Abstract

As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription (Kojima et al., 2001), an N-terminal REST4 isoform caused by alternative splicing, inclusion of an extra exon (N3c) which introduces a pre-mature stop codon, contributes to neurogenesis and tumorigenesis (Palm et al., 1998, 1999; Lee et al., 2000; Raj et al., 2011). Recently, in line with established epigenetic regulation of pre-mRNA splicing (Allo et al., 2010; Luco et al., 2011), we demonstrated that REST undergoes extensive, context-dependent alternative splicing which results in the formation of a large number of mRNA variants predictive of multiple protein isoforms (Chen and Miller, 2013). Supported by the fact that immunoblotting/-staining with different anti-REST antibodies yield different results, alternative splicing allows production of various structurally and functionally different REST protein isoforms in response to shifting physiologic requirements, shedding light on environmental regulation of REST function. However, REST isoforms might be differentially assayed or manipulated, leading to data misinterpretation and controversial findings. For example, in contrast to the proposed neurotoxicity of elevated nuclear REST in ischemia (Noh et al., 2012) and Huntington’s disease (Zuccato et al., 2003; Buckley et al., 2010), Lu et al. recently reported decreased nuclear REST in Alzheimer’s disease and neuroprotection of REST in aging brain (Lu et al., 2014). Unfortunately, alternative REST splicing was largely neglected by Lu et al. (2014), making it necessary for a reevaluation of their findings. As shown in Figure 1 A , human REST gene boundary is now doubled by an alternative …

Highlights

  • Comparison of the Western blotting (WB)/IHC results between different antibodies may hint about the existence of multiple repressor element-1 silencing transcription factor (REST) protein isoforms; no such comparison was shown in Lu et al, while all the presented blots were maximally cropped with only the band of interest available, making it impossible to evaluate the potential existence of multiple REST isoforms

  • Comparison of nuclear REST between young (n ϭ 11), aged (n ϭ 77), AD (n ϭ 72), and MCI (n ϭ 11) groups (Fig. 1E, imaging in Lu et al.) was presumably based on staining of the samples with three different antibodies but not a single antibody, otherwise the remaining two antibodies must have been respectively used for another two sets of samples or occasions of experiments, which were not mentioned in the paper

  • Like the case for IHC, two antibodies (07-579 and ab52850) were previously listed for WB without disclosure of their usage for each independent experiment; based on the addendum, all the presented WB data were obtained using 07-579, while neither the usage nor the result information was disclosed for ab52850, raising the question why this antibody was listed in the paper

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Summary

Introduction

Without evidence of Northern blotting, it is difficult to interpret the full-length REST mRNA expression level with the primer-dependent qRT-PCR data in Lu et al given that most of the previously reported mRNA variants were not tested and that the four qRT-PCR primer sets yielded different results, it is unknown how the total mRNA level and the percentage of REST4 mRNA in brain tissues were calculated.

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