Transcription intermediary factor 1gamma decreases protein expression of the transcriptional cofactor, LIM-domain-binding 1.

LIM-domain-binding 1 (LDB1) is a cofactor that participates in formation of regulatory complexes involving transcription factors containing LIM domains as well as other factors. We have examined the ability of transcriptional intermediary factor 1gamma (TIF1gamma) to decrease LDB1 expression. An expression vector for TIF1gamma was found to decrease expression of LDB1. A mutation which disrupts the ubiquitin ligase activity of TIF1gamma was found to block the ability of TIF1gamma to decrease LDB1 expression. Proteasome inhibitors were also able to block TIF1gamma effects on LDB1. Immunoprecipitation studies provided evidence that LDB1 interacts with TIF1gamma in intact cells. Knockdown of TIF1gamma in zebrafish embryos led to increased expression of LDB1 providing evidence for a physiological role of TIF1gamma in regulating LDB1 expression. Reporter gene assays demonstrated that TIF1gamma can alter the activity of LIM-homeodomain transcription factor-responsive promoters. These studies are consistent with a model in which TIF1gamma acts to ubiquitinate LDB1 leading to degradation of LDB1 and changes in transcription of LDB1-dependent promoters.

Regulation of LIM-domain-binding 1 protein expression by ubiquitination of Lys134.

LDB1 (LIM-domain-binding 1) is a cofactor that participates in formation of transcriptional regulatory complexes involving transcription factors containing LIM domains as well as other factors. The amount of LDB1 protein in cells has previously been shown to be modulated by RNF12 (RING finger protein 12). RNF12 is an E3 ubiquitin ligase that can target LDB1 for poly-ubiquitination and degradation via the proteasome. We find that in HEK (human embryonic kidney)-293 cells expression of RNF12 leads to mono-ubiquitination of LDB1 and increased levels of LDB1 protein. Mutagenesis studies identified Lys134 of LDB1 as the residue that is mono-ubiquitinated by RNF12. Mutation of Lys134 of LDB1 to arginine blocks the formation of mono-ubiquitinated LDB1 and surprisingly also increases LDB1 protein expression in HEK-293 cells. This leads to a model in which Lys134 of LDB1 can be either mono-ubiquitinated, leading to stabilization, or poly-ubiquitinated, leading to degradation by the proteasome pathway. We also find that ubiquitin-LDB1 fusion proteins are stabilized in HEK-293 cells, offering further evidence that mono-ubiquitination stabilizes LDB1 in these cells. Expression in Xenopus laevis embryos of an LDB1 protein in which Lys134 is replaced with arginine leads to enhanced expression of the mutant protein as compared with the wild-type protein. These findings provide evidence that modification of Lys134 can play a major role in regulating LDB1 expression.

Zebrafish melanophilin facilitates melanosome dispersion by regulating dynein.

BACKGROUND: Fish melanocytes aggregate or disperse their melanosomes in response to the level of intracellular cAMP. The role of cAMP is to regulate both melanosome travel along microtubules and their transfer between microtubules and actin. The factors that are downstream of cAMP and that directly modulate the motors responsible for melanosome transport are not known. To identify these factors, we are characterizing melanosome transport mutants in zebrafish. RESULTS: We report that a mutation (allele j120) in the gene encoding zebrafish melanophilin (Mlpha) interferes with melanosome dispersion downstream of cAMP. Based on mouse genetics, the current model of melanophilin function is that melanophilin links myosin V to melanosomes. The residues responsible for this function are conserved in the zebrafish ortholog. However, if linking myosin V to melanosomes was Mlpha's sole function, elevated cAMP would cause mlpha(j120) mutant melanocytes to hyperdisperse their melanosomes. Yet this is not what we observe. Instead, mutant melanocytes disperse their melanosomes much more slowly than normal and less than halfway to the cell margin. This defect is caused by a failure to suppress minus-end (dynein) motility along microtubules, as shown by tracking individual melanosomes. Disrupting the actin cytoskeleton, which causes wild-type melanocytes to hyperdisperse their melanosomes, does not affect dispersion in mutant melanocytes. Therefore, Mlpha regulates dynein independently of its putative linkage to myosin V. CONCLUSIONS: We propose that cAMP-induced melanosome dispersion depends on the actin-independent suppression of dynein by Mlpha and that Mlpha coordinates the early outward movement of melanosomes along microtubules and their later transfer to actin filaments.

The zebrafish moonshine gene encodes transcriptional intermediary factor 1gamma, an essential regulator of hematopoiesis.

Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1gamma (TIF1gamma) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1gamma mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1gamma mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1gamma functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1gamma protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.