LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation
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.