These three mutants are inactive by different mechanisms and may offer differing abilities to trap substrates. To test substrate trapping by the three inactive HDAC1 mutant proteins, we performed a histone binding experiment. Fosaprepitant dimeglumine by HDAC1 is critical for mitotic progression. These findings revealed a previously unknown mechanism of action of HDAC inhibitors including Eg5 acetylation, and provide a persuasive mechanistic hypothesis for HDAC inhibitor-mediated G2/M arrest. employed a substrate trapping strategy to identify mitosis-related protein Eg5 (KIF11) as an HDAC1 substrate. HDAC1 colocalized with Eg5 during mitosis, influenced the ATPase activity of Eg5, and was critical for mitotic progression. These findings reveal a mechanistic model where HDAC inhibitor drugs arrest cells in mitosis through HDAC1-mediated Eg5 acetylation. INTRODUCTION Gene expression is regulated by nucleosomal histone protein modifications, such as acetylation, methylation, and phosphorylation (Khorasanizadeh, 2004). Acetylation is usually catalyzed by histone acetyltransferases and prospects to a less compact chromatin structure, which is associated with transcriptional activation (Kramer et al., 2001). In contrast, histone deacetylase (HDAC) proteins catalyze deacetylation, which induces chromatin condensation and transcriptional repression. Acetylation and HDAC protein activity play important functions in a variety of cellular processes, including proliferation, differentiation, and apoptosis. The unregulated activities of HDAC proteins are associated with a variety of diseases, such as asthma, arthritis, schizophrenia, and malignancy (Kramer et al., 2001). With a causal role in disease, HDAC proteins have emerged as important therapeutic targets for drug development. Currently, four HDAC inhibitors are approved as malignancy therapeutics. Vorinostat (SAHA or Suberoyl Anilide Hydroxamic Acid, Zolinza?) and romidepsin (Depsipeptide, FK-228, Istodax?) are approved for the treatment of cutaneous T-cell lymphoma, whereas belinostat (PXD101, Beleodaq?) and panabinostat (LBH-589, Farydak?) are approved to treat peripheral T-cell lymphoma and multiple myeloma, respectively (Taunton et al., 1996, Yang et al., 1996, Yang et al., 1997, Hu et al., 2000). HDAC inhibitors Fosaprepitant dimeglumine influence proliferation by perturbing cell cycle progression, which ultimately prospects to apoptosis (Marks et al., 2000). HDAC inhibitors arrest cells at G0/G1 and G2/M phases (Richon et al., 2000). HDAC inhibitor-induced G0/G1 cell cycle arrest has been well analyzed and widely attributed to the expression of the p21 (waf1/cip1) and p27 (kip1) proteins after histone hyperacetylation and transcriptional upregulation (Newbold et al., 2014). In contrast, the mechanism accounting for HDAC inhibitor-induced G2/M arrest is usually less understood. Much like G0/G1 arrest, a few reports documented that HDAC inhibitor-induced G2/M arrest is usually accompanied by transcriptional changes, such as increased expression of p21 and decreased expression of cyclins and retinoblastoma (Anh et al., 2012, Wetzel et al., 2005, Peart et al., 2003). In contrast, several studies reported that HDAC inhibitor-induced G2/M arrest does not correlate with transcriptional changes (Ishii et al., 2008, Warrener et al., 2010), suggesting a mechanism impartial of histone acetylation. The limited data suggest that HDAC inhibitor-mediated mitotic arrest entails both histone and non-histone-mediated activities. We hypothesize here that HDAC inhibitors induce mitotic arrest through a mechanism involving non-histone substrates of HDAC proteins. Histones are unquestionably the most analyzed substrate of HDAC proteins (Hassig et al., 1998). By studying histone acetylation, the role of HDAC1 in transcriptional regulation has been well characterized. As discussed earlier, the G0/G1 arrest observed with HDAC inhibitors is usually widely attributed to altered gene expression due to histone acetylation (Peart et al., 2003). However, recent proteomics data revealed that a large number of acetylated proteins exist in cells, in addition to histones (Choudhary et al., 2009, Zhao et al., 2010). Moreover, while many of the eleven HDAC isoform family members are found predominantly in the nucleus near nucleosomal-bound histones, including HDAC1 Fosaprepitant dimeglumine and HDAC2, several HDAC isoforms are found predominantly in the cytoplasm, such as HDAC6, where histones cannot be their predominant substrates. The available data implicate Fosaprepitant dimeglumine an expanded role of HDAC proteins in Fosaprepitant dimeglumine cell biology through non-histone substrates (Zhao et al., 2010, Scholz et al., 2015), which is usually consistent with the hypothesis TNFRSF4 that this mitotic arrest observed with HDAC inhibitors entails nonhistone targets. To characterize the complete role of HDAC proteins in cells, here we sought to identify non-histone substrates. We focused on HDAC1 due to its overexpression in multiple cancers (Weichert et al., 2008a, Miyake et al., 2008, Weichert et al., 2008b, Rikimaru et al., 2007, Sasaki et al.,.