Positive and Negative Regulation of the Transforming Growth Factor β/Activin Target Gene goosecoid by the TFII-I Family of Transcription Factors

Goosecoid (Gsc) is a homeodomain-containing transcription factor present in a wide variety of vertebrate species and known to regulate formation and patterning of embryos. Here we show that in embryonic carcinoma P19 cells, the transcription factor TFII-I forms a complex with Smad2 upon transforming growth factor β (TGFβ)/activin stimulation, is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription. Downregulation of endogenous TFII-I by small inhibitory RNA in P19 cells abolishes the TGFβ-mediated induction of Gsc. Similarly, Xenopus embryos with endogenous TFII-I expression downregulated by injection of TFII-I-specific antisense oligonucleotides exhibit decreased Gsc expression. Unlike TFII-I, the related factor BEN (binding factor for early enhancer) is constitutively recruited to the distal element in the absence of TGFβ/activin signaling and is replaced by the TFII-I/Smad2 complex upon TGFβ/activin stimulation. Overexpression of BEN in P19 cells represses the TGFβ-mediated transcriptional activation of Gsc. These results suggest a model in which TFII-I family proteins have opposing effects in the regulation of the Gsc gene in response to a TGFβ/activin signal

Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxβ

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix–loop–helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxβ, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxβ physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways—histone modification and SUMOylation—brokered in part by TFII-I family proteins

Determination and Functional Analysis of the Consensus Binding Site for TFII-I Family Member BEN, Implicated in Williams-Beuren Syndrome*S⃞

The ubiquitously expressed TFII-I family of multifunctional transcription factors is involved in gene regulation as well as signaling. Despite the fact that they share significant sequence homology, these factors exhibit varied and distinct functions. The lack of knowledge about its binding sites and physiological target genes makes it more difficult to assign a definitive function for the TFII-I-related protein, BEN. We set out to determine its optimal binding site with the notion of predicting its physiological target genes. Here we report the identification of an optimal binding sequence for BEN by SELEX (systematic evolution of ligands by exponential enrichment) and confirm the relevance of this sequence by functional assays. We further performed a data base search to assign genes that have this consensus site(s) and validate several candidate genes by quantitative PCR upon stable silencing of BEN and by chromatin immunoprecipitation assay upon stable expression of BEN. Given that haploinsufficiency in BEN is causative to Williams-Beuren syndrome, these results may further lead to the identification of a set of physiologically relevant target genes for BEN and may help identify molecular determinants of Williams-Beuren syndrome

SUMOylation of GTF2IRD1 regulates protein partner interactions and ubiquitin-mediated degradation

GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxβ, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxβ. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxβ itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell