Two novel and highly related C₂H₂ zinc finger proteins
(CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in
COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and
hematopoietic cell development. However, the precise cellular function(s)
and the contribution of these proteins to neoplastic processes and
hematopoietic cell development remain unknown. The goal of the studies
described herein was to elucidate the molecular mechanisms underlying the
transcriptional repression mediated by these proteins to understand their
biological properties, and ultimately, their cellular function(s).
CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive
manner, suggesting that this repression mechanism(s) may not
involve TSA-sensitive histone deacetylation catalyzed by member(s) of
class I and II HDACs. One possible mechanism is that CTIP proteins may
exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive
HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was
found to interact with CTIP proteins both in vitro and in mammalian cells,
and was recruited to the promoter template in a CTIP-dependent manner.
The proline-rich regions of CTIP proteins and the sirtuin homology domain
of SIRT1 were found to be essential for mediating CTIPs•SIRT1
interactions. Moreover, column chromatography revealed that SIRT1 and
CTIP2 were components of a large complex in Jurkat cell nuclear extracts.
Based on the findings that SIRT1 associates with CTIP proteins in
mammalian cells, SIRT1 may underlie the transcriptional repression activity
of CTIP proteins. The following results support the hypothesis that SIRT1
may underlie the mechanism(s) of CTIP-mediated transcriptional
repression. First, CTIP-mediated transcriptional repression was inhibited,
at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive
HDACs. Second, the decrease in levels of acetylated histones
H3 and/or H4 at the promoter region of a reporter gene was observed upon
overexpression of CTIP proteins, and this effect was inhibited, at least
partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the
promoter template of a reporter gene in mammalian cells upon
overexpression of CTIP proteins. Fourth, SIRT1 enhanced the
transcriptional repression mediated by CTIP proteins and this enhancement
required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected
cells.
In summary, results described herein strongly suggest that CTIP-mediated
transcriptional repression involves the recruitment of SIRT1 to the
template, at which the TSA-insensitive, but nicotinamide-sensitive histone
deacetylase catalyzes deacetylation of promoter-associated histones H3
and/or H4. These results contribute additional understanding to the
molecular mechanisms underlying transcriptional activity of CTIP proteins,
which might be helpful for identification and characterization of the target
genes under the control of CTIP proteins in cells of hematopoietic system
and/or the central nervous system. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30193 |
| Date | 03 March 2004 |
| Creators | Senawong, Thanaset |
| Contributors | Leid, Mark E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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