Selective interaction of mSin3B_SF with NRSF N-terminal domain in yeast. Interaction of NRSF subdomains and mSin3B_SF was examined by using a two-hybrid assay in yeast. (a) Combinations of “bait” and “prey” constructs introduced into the yeast L40 strain. (b) Growth of yeast strains carrying each construct on a his⁻, leu⁻, trp⁻ plate. (c) A filter assay for the β-galactosidase activity on a leu⁻, trp⁻, his⁺ plate. Note that β-galactosidase activity was induced in yeast cells expressing both NRSF-N and mSin3B_SF, but not in the cells expressing mSin3B_SF alone.

Inhibition of HDAC activity by TSA relieves transcriptional repression by RD-1 but not that by RD-2. (A) TSA relieves transcriptional repression mediated by the N-terminal (a) but not by the C-terminal (b) domain. Neuro2a cells were transfected with plasmids expressing either GAL4DBD-NRSF-N-terminal (G4-NR 1–156) or GAL4DBD-NRSF-C-terminal (G4-NR 989-1097) together with a luciferase reporter plasmid pGL-S10PR5GB. After 24 hr, the cells were treated with various concentrations of TSA (0–200 ng/ml) for an additional 24 hr. Relative luciferase activities are calculated relative to the activity obtained by using the GAL4DBD plasmid as an effector. (B) TSA relieves NRS-mediated transcriptional repression by NRSF. Neuro2a cells were transfected with the NRSF expression plasmid pCI-NRSF and the NRS-containing reporter plasmid pGL-S10PRS36+ and treated as described above.

The amino-terminal and carboxyl-terminal domains of NRSF are sufficient to mediate transcriptional repression. Various regions of NRSF, depicted schematically on the left with amino acid residue numbers, were fused in-frame to GAL4-DBD. The resultant series of GAL4 DBD-NRSF (G4-NR) constructs was cotransfected into Neuro2a cells with a luciferase reporter plasmid, pGL-S10PR5GB, containing the SCG10 promoter flanked by five copies of the GAL4-binding site linked to a luciferase gene, together with pRL-TK as an internal control.

NRSF-N forms a complex with mSin3 and HDAC in mammalian cells. (A) Regions of mSin3 involved in the interaction with NRSF-N. Relative strengths of NRSF-N and mSin3 interactions were assessed by quantification of the autoradiograms and scored from background level (−) to strong interaction (+++); n.d., not determined. PAH domains and a unique region in mSin3B_SF are distinguished by hatched and shaded boxes, respectively. Numbers correspond to amino acid positions in the protein. (B) Specific binding of NRSF-N and mSin3 in vitro. GST-NRSF fusion proteins, i.e., GST-NRSF-N (N), GST-NRSF-C (C), GST alone (G), or GST-MAD (M), were incubated with ³⁵S-labeled FLAG-tagged mSin3 deletion constructs. Specifically bound mSin3 subforms then were resolved on an SDS/PAGE gel and detected by autoradiography. An aliquot comprising one-fifth of the total input protein was loaded in lane I as a control. (C) Interaction of NRSF^Myc and mSin3B^FLAG in NIH 3T3 cells. Expression plasmids encoding Myc-tagged NRSF and the long or short isoform of FLAG-tagged mSin3B were cotransfected into NIH 3T3 cells, and the expressed mSin3B^FLAG was immunoprecipitated with anti-FLAG M2 antibody. Proteins that interacted with mSin3B^FLAG were resolved by SDS/PAGE and visualized by Western blotting. Shown are fluorograms of NRSF, visualized by using the anti-myc antibody 9E10 (a), and mSin3B_LF (b) and mSin3B_SF (c), visualized by using the anti-mSin3B antibody A-20. (D) Association of NRSF-N with mSin3A, mSin3B, and HDAC1 in NIH 3T3 cells. GST pull-down experiments show that NRSF-N interacts with mSin3A, mSin3B_LF, and HDAC1. Nuclear extracts of NIH 3T3 cells were incubated with either immobilized GST alone (lane G) or GST-NRSF-N fusion protein (lane N), precipitated, and visualized by using anti-mSin3B (A-20) (a), anti-mSin3A (K-20) (b), or anti-HDAC1 (C-19) (c) antibody. Nuclear extracts were loaded into lane NE as a control.

Ectopic activation of neuron-specific genes in TSA-treated fibroblasts. NIH 3T3 cells were incubated with TSA (100 ng/ml) for the times indicated, and the expression levels of various mRNAs were determined by RT-PCR. (A) Three neuron-specific genes: SCG10, NMDA-receptor type 1 (NMDAR1), and choline acetyltransferase (ChAT), as well as a constitutively expressed control gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH). (B) Immunohistochemical staining using TuJ1 antibody reveals that neuron-specific βIII tubulin also is induced in the TSA-treated NIH 3T3 cells (Lower). Note that cell morphology is changed in the TSA-treated cells (Upper, phase contrast).