The Groucho/Transducin-like Enhancer of split Transcriptional Repressors Interact with the Genetically Defined Amino-terminal Silencing Domain of Histone H3

Abstract

Groucho is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila. We are investigating the molecular mechanisms underlying the functions of Groucho and its mammalian homologs, the transducinlike Enhancer of split (TLE) proteins. We report that Groucho/TLEs are associated with chromatin in live cells and that they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native Groucho/TLE proteins interact specifically with histone H3 and not with other core histones. This interaction is mediated by the H3 aminoterminal domain previously shown by genetic analysis in yeast to be essential for the role of H3 in transcriptional silencing. We also demonstrate that Groucho/ TLEs form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing Groucho/TLEs may associate with chromatin through interactions with the amino terminus of histone H3 and that these interactions may be propagated along the chromosome due to the ability of Groucho/ TLEs to participate in higher order structures.In both invertebrates and vertebrates, signaling mechanisms mediated by the cell-surface receptor Notch control the differentiation of a variety of cell types both by restricting the competence of precursor cells to respond to specific differentiation cues and by providing inductive signals (reviewed in Refs. 1 and 2). Our understanding of the molecular mechanisms underlying the restrictive functions of the Notch pathway points to an important role for proteins involved in transcriptional repression. In particular, molecular genetic analysis of Notch signaling during Drosophila neural development shows that the basic helix-loop-helix (bHLH) 1 proteins encoded by the Enhancer of split complex act at the end point of the Notch cascade by repressing the expression of genes that promote the neural fate (proneural genes) (3-8). This transcriptional function involves the activity of the product of another locus, referred to as groucho (3, 9 -12). This gene was originally implicated in Notch signaling by genetic analysis showing phenotypic interactions between alleles of groucho and other genes involved in the regulation of proneural gene expression (1-3, 13). Molecular studies subsequently showed that the bHLH proteins encoded by the Enhancer of split complex interact with the Groucho protein (10, 12). Groucho also interacts with other bHLH factors related to Enhancer of split proteins; these include the product of the pair rule gene hairy and will be hereafter collectively referred to as Hairy and Enhancer of split-like (HES) proteins (2, 4 -6, 10 -12). It is thought that HES factors recruit the broadly expressed Groucho protein, which lacks DNA-binding ability, to specific DNA sites. Once targeted to DNA, Groucho can repress both basal and activated transcription (12). This particular observation and the demonstration that groucho mutations affect the expression of a variety of ge...

Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation

“…Post-nuclear supernatants and chromatinenriched fractions were obtained as described previously. 17 Subcellular fractions were analyzed by western blotting using anti-FLAG, anti-Gro/TLE or anti-HA antibodies.…”

“…7,16 Complex formation with Hes1 results in Gro/ TLE1 recruitment to DNA, 14,15 where Gro/TLE1 interacts with structural chromatin components. 17 In addition, Hes1 binding causes increased Gro/TLE1 phosphorylation, a process previously termed 'cofactor-activated phosphorylation'. 11,18 Inhibition of cofactor-activated phosphorylation by mutation of selected serine residues within the serine/proline-rich domain of Gro/TLE1 weakens the interaction of Gro/TLE1 with chromatin and impairs both the transcriptional repression ability and anti-neurogenic activity of this protein without affecting its interaction with Hes1.…”

Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates

“…The exact mechanism used by the amino terminus to repress transcription is not known, but the recently described interaction between Groucho proteins and histone H3 may provide a mechanism, especially if the histone H3 interaction domain is found to localize to the amino terminus (Palaparti et al 1997). An interaction with histone H3 could lead to the assembly or stabilization of repressive chromatin on target gene promoters and thereby repress transcription.…”

“…An interaction with histone H3 could lead to the assembly or stabilization of repressive chromatin on target gene promoters and thereby repress transcription. This mechanism is used by the SIR repressors from yeast (Hecht et al 1995), but for the Groucho proteins the role of the interaction with H3 in transcriptional repression is untested (Palaparti et al 1997). The Groucho proteins may also work by other mechanisms in addition to, or instead of, the assembly of chromatin.…”

Transducin-like enhancer protein 1 mediates estrogen receptor binding and transcriptional activity in breast cancer cells

“…The Groucho/transducin-like enhancer of split (TLE) proteins also possesses similar properties to pioneer factors, namely that they can bind to histones (9) within condensed chromatin, independently of other proteins (10). TLE proteins have a well-characterized role as repressor proteins for various transcriptional regulators including Wnt and Notch pathways (reviewed in ref.…”