Journal of Molecular Biology
The Neural Repressor NRSF/REST Binds the PAH1 Domain of the Sin3 Corepressor by Using its Distinct Short Hydrophobic Helix
Introduction
Neural restrictive silencer factor (NRSF),1 also designated as repressor element 1 (RE1) silencing transcription factor (REST),2 is an essential transcriptional repressor for neuron-specific genes in non-neuronal cells and neuronal progenitors. NRSF/REST is a Kruppel-type zinc-finger protein that binds specifically to a 21 bp core sequence of neural-restrictive silencer element, NRSE/RE13, 4 in the regulatory regions of many neuron-specific genes: ion channels, neurotransmitter synthetases, receptors, synaptosomal proteins, neuronal cell adhesion molecules, neuronal cytoskeleton, neurotrophic factors, neuronal growth-associated proteins, and others.5, 6 NRSF/REST mediates transcriptional repression through the association of its N-terminal repressor domain (RD-1) with the mSin3/histone deacetylase-1/2 (HDAC1/2) complex and through the association of its C-terminal repressor domain (RD-2) with the CoREST/HDAC complex.7, 8 Both RD-1 and RD-2 interact directly with TATA-binding protein (TBP) via a chromatin-independent mechanism.9 Several neurological diseases, such as Down's syndrome,10 medulloblastoma11, 12 and Huntington's disease,13 are related, with dysregulation of NRSF/REST and its target genes. Moreover, transgenic mice expressing a dominant-negative mutant of NRSF/REST in their hearts exhibit dilated cardiomyopathy and are highly susceptible to arrythmias.14 An NRSF/REST knockout leads to malformations in several non-neural tissues, as well as apoptosis and embryonic lethality in mice.15 A recent study showed that small, non-coding dsRNA whose sequence is defined by NRSE/RE1 triggers gene expression of neuron-specific genes through interaction with the NRSF/REST transcriptional machinery.16 In addition, NRSF/REST has been shown to regulate the transition from a pluripotent to a neural progenitor cell and from a progenitor to a mature neuron, suggesting the plasticity of neural gene chromatin throughout neurogenesis.17 NRSF/REST has been identified as a tumor suppressor by an RNA interference (RNAi)-based genetic screen for genes that suppress transformation of human mammary epithelial cells.18
The Sin3 transcriptional corepressor complexes with sequence-specific, DNA-binding repressors or nuclear hormone receptor corepressors play crucial roles in eukaryotic gene silencing. Sin3 contains four PAH domains, PAH1, PAH2, PAH3 and PAH4, from its N terminus,19 and the HDAC-interaction domain (HID).20 The PAH domains mediate specific protein–protein interactions, most likely through their independent associations with various repressors. For example, PAH1 interacts with many different transcription factors, such as N-CoR,21, 22 PLZF,23 Opi1,24 HCF-1, Pf1, SMRTER, TIS725 and SAP25,26 while PAH2 interacts with Pf1, Mad family, Ume6, Stb1-5, Sp1-like transcription factors and TIS7.27 For mammalian Sin3 (mSin3), two isoforms, mSin3A and mSin3B, are found to be homologous to yeast corepressor Sin3.28 Both mSin3A and mSin3B sequences are highly conserved, although mSin3B has a shorter amino-terminal region.
Of the four Sin3 PAH domains, the tertiary structures of only mSin3A and mSin3B PAH2 domains have been determined by NMR.29, 30, 31, 32 Although PAH domain stands for paired amphipathic helix domain, which was assumed to contain two helices,19 both PAH2 domains of mSin3A and mSin3B hold a wedged four-helix bundle structure associated with the Sin3-interacting domain (SID) of Mad1, which is involved in mammalian cell proliferation and differentiation, and in both PAH2 domain complexes, the Mad1 SID adopts an amphipathic α-helix. In addition, associated with the SID of HBP1 that acts as a cell-cycle inhibitor and regulator of differentiation, the PAH2 domain of mSin3A holds the wedged four-helix bundle structure that is stabilized by the amphipathic α-helix of the HBP1 SID in the cleft of PAH2, but with a reversed orientation relative to that of the Mad1 helix.32
We reveal that the mSin3B PAH1 domain interacts with the N-terminal repressor domain of NRSF/REST. Moreover, we determine the solution structure of mSin3B PAH1 associated with the NRSF/REST minimal repressor domain. In the complex, PAH1 holds a left-handed four-helix bundle structure followed by a semi-ordered C-terminal tail. The fundamental architecture of the four helices of PAH1 is similar to the corresponding architecture of the PAH2 domains determined thus far; however, they are shorter than the corresponding helices in the PAH2 domains. In addition, the minimal repressor domain of NRSF/REST is folded into a unique hydrophobic short α-helix in the PAH1 complex in contrast to the amphipathic α-helix of Mad1 or HBP1 in the PAH2 complexes.
Section snippets
Interaction domains in mSin3B and NRSF/REST
The NRSF/REST N-terminal repressor domain was found to interact with the mSin3 region containing both PAH1 and PAH2, but there were conflicting results as to which bound to the repressor domain.8, 33, 34 In order to clarify the minimal interacting domain in mSin3B with the NRSF/REST N-terminal repressor domain, we prepared PAH1, PAH2 and PAH1-2 domains, of amino acid residues 28–132, 148–252 and 28–252 of mSin3B, respectively. By using three glutathione-S-transferase (GST)-fused NRSF/REST
Conclusion
In the present study, we have revealed that the NRSF/REST N-terminal repressor domain binds to the mSin3B PAH1 domain and determined the complex structure of the PAH1 domain bound to the NRSF/REST minimal repressor domain by using NMR spectroscopy. It consists of the left-handed, four-helix bundle structure and a semi-ordered C-terminal tail associated with the unique NRSF/REST hydrophobic α-helix. The NMR titration analysis suggested that the putative helical region of N-CoR can interact
Preparation of proteins and peptides
The DNA regions coding for mouse Sin3B PAH1, residues 28–132 and 28–107, mouse Sin3B PAH2, residues 148–252 and mouse Sin3B PAH1-2, residues 28–252 were obtained as described.8 Each of these regions was subcloned into the pET28a vector (Novagen) to produce a His6-tagged protein with a recombinant thrombin protease recognition site. Each protein was expressed in Escherichia coli strain BL21 (DE3) grown in LB medium. Each of the 15N-labeled or 13C/15N-labeled proteins was expressed in M9 minimal
Acknowledgements
We thank Dr Saburo Aimoto and Dr Kenichi Akaji at Osaka University for their kind preparation of two synthetic peptide fragments. This work was supported by a Project of Protein 3000, Transcription and Translation, Grants in Aid for Scientific Research from MEXT and a Collaborative of Regional Entities for the Advancement of Technological Excellence (CREATE) from JST.
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Present addresses: K. Murai, Division of Neuroscience, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA; N. Mori, Department of Anatomy and Neurobiology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.