The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

• Keywords
• CD, circular dichroism, CoIP, coimmunoprecipitation, DLS, dynamic light scattering, Differentiation, EM, EM, electron microscopy, Electron microscopy, IDRs, intrinsically disordered regions, NRSE, neuron-restrictive silencer element, NRSF, NRSF, neuron-restrictive silencer factor, Neuron-restrictive silencer factor, Neuronal, PCNA, proliferating cell nuclear antigen, RD1/2, repressor domain 1/2, RE1, repressor element-1, RE1-silencing transcription factor, REST, REST, RE1-silencing transcription factor, REST-FL, full-length REST, REST-N62, REST-N62, splicing isoform of REST, also known as REST4 or REST4-S3, REST4, ZF, zinc finger, aa, amino acid(s), bp, base pair(s), kDa, kilodaltons,
• Publication type
• Journal Article MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• DNA Damage MeSH
• Telomeric Repeat Binding Protein 2 * MeSH
• Telomere-Binding Proteins * metabolism   MeSH
• Proteomics MeSH
• Shelterin Complex * MeSH
• Telomere metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Telomeric Repeat Binding Protein 2 * MeSH
• Telomere-Binding Proteins * MeSH
• Shelterin Complex * MeSH
• TINF2 protein, human MeSH Browser