Journal
PROTEIN SCIENCE
Volume 26, Issue 5, Pages 997-1011Publisher
WILEY
DOI: 10.1002/pro.3142
Keywords
REST; NRSF; CoREST; LSD1; HDAC1; DNA transcription; transcriptional repression; histone deacetylation; histone demethylation; drug mechanism; medulloblastoma
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Funding
- Lundbeck Foundation's Fellowship program
- Sapere Aude Program of the Danish Council for Independent Research
- Danish Cancer Society
- Carlsberg Foundation
- A.P. Moller Foundation for the Advancement of Medical Sciences
- Fabrikant Einar Willumsens Mindelegat
- Helga og Peter Kornings Fond
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In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.
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