The Neural Repressor NRSF/REST Binds the PAH1 Domain of the Sin3 Corepressor by Using its Distinct Short Hydrophobic Helix

doi:10.1016/j.jmb.2005.10.008

Journal of Molecular Biology

Volume 354, Issue 4, 9 December 2005, Pages 903-915

https://doi.org/10.1016/j.jmb.2005.10.008 Get rights and content

In non-neuronal cells and neuronal progenitors, many neuron-specific genes are repressed by a neural restrictive silencer factor (NRSF)/repressor element 1 silencing transcription factor (REST), which is an essential transcriptional repressor recruiting the Sin3-HDAC complex. Sin3 contains four paired amphipathic helix (PAH) domains, PAH1, PAH2, PAH3 and PAH4. A specific target repressor for Sin3 is likely to bind to one of them independently. So far, only the tertiary structures of PAH2 domain complexes, when bound to the Sin3-interacting domains of Mad1 and HBP1, have been determined. Here, we reveal that the N-terminal repressor domain of NRSF/REST binds to the PAH1 domain of mSin3B, and determine the structure of the PAH1 domain associated with the NRSF/REST minimal repressor domain. Compared to the PAH2 structure, PAH1 holds a rather globular four-helix bundle structure with a semi-ordered C-terminal tail. In contrast to the amphipathic α-helix of Mad1 or HBP1 bound to PAH2, the short hydrophobic α-helix of NRSF/REST is captured in the cleft of PAH1. A nuclear hormone receptor corepressor, N-CoR has been found to bind to the PAH1 domain with a lower affinity than NRSF/REST by using its C-terminal region, which contains fewer hydrophobic amino acid residues than the NRSF/REST helix. For strong binding to a repressor, PAH1 seems to require a short α-helix consisting of mostly hydrophobic amino acid residues within the repressor. Each of the four PAH domains of Sin3 seems to interact with a characteristic helix of a specific repressor; PAH1 needs a mostly hydrophobic helix and PAH2 needs an amphipathic helix in each target repressor.

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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Late-onset Alzheimer disease (LOAD) is the most frequent cause of dementia in elderly adults. However, the factors determining disease onset remain unclear. In the elderly, the activation and expression of the gene encoding RE-1 silencing transcription factor (REST) may be a determinant of neuroprotective mechanisms and good amyloidogenic pathway management. In the present study, the minimal promoter region of REST1 was genetically and epigenetically analyzed in blood samples from 21 subjects with LOAD and 20 cognitively healthy elderly subjects. Genomic DNA was isolated, treated with bisulfite and pyrosequenced, and gene expression was determined using real-time PCR. Notably, subjects with LOAD exhibited hypermethylation and significantly diminished expression of REST1 compared with healthy subjects (p = 0.001). In the LOAD group, the gene expression of CAT, SOD2 and GPX also showed a significant decrease and an increase in malondialdehyde. A docking analysis revealed that the first zinc finger protein Sp1 recognized and bound the methylated sequence in subjects with LOAD differently than the binding observed in control subjects. These results reveal that in patients with LOAD the methylation of specific sites in the promoter sequence of REST suppresses its expression and this could be regulating the decreased expression of CAT, SOD and GPX, besides interfering with the action of transcription factors as Sp1.
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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.

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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

Section snippets

Interaction domains in mSin3B and NRSF/REST

Conclusion

Preparation of proteins and peptides

Acknowledgements

Cell

Neuron

Neuron

Neuron

Lancet

Cell

Cell

Cell

Cell

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Graph.

J. Mol. Biol.

J. Mol. Biol.

The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes

Science

Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes

Proc. Natl Acad. Sci. USA

Corepressor-dependent silencing of chromosomal regions encoding neuronal genes

Science

Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Proc. Natl Acad. Sci. USA

Direct interaction of NRSF with TBP: chromatin reorganization and core promoter repression for neuron-specific gene transcription

Nucl. Acids Res.

Many human medulloblastoma tumors overexpress repressor element-1 silencing transcription (REST)/neuron-restrictive silencer factor, which can be functionally countered by REST-VP16

Mol. Cancer Ther.

The neuronal repressor REST/NRSF is an essential regulator in medulloblastoma cells

Nature Med.

Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes

Nature Genet.

NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function

EMBO J.

Knockout of REST/NRSF shows that the protein is a potent repressor of neuronally expressed genes in non-neural tissues

BioEssays