Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17.

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.

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.

Generation of mice with a conditional allele for Ift172.

Ift172 encodes a gene product that is part of a complex that mediates intraflagellar transport (IFT), a process necessary for the genesis and maintenance of cilia. Genetic studies in mice have offered evidence that Ift172 also plays a role in hedgehog signaling. Disruption of Ift172 in mice is associated with lethality at about embryonic day 11, limiting studies to understand the role for Ift172 in later development and the adult. To further our understanding of the later roles of Ift172, we have generated mice with a conditional allele for Ift172. We have confirmed the phenotype of the disrupted allele by using CRE expression directed by the prx1 enhancer to disrupt the conditional Ift172 allele in the developing limb.

Expression of the synaptotagmin I gene is enhanced by binding of the pituitary-specific transcription factor, POU1F1.

The POU1F1 transcription factor (also known as Pit-1/GHF1) is required for development of pituitary cells that secrete prolactin, GH, and TSH. Presumably, POU1F1 regulates the expression of multiple genes required for expansion and differentiation of these pituitary cell lineages. However, only a few genes regulated by POU1F1 have been identified. In the present studies we have identified synaptotagmin I (Syt1) as a target gene for POU1F1 in GH(3) pituitary cells. Chromatin immunoprecipitation assays have provided evidence that POU1F1 binds close to the Syt1 exon that contains the initiation codon. Although this exon has previously been considered to be located far from the transcription initiation site, transcript mapping in GH(3) cells indicates that Syt1 mRNA synthesis is initiated close to the mapped POU1F1-binding site. POU1F1 knockdown studies using a short hairpin RNA vector have provided evidence that POU1F1 plays a role in stimulating expression of the endogenous Syt1 gene. Transfection studies with a Syt1-luciferase reporter gene are consistent with the presence of an internal, POU1F1-regulated promoter in the Syt1 gene. In vitro binding studies have provided further evidence for a POU1F1-binding site within this region of the Syt1 gene. Overall the studies provide evidence that Syt1 is a target gene regulated by POU1F1 in GH(3) pituitary cells. Because SYT1 has been extensively studied as an important transducer of Ca(2+) signaling in regulated secretion, it seems likely that activation of Syt1 gene expression is part of a mechanism mediating POU1F-induced differentiation of pituitary cells.

The pituitary-specific transcription factor, Pit-1, can direct changes in the chromatin structure of the prolactin promoter.

The chromatin structure of a promoter is an important determinant of its transcriptional activity. Many promoters are assembled into repressive polynucleosomal arrays that are subsequently remodeled to allow for the activation of gene expression. This study addresses the contribution of a single transcription factor, Pit-1, in orchestrating the chromatin structure of the prolactin gene. Utilizing an in vivo reconstitution system, we found that Pit-1 can bind to multiple sites in the chromatin-assembled 5'-flanking region of the prolactin gene and activate transcription from the chromatin-assembled template. Interestingly, Pit-1 was able to substantially alter micrococcal nuclease digestion of the prolactin 5'-flanking region, and the results are consistent with presence of a translationally positioned nucleosome on the prolactin promoter. Changes in micrococcal nuclease digestion were also observed with a truncated Pit-1 mutant containing only the DNA-binding domain. As the truncation mutant was unable to activate transcription from the chromatin-assembled template, the ability of Pit-1 to alter chromatin structure of the prolactin gene is not dependent on transcriptional activation. We propose that Pit-1 likely plays a role in altering chromatin to facilitate recruitment and subsequent transcriptional activation by additional factors.

Gonadotropin-releasing hormone-induced stimulation of the rat secretogranin II promoter involves activation of CREB.

To investigate the events involved in regulation of the secretogranin II (SgII) gene, luciferase reporter constructs were transfected into gonadotrope-derived, alphaT3-1 cells. DNA between -91 and -60 relative to the transcription start site was found to be required for GnRH induced SgII reporter gene activation. This region contains a consensus cAMP response element (CRE) and disruption of this CRE reduced GnRH responsiveness of the SgII promoter. CREB was shown to bind to the SgII CRE and transfection studies with a dominant-negative CREB mutant provided evidence that CREB is required for GnRH responsiveness of the SgII promoter. An expression vector for an inhibitor of the cAMP-dependent protein kinase was found to reduce the ability of cAMP or GnRH to activate the SgII-luciferase reporter gene. These studies offer evidence that GnRH-induced activation of the SgII promoter in the alphaT3-1 cell line requires cAMP-dependent protein kinase activity and a functional CRE within the 5'-flanking region of the gene.