QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brickman, J. M. Articles by Dattani, M. Search for Related Content PubMed PubMed Citation Articles by Brickman, J. M. Articles by Dattani, M. Social Bookmarking                         What's this?

Molecular effects of novel mutations in Hesx1/HESX1 associated with human pituitary disorders

Joshua M. Brickman^1,*,, Melanie Clements¹, Richard Tyrell², David McNay³, Kathryn Woods³, Justin Warner⁴, Andrew Stewart¹, Rosa S. P. Beddington¹ and Mehul Dattani^1,3,

¹ Division of Mammalian Development,
² Division of Protein Structure, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK
³ Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
⁴ Department of Paediatric Endocrinology, John Radcliffe Hospital, Headley Way, Headington, Oxford OX3 9DU, UK
^* Present address: Centre for Genome Research, The University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

View larger version (67K): [in a new window]   Fig. 1. (A) Sagittal MRI scan of the head of a normal child showing the corpus callosum (cc), the optic chiasm (oc), the anterior pituitary (ap), the pituitary stalk (ps) and posterior pituitary (pp) in the normal sella turcica. Note the well-formed corpus callosum and the optic chiasm and the posterior pituitary which appears as a bright fossa within the sella turcica. (B) Sagittal MRI scan of sibling 1 with a homozygous R160C mutation in HESX1. Note that the splenium of the corpus callosum is more hypoplastic than the rest of the structure and that the sella turcica is shallow as compared with the MRI scan of sibling 2 (C). The posterior pituitary (pp) appears to be partially descended. (C) Sagittal MRI scan of sibling 2 with a homozygous R160C mutation in HESX1. The corpus callosum (cc) is severely hypoplastic, as is the optic chiasm (oc) and the anterior pituitary (ap) located in a well-formed but empty sella turcica. Note the ectopic posterior pituitary (pp) and the lack of a visible pituitary stalk. (D) Sagittal MRI of a patient with S170L mutation in HESX1. Note the atrophic posterior pituitary (pp) that has not descended completely into the fossa and an anterior pituitary (ap) gland that is hypoplastic and does not enhance well. The pituitary stalk is thin, but the optic chiasm (oc) is normal as is the corpus callosum (cc).

View larger version (58K): [in a new window]   Fig. 2. DNA binding by Hesx1/HESX1 and HESX1 mutants. EMSA with wild-type and mutant proteins binding to dimeric P3 and monomeric GBS sites. (A) HESX1(S170L) binding affinity is defective relative to wild-type HESX1. Increasing concentrations (0.9-240 nM) of purified recombinant WT HESX1 or HESX1(S170L) were added to the labelled binding sites indicated. (B) HESX1(N125S) binds DNA with at least wild-type affinity. Increasing concentrations (0.78-25 nM) of purified recombinant wt HESX1 or HESX1 (N125S) were added to the labelled P3 and monomeric GBS sites respectively. (C) Wild-type HESX1 and HESX1(S170L), but not HESX1(R160C) and HESX1(N125S), could form ternary complexes with an antibody to the amino-terminal His tag. All proteins were added at concentrations of 25 nM.

View larger version (27K): [in a new window]   Fig. 3. Hesx1 is a transcriptional repressor with a 36 amino acid repression domain. Increasing concentrations of plasmids expressing the depicted GAL4-Hesx1 fusions were co-transfected with a reporter containing five GAL4 sites upstream of the SV40 promoter (A-C) or E4 promoter (D) driving luciferase into ES cells. Arrows indicate increasing concentrations of transfected GAL4 fusion. In A, B and C, 200 and 500 ng of expression vector were used respectively. In D increasing concentrations of GAL4-Hesx1(1-49) from 50 ng to 500 ng were co-transfected with the E4 promoter fragment (50, 100, 250, 400, 500).

View larger version (42K): [in a new window]   Fig. 4. Hesx1 fusion proteins are all expressed in COS cells. Western blot analysis using Hesx1-VP16 fusions with a monoclonal antibody directed at the minimal VP16 activation region. Both mouse and human proteins are expressed at equivalent levels and both mutations in the homeodomain and truncation of the amino-terminal repression domain do not appear to affect these levels.

View larger version (16K): [in a new window]   Fig. 5. Hesx1 represses transcription induced by paired class activator proteins. (A) Hesx1 but not Hesx1(50-185) can repress transcription stimulated by Bix, Mix-1, and Mixer. Expression vectors for these paired class activators were co-transfected along with the indicated reporter, (P3)6E4, which contains six dimeric paired class binding sites upstream of the minimal E4 promoter and expression vectors for either GAL4(1-147), GAL4(1-147)-Hesx1 or GAL4(1-147)-Hesx1(50-185). Increasing concentrations of expression vectors (25 and 100 ng) for Bix and Mix and 100 ng for Mixer were co-transfected with 25 ng of the indicated Hesx1 derivative. (B) Cooperative repression by Hesx1 but not Hesx1(50-185). A Bix expression vector was co-transfected with the (P3)6E4 as in A. Increasing amounts of GAL4-Hesx1 or GAL4-Hesx1(50-185) were co-transfected with the Bix reporter. Titrations of the Bix expression vector were used to determine the optimal levels of induction of the (P3)6E4 reporter. Optimal levels of induction were found to be between 220- and 380-fold depending on the experiment. The addition of expression vector encoding either full length Hesx1 or Hesx1(50-185) always produced the same repressed level of transcription (i.e. 20-fold when Bix was co-transfected with 25 ng of vector encoding full length GAL4-Hesx1 compared to 225-fold when Bix was co-transfected with vector encoding GAL4-Hesx1(50-185)).

View larger version (15K): [in a new window]   Fig. 6. Defect in repression by Hesx1(50-185) is not due to a defect in its DNA binding affinity or overall stability of Hesx1 fusions. (A) Relative activity of GAL4-Hesx1, GAL4-Hesx1(50-185), GAL4-Hesx1-VP4 and GAL4-Hesx1(50-185)-VP4 from GAL4 sites. Vectors expressing these fusion proteins were co-transfected with the indicated reporters as in Fig. 3. B) Relative activity of GAL4-Hesx1-VP4 and GAL4-Hesx1(50-185)-VP4 on P3 sites. Transfections were performed as in A except the reporter is (P3)6E4.

View larger version (22K): [in a new window]   Fig. 7. Dominant negative activity of HESX1(R160C) requires amino terminal repression domain. (A) Dominant negative activity of HESX1(R160C) in vitro. Recombinant HESX1(R160C) (50-200 nM) was mixed together with wild-type HESX1 protein (100 nM) and incubated with radiolabeled DNA containing a P3 binding site. The numbers above the lanes indicate the molar ratio of HESX1(R160C) or heat denatured (denoted by *) HESX1(R160C) to wild-type protein. (B) Dominant negative activity of HESX1 in vivo depends on the amino terminal repression domain. Increasing concentrations of wild-type HESX1-VP4 expression vector (20, 50, 150 and 400 ng) were co-transfected with the P3 luciferase reporter. Wild-type HESX1-VP4 expression vector was co-transfected with 400 ng of either HESX1(R160C)-VP4 or HESX1(R160C)(50-185)-VP4.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter