Ajuba associates with Gfi1. A, immunoprecipitation of Ajuba from Jurkat nuclear extracts with two different antisera against Ajuba, control antisera against LPP, or normal rabbit antisera, followed by immunoblot analysis to detect Gfi1. B, schematic of Ajuba and Gfi1 domains. C, immunoprecipitation of FLAG-epitope-tagged Gfi1 or the SV40SWAP mutant, followed by immunoblot analysis for myc-epitope-tagged Ajuba (I.P., upper panels). The blots were stripped and reprobed for FLAG to demonstrate the immunoprecipitated FLAG-tagged proteins (I.P., lower panel). Immunoblot analysis of epitope-tagged proteins from 10% of input whole cell extracts of 293T cells co-transfected with expression constructs encoding myc-Ajuba (Input, upper panel) and either Gfi1-FLAG or SV40SWAP-FLAG constructs (Input, lower panel). D, immunoprecipitation of FLAG-epitope-tagged Gfi1, followed by immunoblot analysis for myc-epitope-tagged Ajuba or the N-terminal pre-LIM region of Ajuba (upper panels). Immunoblot analysis of epitope-tagged proteins from 10% of input whole cell extracts of 293T cells co-transfected with expression constructs encoding Gfi1-FLAG and either myc-Ajuba or myc-Ajuba-preLIM (lower panels). E, immunoprecipitation of FLAG-epitope-tagged Gfi1, followed by immunoblot analysis for myc-epitope-tagged C-terminal LIM domains of Ajuba (upper panels). Immunoblot analysis of epitope-tagged proteins from 10% of input whole cell extracts of 293T cells co-transfected with expression constructs (lower panels).

GFI1 and Ajuba associate with histone deacetylase enzymes. A, immunoblot analysis of Sephacryl S-300 gel-filtration fractions from Jurkat T cell nuclear extracts, with antisera against Gfi1, HDAC1, -2, or -3 and Ajuba. Elution peaks for calibration controls are indicated by arrows along with their molecular weight. B, co-immunoprecipitation of FLAG-epitope tagged HDAC1, -2, or -3 with antisera specific to a myc-epitope-tagged Ajuba, but not control IgG (top). C, co-immunoprecipitation of myc-epitope-tagged Ajuba with antisera specific to FLAG-epitope tagged HDAC1, -2, or -3 (top left panel), but not control IgG (top right panel). Immunoblot analysis reveals levels of 10% of input proteins (bottom panels).

Ajuba acts a Gfi1 co-repressor in living cells. A, ChIP analysis in HL60 cells with a Gfi1-specific monoclonal antibody (Gfi1 mAb), Ajuba-specific rabbit antisera (Ajuba pAb), or isotype IgG controls (Con M IgG and Con R IgG, respectively), and primers specific for Gfi1 target genes GFI1, GFI1B, and CSF1. β-ACTIN is used as the negative control. B, TaqMan analyses on HL60 cells transduced with three independent lentiviral shRNA specific for GFI1 or AJUBA or a non-targeting control. Representative immunoblot demonstrates lower levels of GFI1 and AJUBA with one of three independent shRNA constructs. C, schematic representation of GFI1 autoregulation by GFI1, AJUBA, and HDACs.

Ajuba increases Gfi1 transcriptional repression. A, map of the reporter constructs employed in transient transcription experiments. TK, herpes simplex virus thymidine kinase minimal promoter; CAT, chloramphenicol acetyltransferase; poly(A)+, polyadenylation sequences; B30, synthetic Gfi1 binding site (18). B, transient transcription assay in 293T cells with reporters in A, and titration of expression vectors encoding FLAG-epitope-tagged Gfi1 (top panel) or the SV40SWAP (Gfi1·SNAG domain) mutant (bottom panel). Data are expressed as -fold repression (values from TKCAT/values from B30TKCAT) ± S.E. Statistical analysis compared the -fold repression of samples to that of the preceding sample. C, immunoblot analysis of whole cell lysates corresponding to B with an anti-FLAG monoclonal antibody. D, transient transcription assay in 293T cells co-transfected with reporters and expression vectors encoding Gfi1 and Ajuba. The values for CAT activity were corrected for transfection efficiency, and the data are expressed as relative CAT activity with -fold repression. E, transient transcription assay in 293T cells co-transfected with the indicated constructs. F, immunoblot analysis of whole cell lysate corresponding to E with anti-FLAG and anti-Myc-monoclonal antibodies. G, transient transcription assay in 293T cells first transduced with non-targeting or Ajuba-targeting shRNA, then transfected with a control Renilla luciferase reporter, and TK- or B30 × 2-Firefly-luciferase reporter vectors. The values for Firefly luciferase activity were corrected for Renilla activity (for transfection efficiency). Data are expressed as relative Firefly luciferase activity and -fold repression. ^*, p < 0.05; ^**, p < 0.001.

Ajuba LIM domains function as a co-repressor that synergizes with Gfi1. A, map of the Ajuba and Gfi1 expression and reporter constructs (B) employed in transient transcription experiments. “Lex” = LexA DNA binding site. C, immunoblot analysis with LexA-specific antisera on lysates from 293T cells transfected with expression constructs encoding LexA, or LexA fused to full-length Ajuba, the Ajuba-pre-LIM, Ajuba-LIM domains or the Gfi1 N terminus. D, transient transcription assay in 293T cells with the reporter and expression constructs in A and B encoding LexA, LexA-Ajuba, and Gfi1-N terminus-LexA fusions. Vector (ng) refers to the quantity of each expression vector plasmid transfected. Note that where only single expression vectors are indicated, the total amount of expression vector transfected has been controlled by the addition of equivalent amounts of empty vector control DNA. E, transient transcription assay in 293T cells with expression constructs encoding LexA, or LexA-Ajuba-LIM and LexA-Ajuba-pre-LIM region fusions. Vector (ng) as in D. F, transient transcription assay in 293T cells with expression constructs encoding LexA, Gfi1-LexA, LexA-Ajuba-LIM, and LexA-Ajuba-pre-LIM region fusions. The -fold repression of LexA-Gfi1 with LexA-Ajuba-LIM is compared with that of Gfi1-LexA with LexA control, or Gfi1-LexA with LexA-Ajuba-preLIM. Vector (ng) as in D. ^*, p < 0.05; ^**, p < 0.001.

HDAC activity is required for Gfi1 and Ajuba-mediated transcriptional repression. A, fluorescent histone-deacetylase enzymatic analysis of FLAG-monoclonal antibody or isotype-matched IgG-control immunoprecipitants from a Jurkat T cell clone stably expressing low levels of Gfi1-FLAG. TSA-sensitive relative-fluorescent units (values for triplicate immunoprecipitations treated with TSA subtracted from values for triplicate immunoprecipitations treated with vehicle control) ± absolute error are displayed with values from Gfi1-FLAG samples arbitrarily set to 100. B, fluorescent histone-deacetylase enzymatic analysis of FLAG monoclonal antibody or isotype-matched IgG-control immunoprecipitants from Jurkat T cells stably expressing Ajuba-FLAG. TSA-sensitive relative-fluorescent units (values calculated as in A) ± absolute error are displayed with values from Ajuba-FLAG samples arbitrarily set to 100. C, transient transcription assay in 293T cells transfected with the reporter and expression constructs in Fig. 3 (A and B) encoding LexA, LexA-Gfi1, or LexA-Ajuba, and treated with either TSA, sodium butyrate, or vehicle control. The relative CAT activity and -fold repression of TSA- or sodium butyrate-treated Gfi1-LexA with LexA-Ajuba are compared with those of vehicle-treated GFI1-LexA with LexA-Ajuba. D, transient transcription assay in 293T cells transfected with expression constructs encoding LexA, Gfi1-LexA, or LexA-Ajuba-LIM domains, and treated with TSA or vehicle control. The relative CAT activity and -fold repression of TSA-treated LexA-LIM, Gfi1-LexA, or Gfi1-LexA with LexA-LIM is compared with the corresponding samples treated with vehicle control. ^*, p < 0.05; ^**, p < 0.001.