A Role for Cell Cycle-regulated Phosphorylation in Groucho-mediated Transcriptional Repression

Abstract

Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family are involved in a variety of cell differentiation mechanisms in both invertebrates and vertebrates. They become recruited to specific promoter regions by forming complexes with a number of different DNA-binding proteins thereby contributing to the regulation of multiple genes. To understand how the functions of Gro/TLE proteins are regulated, it was asked whether their ability to mediate transcriptional repression might be controlled by cell cycle-dependent phosphorylation events. It is shown here that activation of p34 cdc2 kinase (cdc2) with okadaic acid is correlated with hyperphosphorylation of Gro/TLEs. Moreover, pharmacological inhibition of cdc2 activity results in Gro/TLE dephosphorylation. In agreement with these findings, a purified cdc2-cyclin B complex can directly phosphorylate Gro/TLEs in vitro. Two separate Gro/TLE domains, the CcN and SP regions, contain sequences that are phosphorylated by cdc2. Deletion of these sequences is correlated with loss of Gro/ TLE phosphorylation by cdc2 in vitro and okadaic acidinduced Gro/TLE hyperphosphorylation in vivo. In addition, Gro/TLEs are phosphorylated during the G 2 /M phase of the cell cycle, and this is correlated with a decreased nuclear interaction. Finally, the transcription repression ability of Gro/TLEs is enhanced by pharmacological inhibition of cdc2. Taken together, these results demonstrate that Gro/TLE proteins are phosphorylated as a function of the cell cycle and implicate phosphorylation events occurring during mitosis in the negative regulation of Gro/TLE activity.In both invertebrates and vertebrates, transcriptional corepressor proteins of the Groucho/transducin-like Enhancer of split (Gro/TLE) 1 family play crucial roles in the regulation of a variety of cell differentiation mechanisms. In particular, Drosophila Gro is required for sex determination, segmentation, and neural development (1). Vertebrate Gro/TLE proteins contribute to the regulation of neuronal development (2), patterning of the neural tube (3, 4), skeletogenesis (5, 6), hematopoiesis (7-9), and myogenesis (10, 11). Gro/TLE proteins have no intrinsic DNA binding activity but can be targeted to specific gene regulatory regions due to their ability to interact with a number of different DNA-binding transcription factors. Examples of these include basic helixloop-helix proteins of the Hes family (12-17), Runt homology domain proteins of the Runt/RUNX family (7,18,19), homeodomain factors containing engrailed homology region 1 motifs (3, 20 -24), winged-helix domain transcription factors (23, 25), and high mobility group box proteins (7, 9). By virtue of these multiple interactions, the general transcription repression activity of Gro/TLE proteins can be recruited in context-dependent manners to specific target genes.Transcriptional repression by Gro/TLE family members is thought to be mediated by at least two mechanisms. Oligomeric Gro/TLEs can interact with both histones (26, 27) ...

Mitotic partitioning of transcription factors

“…Thus, mitotic phosphorylation and degradation in the case of the hBrm-based SWI/SNF complex are thought to contribute to mitotic transcription silencing [Muchardt et al, 1996;Sif et al, 1998]. Mitotic phosphorylation of transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family is also correlated with a decreased nuclear interaction [Nuthall et al, 2002].…”

“…Thus, mitotic phosphorylation and degradation in the case of the hBrm-based SWI/SNF complex are thought to contribute to mitotic transcription silencing [Muchardt et al, 1996;Sif et al, 1998]. Mitotic phosphorylation of transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family is also correlated with a decreased nuclear interaction [Nuthall et al, 2002].C 2 H 2 zinc finger proteins constitute a predominant family among mammalian transcription factors, and the linkers separating the finger motifs in the DNA-binding domain are highly conserved, as they are likely to contain the consensus amino acid sequence TGEKP. Among this family, Ikaros and Sp1 are not only excluded from chromatin in early mitosis, but it has also been shown that the three linkers of Ikaros and at least one of the Sp1 linkers are phosphorylated [Dovat et al, 2002].…”

Characterization of Aes nuclear foci in colorectal cancer cells

“…These results may appear contradictory to the 'wine tannin hypothesis' if the soluble form of Aes-TLE1 complex is responsible for transcriptional inhibition. We speculate that being soluble is a necessary but not the sufficient feature for Aes-TLE1 to inhibit transcription, and that TLE1(S239A) is unable to interact with the Rbpj transcription factor complex as with other transcription factors (17,28).…”

“…3A left) (17,28). Therefore, we studied whether phosphorylation of TLE1(S239) was critical for the inhibition of Notch signalling-mediated transcription and/or focus formation with Aes.…”

“…Therefore, we studied whether phosphorylation of TLE1(S239) was critical for the inhibition of Notch signalling-mediated transcription and/or focus formation with Aes. To this end, we overexpressed a mutant form of TLE1 of which S239 was converted to a structurally similar but unphosphorylatable amino acid alanine (S239A) (28) in HCT116 CRC cells. Compared with wild-type TLE1, S239A mutant did not show inhibition of Notch signalling transcription when co-expressed with Aes (Fig.…”