Azman, Daruliza Kernain Mohd
(2016)
Functional characterization of the interactions between CTCF truncated transcriptional factor with y-box binding protein-1 and the CTD of POL II.
PhD thesis, Universiti Sains Malaysia.
Abstract
CCCTC sequence binding factor (CTCF) involved in the regulation of transcription,
insulator function, control of imprinting and the X-chromosome inactivation. Two of the
CTCF protein interacting partners were the transcriptional factor YB-1, a member of the
Y-box binding protein 1 and the second protein was the large subunit of RNA
polymerase II (LS Pol II) the principal enzyme for transcription. Previous studies have
shown the interaction between CTCF and YB-1 occurred at the Zinc finger (ZF) and
Cold Shock Domain (CSD) respectively whereas the interaction between CTCF and
RNA Pol II occurred at the C-terminal domains (CTD) for both proteins. The objectives
of this study are to determine the interaction between CTCF and YB-1 in Recurrent
Glioblastoma Multiforme (RGBM) cell line and to identify the motif within CTCF CTD
that is critical for binding with RNA Pol II CTD. CTCF truncated proteins were
produced and used to map the interaction to YB-1 in RGBM cell line. The interaction of
these truncated proteins was mapped using co-immunoprecipitation (Co-IP) and pulldown
assay and the functional significant of the interaction was characterized via
mammalian two hybrid system. On the other hand, CTCF and RNA Poll II interactionswere characterized by identifying two sites within CTCF C-terminal domain important
for the interaction and seven candidates CTCF C mutant variants named 1A, 1B, 1A1B,
2A, 2B, 2A2B and CM deficient for binding with Poll II CTD were produced.
The interaction of these mutants to Poll II CTD was characterized via Coimmunoprecipitation
(Co-IP) and pull-down assay, while the ability of these mutants to
induce apoptosis in recurrent glioblastoma multiforme (RGBM) cell line was also
analysed using Caspase 3/7 Glo assay. From the results obtained for CTCF and YB-1
interactions, the co-immunoprecipitation (co-IP) showed that CTCF was able to form a
complex with YB-1 in the RGBM total cell lysate. From pull down assay results, CTCFZF
was the only domain binds with YB-1 Cold Shock Domain (CSD) and the rest of
domains fail to interact. As for CTCF and RNA Pol II interactions, the Co-IP results
shown positive interaction for CTCF wild-type and all the mutant variants in RGBM cell
line. The interaction was further characterized using pull-down assay and EEEE motif
was identified as a critical motif for binding to Pol II CTD. The effects of CTCF C
mutant variants on cell death by apoptosis assay showed that CTCF complete mutant
(CM) induced highest apoptosis percentage in the cell as compared to the wild-type
CTCF. As conclusion, CTCF ZF domain was found to interact with YB-1 CSD in
RGBM cell line and the important motif in CTCF C terminal region critical for Poll II
CTD binding was successfully identified. The EEEE motif was found to be a critical
motif for binding; hence further study is needed to determine the significant of these
motifs in CTCF and RNA Poll II interactions.
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