Figure 1.
LHX3-interacting proteins isolated from pituitary cell extracts.
(A) To identify proteins that interact with the LHX3 C-terminus, glutathione-S-transferase (GST)-human LHX3a C-terminus (residues 225–397) fusion protein was generated as “bait” and resins containing either GST-LHX3 C-terminus or GST alone (as negative control) were incubated with precleared protein extracts from cultured mouse pituitary cell lines or adult mouse pituitary gland extracts. After washing, SDS-PAGE and Coomassie staining was used to visualize proteins specifically retained by the GST-LHX3 C-terminus resins in comparison to GST negative control. Unique protein “bands” were extracted from gels and identified by mass spectrometry (mass spec.). The mass spectrometry data identified three proteins: leucine rich acidic nuclear protein (LANP), template-activating factor-1β (TAF-1β), and ataxin-3. (B) The interaction screens were then repeated and the recovered proteins were analyzed by western blotting. Experiments using antibodies recognizing LANP and TAF-1β confirmed enrichment for these proteins following interaction with the LHX3 C-terminus-containing resins compared to GST-alone negative control. (C) INHAT proteins are expressed in pituitary cells. Polyclonal antibodies against INHAT proteins were used to probe whole cell extracts of pituitary and non-pituitary cell lines. 293T = human embryonic kidney cells, Pit = pituitary lysate from pooled wild type C57/BL6 adult mouse pituitaries, LβT2 = mouse pre-gonadotrope cell line, αTSH = mouse thyrotrope cell line. (D) Binding of INHAT proteins to LHX3a proteins. GST-fusion resins were incubated with 35S radiolabeled interactor proteins in interaction buffer. After incubation, resins were washed and separated by SDS-PAGE. Gels were treated with destain/fixative then Amplify fluorography reagent (Amersham). Dried gels were exposed to Biomax MR film. Control interactions received equivalent amounts of GST alone. Similar data were obtained with full-length and C-terminal LHX3 proteins.
Figure 2.
Mapping of LHX3-INHAT interactions using radiolabeled deletion constructs.
(A) LHX3-LANP interaction was mapped using radiolabeled deletion constructs. The indicated in vitro translated 35S-labeled LANP expression constructs were incubated with either GST alone (control) or GST-LHX3 resins. Resins were washed at least four times in interaction buffer and associated proteins separated by SDS-PAGE. Gels were fixed and incubated in Amplify fluorography reagent (Amersham). Dried gels were exposed to Biomax MR film (Kodak). Input = 10% of input radiolabeled LANP lysate. (B) Mapping of LHX3-TAF-1β interactions using radiolabeled deletion constructs. The indicated in vitro translated 35S-labeled TAF-1β expression constructs were incubated with either GST alone (control) or GST-LHX3 resins. Reactions were washed at least four times in interaction buffer and separated by SDS-PAGE. Gels were fixed and incubated in Amplify fluorography reagent (Amersham). Dried gels were exposed to Biomax MR film (Kodak). Input = 10% of radiolabeled TAF-1β lysate.
Figure 3.
The LANP and TAF-1β INHAT proteins are associated with the LHX3-bound αGSU/Cga gene promoter.
Chromatin immunoprecipitation (ChIP) was used to probe occupancy of the pituitary glycoprotein binding element (PGBE) region of the mouse αGSU/Cga promoter [12], [48] by LANP, TAF-1β (A) or LHX3 (B) proteins in LβT2 pituitary cells. ChIP enrichment was measured by quantitative PCR and represented as percent input, calculated by 100*2∧(input - Ct (IP)). Values are mean ± SEM for three independent experiments. Immunoprecipitation with non-immune species-matched IgG were carried out as negative controls. a = significantly different to the IgG control value (P<0.05).
Figure 4.
Effects of INHAT proteins on LHX3a trans-activation of the mouse αGSU/Cga promoter.
(A, B) The indicated combinations LHX3, INHAT, CBP, or control expression vectors were transiently co-transfected into mouse pituitary GHFT1 cells with an αGSU (Cga) promoter luciferase reporter gene. Promoter activity was assayed by measurement of luciferase activity after 48 hours. Activities are mean [light units/10 seconds/µg total protein] of triplicate assays ± S.E.M. a = significantly different to the LHX3+ pcDNA3.1 value (P<0.05); b = significantly different to the LHX3+ CBP value (P<0.05).
Figure 5.
LANP represses LHX3 target promoters in pituitary GHFT1 cells.
(A) A Prl enhancer/promoter luciferase reporter gene was transiently co-transfected into GHFT1 cells with the indicated LHX3 and LANP expression vectors. a = significantly different to the LHX3+ pcDNA3.1 value (P<0.05). (B) The mouse thyroid-stimulating hormone beta subunit (TSHβ/Tshb) gene promoter reporter construct was transiently co-transfected into pituitary GHFT1 cells with the indicated LHX3 and LANP expression vectors. LHX3 and empty pcDNA3.1 vectors were co-transfected with the reporters to control for vector effects on LHX3 activity. Promoter activity was assayed by measurement of luciferase activity after 48 hours. Activities are mean [light units/10 seconds/µg total protein] of triplicate assays ± S.E.M. a = significantly different to the LHX3+ pcDNA3.1 value (P<0.05).
Figure 6.
LANP represses LHX3-PIT1 synergism on target reporter genes.
(A) LANP influence on trans-activation of the Prl promoter by LHX3 and PIT1. A Prl enhancer/promoter luciferase reporter gene was transiently transfected into 293T cells with the indicated LHX3 and/or PIT1 expression vectors. Promoter activity was assayed by measurement of luciferase activity after 48 hours. Activities are mean [light units/10 seconds/µg total protein] of triplicate assays ± S.E.M. WT = wild type. a = significantly different to the LHX3+ PIT1+ pcDNA3.1 value (P<0.05). (B) Effects of LANP on the trans-activation of a luciferase reporter gene containing the mouse thyroid-stimulating hormone beta subunit (TSHβ/Tshb) gene promoter by LHX3 and PIT1. The indicated combinations of LHX3, PIT1, LANP or control expression vectors were transiently co-transfected into 293T cells. Promoter activity was assayed by measurement of luciferase activity after 48 hours. Activities are mean [light units/10 seconds/µg total protein] of triplicate assays ± S.E.M. a = significantly different to the LHX3+ PIT1+ pcDNA3.1 value (P<0.05).