The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.

Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression.

The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.

A recessive mutation named Justy was found that abolishes B lymphopoiesis but does not impair other major aspects of hematopoiesis. Transplantation experiments showed that homozygosity for Justy prevented hematopoietic progenitors from generating B cells but did not affect the ability of bone marrow stroma to support B lymphopoiesis. In bone marrow from mutant mice, common lymphoid progenitors and pre-pro-B cells appeared normal, but cells at subsequent stages of B lymphopoiesis were dramatically reduced in number. Under culture conditions that promoted B lymphopoiesis, mutant pre-pro-B cells remained alive and began expressing the B cell marker CD19 but failed to proliferate. In contrast, these cells were able to generate myeloid or T/NK precursors. Genetic and molecular analysis demonstrated that Justy is a point mutation within the Gon4-like (Gon4l) gene, which encodes a protein with homology to transcriptional regulators. This mutation was found to disrupt Gon4l pre-mRNA splicing and dramatically reduce expression of wild-type Gon4l RNA and protein. Consistent with a role for Gon4l in transcriptional regulation, the levels of RNA encoding C/EBPalpha and PU.1 were abnormally high in mutant B cell progenitors. Our findings indicate that the Gon4l protein is required for B lymphopoiesis and may function to regulate gene expression during this process.

Erythropoietin receptors associate with a ubiquitin ligase, p33RUL, and require its activity for erythropoietin-induced proliferation.

The proliferation and survival of hematopoietic cells is strictly regulated by cytokine growth factors that act through receptors of the Type I cytokine receptor family, including erythropoietin (Epo) and its receptor, EpoR. Mitogenic signaling by these receptors depends on activation of Jak tyrosine kinases. However, other required components of this pathway have not been fully identified. In a screen for proteins that interact with EpoR and Jak2, we identified a novel member of the U-box family of ubiquitin ligases. This receptor-associated ubiquitin ligase, RUL, co-precipitated with EpoR from mammalian cells and mediated ubiquitination of EpoR. Also, endogenously expressed RUL was rapidly and transiently phosphorylated on serine after cytokine treatment of factor-dependent hematopoietic cells. Expression of ubiquitin ligase-deficient mutants of RUL inhibited Epo-induced expression of c-myc and bcl-2, two immediate-early genes normally associated with Epo-induced cell growth. Consistent with that finding, expression of mutant RUL also inhibited Epo-dependent proliferation and survival of factor-dependent cells. Together, these observations suggest that RUL is a required component of mitogenic signaling by EpoR. We also show that RUL is phosphorylated in response to growth factors that act through non-cytokine receptors, suggesting that RUL may function as a common regulator of mitogenesis.