Ajuba Functions as An HDAC-Dependent Co-Repressor for Autoregulation of the Growth Factor Independent-1 Transcription Factor

Growth factor independent-1 (Gfi1) is mutated in patients with Severe Congenital Neutropenia. Gfi1 is a zinc finger protein with a SNAG-transcriptional repressor domain. The SCN-associated Gfi1 mutant proteins function as dominant negatives to sequester limiting SNAG-dependent corepressor proteins from the remaining wild type Gfi1 protein, and deregulate a subset of Gfi1 target genes such as CSF1. The identity of the critical Gfi1 SNAG-domain-associated co-repressor proteins is unknown. Ajuba is a LIM-domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor-domain containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Coimmunoprecipitation of synthetic and endogenous proteins, and co-elution with gel filtration identified an endogenous Ajuba-Gfi1-HDAC multiprotein complex. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain dependent. ChIP analysis and reciprocal knock-down experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on a subset of Gfi1 target genes, but is not the critical SNAG-dependent cofactor sequestered by SCN-associated Gfi1 mutant proteins.