Dr Bruce King MBBS FRACP PhD
Contact Details
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Paediatric Endocrinologist Paediatrics
Postal Address.
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.........61 (0) 249 855634
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Current Research
NOVEL INTERACTIONS BETWEEN REGULATORY ELEMENTS OF THE CRH GENE.
CRH production in the hypothalamus is suppressed by glucocorticoids,
whereas in the placenta CRH production is stimulated by glucocorticoids. The
human genome contains only one copy of the CRH gene, which has only one initiation
site. We hypothesize that the observed differences in transcription regulation
are due to tissue specific alterations in expression of transcription factors.
Therefore, working in Prof Rick Nicholson's group,
we sought to identify the key cis-acting elements and the transcription factors
which regulate the human CRH gene.
We have used AtT20 cells as a model for inhibition of
CRH expression by glucocorticoids in the hypothalamus. A human CRH promoter-reporter
construct was developed and a series of deletions of the CRH promoter were
used to identify cis-acting elements important for glucocorticoid and/or
cAMP responsiveness. To identify transcription factors acting at these
regulatory elements nuclear proteins were extracted and electrophorectic
mobility shift assays (EMSA) performed. Mutational analyses were conducted
to clarify the key cis-acting regulatory elements and the requirements
for transcription factor binding.
In AtT20 cells two cAMP responsive regions were identified:
(1) a consensus cAMP response element (CRE) and (2) a previously unrecognized
caudal type homeobox response element (CDXRE). Overall cAMP-stimulated
activity is due to the sum of the actions at these two sites. Glucocorticoids
inhibited the component of the cAMP-stimulation occurring via the CRE (this
action involves a negative glucocorticoid response element, nGRE, in the
CRH promoter) but not that acting via the CDXRE. We also identified
two regions of the CRH promoter that can be stimulated by glucocorticoids
in AtT20 cells: (1) the CRE and (2) a region between -213 and -99bps. EMSA,
using the human CRE, compared nuclear protein extracts from AtT20 with
those from human primary placental cells (in which we have previously shown
the CRE to be involved in stimulation of CRH by cAMP and by glucocorticoids).
The transcription factors Jun and CREB were detected by EMSA-supershift
in placental cells while in AtT20 extracts Fos and CREB were present. Furthermore,
using a yeast one hybrid system a novel transcription factor with a leucine
zipper motif was identified which bound to the CRE and is present in placental
extracts.
Our results identify novel interactions between four cis
regulatory sites in the CRH promoter important for regulation by cAMP and
glucocorticoids, and indicate that the nuclear transcription factors binding
to the CRE differ between the AtT20 cells and the placenta. We show that
in the absence of specific interactions involving the nGRE the CRH promoter
can be stimulated by glucocorticoids in AtT20 cells. The response of the
CRH gene to factors acting via glucocorticoid receptors and cAMP is a reflection
of the interactions of tissue specific transcription factors at the sites
that have been identified.
Memberships
Publications
King BR, Nicholson RC (2007) "Advances in understanding Corticotrophin-Releasing Hormone gene expression." Frontiers in Bioscience 12, 581-590.
Shipman KL, Robinson PJ, King BR, Smith R, Nicholson RC. (2006) "Identification of a Family of DNA-Binding Proteins with Homology to RNA Splicing Factors." Biochemistry & Cell Biology, 84: 9-19.
Ni, X., Hou, Y., King, B.R., Tang, X., Read, M.A., Smith, R., Nicholson, R.C. (2004) "Estrogen Receptor Mediated Down-regulation of CRH Gene Expression is Dependent on a cAMP Regulatory Element in Human Placental Syncytiotrophoblast Cells." J Clinical Endocrinology & Metabolism, 89:2312-2318.
Nicholson, R.C., King, B.R., Smith, R. (2004). "Complex regulatory interactions control CRH gene expression". Frontiers in Bioscience, 9:32-39.
King, B.R., Smith, R., Nicholson, RC. (2002). "Novel glucocorticoid and cAMP interactions on the CRH gene promoter". Molecular and Cellular Endocrinology, 194:19-28.
Ni, X., Nicholson, R.C., King, B.R., Chan, E-C., Read, M.A., Smith, R. (2002).
"Estrogen Represses Whereas the Estrogen-Antagonist ICI 182780 Stimulates
Placental Corticotropin-Releasing Hormone Gene Expression".
J.
Clinical Endocrinology & Metabolism, 87:3774-3778.
Nicholson, R.C., King, B.R. (2001). “Regulation of
CRH gene expression in the placenta.” The endocrinology of parturition. Basic
science and clinical application.
S Karger AG Publishers. Frontiers
of Hormone Research 27: 246-257.
King, B.R., Smith, R., Nicholson, R.C. (2001). “The regulation of human corticotropin-releasing hormone gene expression in the placenta”. Peptides 22: 1941-1947.
King, B.R., Nicholson, R., Smith, R. (2001). “Placental Corticotrophin-Releasing Hormone, local effects and fetomaturnal endocrinology”. Stress 4: 219-233.
Cheng, Y-H.., Nicholson, R.C., King, B., Chan, E-C., Fitter, J.T., Smith, R. (2000). “Glucocorticoid stimulation of corticotropin-releasing hormone gene expression requires a 3’,5’-cyclic AMP regulatory element in human primary placental cytotrophoblast cells”. J. Clinical Endocrinology & Metabolism 85: 1937-1945.
Cheng, Y-H.., Nicholson, R.C., King, B., Chan,
E-C., Fitter, J.T., Smith, R. (2000). “CRH gene expression in primary placental
cells is modulated by cAMP”. J.
Clinical Endocrinology & Metabolism 85: 1239-1244.
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