The regulation of gene expression at the transcriptional level is one of the paramount
mechanisms for maintaining control of growth, development and metabolic homeostasis.
The Liver X Receptor (LXRa) is a novel member of the nuclear hormone receptor
superfamily of transcription factors, which was originally isolated in our laboratory.
Subsequent studies have revealed that LXRa is an essential transcriptional regulator of
cholesterol homeostasis and a number of potent LXRa activators, including the oxysterol
22(R)-hydroxycholesterol have also been identified. As other members of the
superfamily, LXRa exerts its regulatory control of target genes directly by binding to
LXRa-responsive enhancer elements (LXREs), located upstream of the target gene
promoter. Our laboratory initially demonstrated that LXRa heterodimerizes with the
Retinoid X Receptor (RXRa) and cooperatively binds to a synthetic LXRE (DR4-
LXRE), which consists of direct repeats of the hexad core consensus sequence spaced by
four nucleotides. Tc date, two naturally occurring LXREs have been identified,
including the LXRE--L\MTV element, located in the promoter region of the mouse
mammary tumor vims long terminal repeat and the CYP7 A-LXRE element, located in
the proximal promoter region of the rat cholesterol a-hydroxylase gene.
In order to delineate the mechanism by which LXRa mediates the transcriptional
regulation of target genes, a series of highly integrated characterization studies were
initiated. Our initial interest was identifying the transactivation properties ofLXRa.
Thus, a series of tramient transfection studies were performed, which investigated the
effect of various LXREs, ligands/activators and cell lines on LXRa.-mediated
transactivation. Ultimately, these studies revealed that the LXRa.-mediated
transcriptional response was highly varied and specifically dependent upon the response
element, ligand and cell line employed. Thus, these investigations indicate the specificity
and great diversity in the nuclear hormone receptor-mediated transcriptional regulation of
target genes. Furthermore, these studies resulted in the establishment of a viable and
efficient transient transfection assay for further LXRa. in vivo investigations.
Nuclear hormone receptors, including LXRa., are comprised of several modular
domains termed AlB, C, D and E. A number of recent studies have implicated the highly
divergent AlB domain of variety of nuclear receptors, and their isoforms, as a participant
in transactivation. Specifically, these nuclear receptors have been shown to posses,
within their respective AlB domains, an autonomous ligand-independent transactivation
function termed the AF-1 domain, which can either function independently or can
synergize with the E domain of the same receptor. Thus, determination of whether or not
the 97 amino acid AI B domain of LXRa. participated in LXRa.-mediated transactivation
became a main focus; in our investigation of LXRa.. In vitro EMSA analysis revealed
that deletion of up to 63 amino acids of the N-terminal region of the LXRa. AlB domain
did not effect either LXRa./ RXR.a. heterodimerization nor cooperative binding to
LXREs. In vivo transient transfection assays further illustrated that theN-terminal 63
amino acids of the LXR.a. AlB domain were dispensable for LXR.a./RXR.a.-mediated
transactivation. Therefore, as determined by the limitations of these assays, theNIV
terminal63 amino acids of the LXRa AlB domain do not participate in neither
transactivation nor heterodimerization and subsequent binding to LXR.Es.
Transcriptional regulation, mediated by members of the nuclear hormone receptor
superfamily, has been shown to involve multiple auxiliary co-factors, which modulate
receptor-mediated tnmsactivation. These co-factors can either serve to repress (corepressors)
or activate (co-activators) transcription not only through blocking or
facilitating interactio r1s, respectively, between receptors and the basal transcription
machinery but also through chromatin remodeling. Thus, the identification of LXRainteracting
co-facton and the subsequent investigation of their ability to modulate LXRamediated
transactiva1ion, were of particular interest. We demonstrated, via utilization of
in vitro GST-binding assays, that LXRa interacts with RIP 140, SRC-1a and SMRT cofactors
in a ligand-independent manner. Furthermore, these studies illustrate that the
LXRa AF-2 core domain is necessary for efficient RIP 140 and SRC-1a binding.
Surprisingly, this domain appears to impede, although not absolutely, the SMRTILXRa
interaction, which has also been observed for the Retinoic Acid Receptor (RAR)/SMRT
interaction. Functional studies ofLXRa, RXRa and RIP 140 indicate that RIP 140
antagonizes LXRa/RXR.a-mediated transactivation, which suggests that RIP 140 may
serve to attenuate the transcriptional response of nuclear receptors modulated by other,
more potent co-activators, as previously suggested in Peroxisome Proliferator-activated
receptor a (PPARa);RIP 140 studies. As well, it is apparent that neither'the RIP
140/LXRa interaction nor the RIP 140-mediated repression of LXRa activity is effected
upon deletion of the N-terminal 63 amino acids of the LXR.a. AlB domain. Interestingly,
functional studies of LXR.a., RXR.a. and the partial SRC-1a clone, which lacks the Nterminal
PAS-bHLH domain, indicate that this SRC-1a clone antagonized LXR.a.IRXR.a.mediated
transactivation. While this result may simply demonstrate the necessity for a
full length SRC-1a clone it may also indicate SRC-1 isoform-specific differences as
previously illustrated in Estrogen Receptor (ER)/SRC-1 studies. Lastly, preliminary
functional studies of LXR.a., RXR.a. and S:MR.T indicate that S:MR.T has no significant
effect on LXR.a./RXR-mediated transactivation. These tentative results indicate that
while LXR.a. and SMRT interaction in solution, S:MR.T may not be able to interact with
LXR.a. when bound to DNA, and is thus unable to modulate LXR.a.-mediated
transcriptional activation as previously demonstrated for the PP ARy and the orphan
receptor Rev Erb. Taken together, the investigations presented in this study of LXR.a., further our understanding of not only the mechanism by which LXR.a. mediates its transcriptional
response, but also hew nuclear receptors achieve specificity and diversity in the
activation of target gene expression. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23536 |
| Date | 03 1900 |
| Creators | McCaw, Shannon E. |
| Contributors | Capone, John P., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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