Abstract
Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.
Highlights
In 1992, two groups found repressor element-1 (RE-1) and bound it to a zinc finger transcription repressor in the SCN2A, SCG10 and synapsin genes [1,2]
PAH1 and RE-1 silencing transcription factor (REST) peptides derived from whole Sin3B and REST molecules
We posit that PAH1 and REST peptides, consisting of the binding
Summary
In 1992, two groups found repressor element-1 (RE-1) and bound it to a zinc finger transcription repressor in the SCN2A, SCG10 and synapsin genes [1,2]. RE-1 silencing transcription factor (REST), which is known as the neural restrictive silencer factor (NRSF) [3], was found to be an elemental repressor of transcription when binding to RE1 [4] or neural restrictive silencer element (NRSE) [1] consisting of a 21-bp DNA element in many genes, including neuronal genes [3,4,5]. Genes regulated by REST/NRSF were reported to have functions in synaptic transmission [5,6], neurotransmitter signaling [7,8,9], ion channeling [4,10] etc. REST/NRSF plays a critical role in neuronal differentiation and functions and regulates other types of cells that do not express neuronal-specific genes. REST has two repressor domains at both N- and C-termini, respectively, and two co-repressor complexes [17,18].
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