Nucleus Accumbens-Associated Protein 1 Binds DNA Directly through the BEN Domain in a Sequence-Specific Manner.

Eiji Obayashi,Naohiro Kobayashi,Kentaro Nakayama,Gyosuke Sakashita,Takeshi Urano,Hiroaki Kato,Naomi Nakayama,Yuko Nariai,Takashi Nagata,Hisashi Yoshida,Satoru Kyo,Sam-Yong Park

doi:10.3390/biomedicines8120608

Abstract

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short β sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

Highlights

Nucleus accumbens-associated protein 1 (NAC1), encoded by the NACC1 gene, is a nuclear protein that encompasses an amino-terminal BTB and a carboxyl-terminal BEN (BANP, E5R and NAC1) domain
It was recently demonstrated that NAC1 promotes mesendodermal and represses neuroectodermal fate selection in embryonic stem cells, in cooperation with the pluripotency transcription factors, Oct4, Sox2, and Tcf3 [18,19] and that NAC1 is critical for efficient iPSC generation [20]
We have recently shown that NAC1 harbors an unusual bipartite-type nuclear localization signal to endow transcriptional regulator functions (Figure 1A) [58], and used live cell photobleaching analysis to show that a substantial fraction of NAC1 in the nucleus is associated with or interacts with nuclear proteins or chromatin [59]

Summary

Introduction

Nucleus accumbens-associated protein 1 (NAC1), encoded by the NACC1 gene, is a nuclear protein that encompasses an amino-terminal BTB (broad complex, tramtrack, bric-à-brac) and a carboxyl-terminal BEN (BANP, E5R and NAC1) domain. NACC1 was identified as a cancer-associated BTB. NAC1 was shown to be important for the pluripotency of embryonic stem cells [15,16] through direct transcriptional regulation of c-Myc [17]. It was recently demonstrated that NAC1 promotes mesendodermal and represses neuroectodermal fate selection in embryonic stem cells, in cooperation with the pluripotency transcription factors, Oct, Sox, and Tcf3 [18,19] and that NAC1 is critical for efficient iPSC generation [20]. NAC1 has been reported to be an important component of RIG-I-like receptor mediated innate immune responses against viral infection [22]

Methods

Results

Conclusion