present in xHP1γ. Moreover, xHP1α shows a binding preference for histone H1 in a far-western assay. Additional analysis using salt-dependent folded chicken chromatin indicates that xHP1α favours binding to a highly compacted chromatin fibre containing linker histone. Taken together, it could be that this specific interaction is related to a particular type of chromatin, perhaps heterochromatin. It is also possible that this functional different between xHP1α and xHP1γ is responsible for their differential sub-nuclear localisation. RT-PCR analysis revealed that both xHP1α and xHP1γ are maternally abundant and their expression increases to a high level during neuralation stages, RNA in situ hybridisation shows that xHP1α shares an overlapping pattern expression with xPolycomb 2. The expression was observed in the head and neural tube. To study the functional significance of HP1 in the development, we manipulated the expression level of xHP1α and xHP1γ. The data point towards an early development role for xHP1α and xHP1γ. Both the depletion and overexpression experiments clearly show that xHP1s are essential for early development after the mid-blastula transition (MBT). Moreover, when embryo overexpressed xHP1α and xHP1γ, or both xHP1α and xHP1γ together, these embryos also incorporated a significantly higher level of 32S-UTP after MBT. These observations are consistent with the prediction that HP1 acts as an essential player in the dynamic organisation of nuclear architecture.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available