Abstract
RE-1 silencing transcription factor (REST) is a transcriptional repressor that regulates gene expression by binding to repressor element 1. However, despite its critical function in physiology, little is known about its interaction proteins. Here we identified 204 REST-interacting proteins using affinity purification and mass spectrometry. The interactome included proteins associated with mRNA processing/splicing, chromatin organization, and transcription. The interactions of these REST-interacting proteins, which included TRIM28, were confirmed by co-immunoprecipitation and immunocytochemistry, respectively. Gene Ontology (GO) analysis revealed that neuronal differentiation-related GO terms were enriched among target genes that were co-regulated by REST and TRIM28, while the level of CTNND2 was increased by the knockdown of REST and TRIM28. Consistently, the level of CTNND2 increased while those of REST and TRIM28 decreased during neuronal differentiation in the primary neurons, suggesting that CTNND2 expression may be co-regulated by both. Furthermore, neurite outgrowth was increased by depletion of REST or TRIM28, implying that reduction of both REST and TRIM28 could promote neuronal differentiation via induction of CTNND2 expression. In conclusion, our study of REST reveals novel interacting proteins which could be a valuable resource for investigating unidentified functions of REST and also suggested functional links between REST and TRIM28 during neuronal development.
Highlights
RE-1 silencing transcription factor (REST), which is known as neuron-restrictive silencer factor (NRSF), is a transcription repressor that binds to the 21-bp RE1 sites in the regulatory regions of its target genes[1]
Neurite outgrowth was increased by knockdown of REST or Tripartite motif-containing 28 (TRIM28), suggesting that downregulation of both REST and TRIM28 might promote neuronal differentiation via induction of CTNND2 expression
Since artificial tagging can sometimes cause proteins to mislocalize in cells, we checked the localization of V5-tagged REST proteins using immunofluorescence staining, which showed that exogenous REST proteins were mainly localized in the nucleus, as previously reported (Fig. 1B 12)
Summary
RE-1 silencing transcription factor (REST), which is known as neuron-restrictive silencer factor (NRSF), is a transcription repressor that binds to the 21-bp RE1 sites in the regulatory regions of its target genes[1]. REST is known to repress its target genes by interacting with subunits of several transcription regulatory complexes, including CoREST and mSin[3] corepressor complexes, the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, and polycomb repressive complex 1 (PRC1) and PRC21,5,6. These REST-interacting proteins were independently identified using yeast two-hybrid screening or co-immunoprecipitation under different experimental conditions. Neurite outgrowth was increased by knockdown of REST or TRIM28, suggesting that downregulation of both REST and TRIM28 might promote neuronal differentiation via induction of CTNND2 expression This interactomic study revealed novel interacting proteins that may represent a valuable resource for investigating additional functions of REST and suggested functional links between REST and TRIM28 during neuronal development
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