Expression of the Neural REST/NRSF–SIN3 Transcriptional Corepressor Complex as a Target for Small‑Molecule Inhibitors

Sakthidasan Jayaprakash1 · Le T. M. Le2 · Bjoern Sander2,3,4 . Monika M. Golas1,5
Accepted: 21 October 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract

The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacety- lase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF–SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

Keywords SIN3 · REST/NRSF · HDAC · Transcriptional repression · Small-molecule inhibitors

Abbreviations

Aa Amino acid
CE Cytoplasmic extract
EDTA Ethylenediaminetetraacetic acid
HAT Histone acetyl transferase
HDAC Histone deacetylase
kDa Kilo Dalton
NE Nuclear extract
NRSE Neuron-restrictive silencing element
NRSF Neuron-restrictive silencer factor
PAGE Polyacrylamide gel electrophoresis PAH Paired amphipathic helix
RBAP46 Retinoblastoma-associated protein 46
RBBP7 Retinoblastoma-binding protein 7
RBBP4 Retinoblastoma-binding protein 4
RE1 Repressor element 1
REST Repressor element 1 silencing transcription factor
SAP30 SIN3-associated protein 30 SID Sin3 interacting domain SIN3 Switch independent 3
SDS Sodium dodecyl sulfate
SDS3 Suppressor of defective silencing 3 TBE Tris/borate/EDTA
WD Tryptophan-aspartic acid
ZF Zinc finger

Introduction

In eukaryotes, chromatin is remodeled by different factors [1] including ATP-dependent chromatin remodelers [2] as well as histone modifying enzymes such as histone acetyl transferases (HAT) and histone deacetylases (HDAC) [3]. For example, histone acetylation favors active transcription of genes [4, 5], while deacetylation of histone proteins is associated with transcriptional repression [6]. HDACs are divided into dif- ferent classes [7], i.e., classes I, IIA, IIB, III, and IV HDACs. HDAC1 and HDAC2 are members of the zinc-dependent HDAC class I and associate with additional proteins to form the SIN3 complex [3], the Nucleosome remodeling and dea- cetylating (NuRD) complex [8] and the CoREST protein com- plex [9].
Two of these HDAC complexes, SIN3 and CoREST, bind to the N and C terminus of the repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing fac- tor (REST/NRSF), respectively [10]. REST/NRSF is a multi- domain protein that belongs to the Krüppel-type zinc finger (ZF) family [11]. REST/NRSF is involved in regulating gene expression by binding to RE1/neuron-restrictive silencing ele- ment (NRSE) sites [12]. In embryonic stem cell (ESC) and non-neuronal cells, REST/NRSF acts as the principle repres- sor of a large set of neuronal genes, among others [13, 14]. The SIN3 complex harbors—in addition to the eponymous paralogues, SIN3A and SIN3B [15] and the enzymatic activi- ties HDAC1 or HDAC2 [3]—a set of core proteins includ- ing the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, suppressor of defective silencing 3 (SDS3) with putative dimerization function as well as SIN3-associated protein 30 (SAP30) [6, 16–19].
The SIN3 complex is a druggable assembly. In particular, a class of small-molecule inhibitors has been developed that interferes with the functional activity of HDACs [20–22], some of which have been introduced into the treatment reper- toire of cancer [23]. Furthermore, a limited number of inhibi- tors have been identified that target the SIN3 protein [24]. For the characterization of small-molecule inhibitors, protein fragments and individual proteins are typically used; how- ever, growing evidence suggests that the inhibitory activity of a given drug may differ, once the protein targeted resides in a macromolecular complex [25]. Here, we thus recombinantly expressed the human REST/NRSF–SIN3 transcriptional core- pressor complex and its subcomplexes and purify the multi- protein assemblies for molecular and pharmacological studies.

Materials and Methods

cDNA Library Synthesis
A complementary DNA (cDNA) library was prepared from RNA [26] that was extracted from human neural progenitor cells as outlined [27]. The Uniprot resource (https://www. uniprot.org) was used to identify canonical isoforms.

Amplification of SIN3 Genes and Expression in a Baculovirus Insect Cell System
Genes encoding components of the SIN3 complex were amplified using the cDNA library generated. Primers are given in Table S1 of the electronic supplementary mate- rial. SIN3A and SIN3B genes were inserted into the accep- tor vector pGS-BacA-21222 [28]. REST/NRSF [29] as well as HDAC1, RBAP46/RBBP7, RBAP48/RBBP4, SDS3, and SAP30 genes were subcloned into the donor vectors pIDS, pIDK, and pIDC (Geneva Biotech, Geneva, Switzerland). Sequences were confirmed by DNA sequencing (Macrogen Europe, Amsterdam, Netherlands). Plasmids are listed in Table S2 of the electronic supplementary material. Cre/LoxP based recombination, bacmid preparation, virus amplifica- tion and protein expression were performed as described [28, 29].

FLAG Purification of SIN3
SIN3 complexes were overexpressed in a starting cul- ture of 5 × 108 Sf9 insect cells (Thermo Fisher Scientific, Waltham, USA). A volume of 20 mL of the nuclear extract was obtained and used for anti-FLAG affinity selection as outlined [28], except that the salt concentration (400 mM NaCl) was kept constant during the purification.

Western Blotting
The denatured protein samples were separated by SDS-PAGE electrophoresis, and the gel was transferred onto a nitrocellu- lose membrane at 4 °C overnight for western blot analysis. All subsequent steps were performed at room temperature. Blot- ting was verified by staining the nitrocellulose membrane with Ponceau S stain (0.1% Ponceau S, 5% acetic acid) for 1 min. Tris buffered saline (1 × TBS) supplemented with 0.1% Tween- 20 and 5% non-fat milk was used for blocking for 60 min. Primary antibodies were diluted in a 1 × TBS/0.05% Tween- 20/5% non-fat milk mixture, and the membrane was incubated for 60 min using this mixture. Following washing of the mem- brane using TBST buffer (20 mM Tris, pH 7.6 and 150 mM NaCl, 0.1% Tween-20) twice for 5 min, each, the membrane was treated with an appropriate secondary antibody mixture (diluted in 1 × TBS/0.05% Tween-20/3% non-fat milk) for 60 min. Finally, the membrane was washed again and imaged in an ImageQuant (GE Healthcare, Chicago, IL, USA) using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). We used acidic buffer (0.2 M glycine, pH 2.2, 0.1% SDS, 1% Tween-20) for stripping. The antibodies used are listed in Table S3 of the electronic supplementary material.

Glycerol Gradient Ultracentrifugation
Anti-FLAG eluates of the REST/NRSF–SIN3 transcriptional corepressor complex were ultracentrifuged using a 10–30% glycerol gradient in gradient buffer [20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM DTT] at 239,400× g and 4 °C for 17 h. Eleven fractions of 400 μL volume, each, were collected from the bottom.

Electrophoretic Mobility Shift Assay (EMSA)
Reactions using a cyanine 5 (Cy5)-labeled probe were set up as outlined [29]. Anti-FLAG eluates of the REST/NRSF–SIN3B and the REST/NRSF–SIN3 transcriptional corepressor com- plex were used directly in undiluted form and following serial dilution as indicated. The final reaction mix contained 28.6 mM HEPES–NaOH pH 7.5, 3% Ficoll, 172 mM NaCl, 0.43 mM EDTA, and 0.43 mM DTT.

HDAC Assay
HDAC assays were performed using the fully assembled REST/NRSF–SIN3 transcriptional corepressor complex iso- lates, which was based on the use of the fluorogenic substrate BOC-Ac-Lys-AMC and measurement of the fluorescence [30]. Dimethyl sulfoxide (DMSO), suberoylanilide hydroxamic (SAHA), mocetinostat, and romidepsin were added as indi- cated. The assay was performed for three biological replicates (n = 3).

Statistical Analysis
Data were analyzed by one-way analysis of variance (ANOVA) with Tukey post hoc HSD test in the framework of R [31]. Significance levels were set to *p < 0.05, **p < 0.01, and
***p < 0.001. Data are shown as the mean ±standard error of the mean (SEM).

Results

Expression of Neural‑Specific, Human SIN3 Complexes
SIN3-related isoforms were isolated from a cDNA library synthesized from neural progenitor cells. The encoded protein sequences are given in Fig. S1 of the electronic supplementary material. The SIN3A and SIN3B isoforms encode proteins of 1273 amino acids (aa) and 1130 aa, respectively (Fig. 1a). Both SIN3A/B proteins contain three paired amphipathic helix (PAH) domains in the N-terminal half. For SIN3B, we obtained a splicing vari- ant, which lacks an in-frame exon (exon 10 of the longest, canonical SIN3B isoform NM_015260.3). This in-frame exon is found in primates but is absent from the vast majority of organisms.
For RBAP46/RBBP7 and RBAP48/RBBP4, proteins of 469 aa and 425 aa, respectively, are encoded by the mRNA isoforms extracted from the neural progenitor cell cDNA library. The encoded proteins encompass seven and six tryptophan-aspartic acid (WD) repeats, respectively (Fig. 1a). The isoform encoded by RBAP46/RBBP7 extracted from the neural progenitor cells differs from the canonical isoform by the encoded N terminus, which is longer and distinct in the isoform isolated in comparison to the canonical isoform (canonical RBAP46/RBBP7 isoform NM_002893.4). For SDS3, a 328 aa protein that harbors a coiled-coiled domain and a Sin3 interacting domain (SID) motif is encoded by the isoform obtained, and for the Zinc finger (ZF) motif containing SAP30, a 220 aa protein with SID motif was encoded by the isoform (Fig. 1a). Both, the SDS3 and SAP30 isoforms, represent the canonical iso- forms of the respective protein. For the histone deactylase HDAC1 and the ZF domain protein REST/NRSF, we used the canonical isoforms of the proteins, which are the pre- dominant isoforms expressed in the brain [32]. However, we took advantage of the Cys402Ser REST/NRSF mutant. The corresponding murine Rest/Nrsf Cys397Ser variant has been shown to facilitate folding of the ZF domain 8, which in the wildtype human and murine protein harbors a third cysteine residue in addition to the cysteine residues coordinating the zinc ion of the ZF domain [33].

SIN3A and SIN3B Bind to REST/NRSF
Human SIN3A or SIN3B proteins tagged with 3xFLAG on the C terminus were co-expressed with the human Cys402Ser REST/NRSF in a baculovirus insect cell expression system (Fig. 1b, first and second complex), and cytoplasmic and nuclear extracts were prepared (Fig. 2a,

Fig. 1 SIN3 complexes studied. a Domain organization of the human, neural SIN3 complex proteins. For SIN3B and RBAP46/RBBP7, var- iant isoforms were obtained with respect to the canonical isoforms. PAH paired amphipathic helix, WD tryptophan-aspartic acid, CC coiled-coil, SID Sin3 interacting domain, ZF zinc finger, HDA histone deacetylase domain, NR N-terminal repressor domain, CR C-terminal repressor domain. b Schematic drawing of SIN3 complexes expressed in this study. For simplicity, each SIN3-associated protein expressed is shown as single protein
b). Pre-experiments indicated that protein degradation was minimized using nuclear extracts for purification only and thus improved the purification quality. Using anti-FLAG affinity selection, we co-purified REST/NRSF with both 3xFLAG-tagged SIN3A and SIN3B, respectively as shown by Coomassie-stained SDS–polyacrylamide gel electro- phoresis (PAGE) and western blotting (Fig. 2a, b). As observed previously [29], the high-molecular weight form of REST/NRSF was co-purified with SIN3A and SIN3B. Thus, SIN3A and SIN3B form stable protein complexes with REST/NRSF.
Assembly of the SIN3 Complex
Next, we co-expressed SIN3A-3xFLAG, HDAC1, and RBAP46/RBBP7 along with REST/NRSF (Fig. 1b, third complex). As for the REST/NRSF–SIN3A/B complexes, we purified SIN3A-associated proteins by anti-FLAG immunoaffinity chromatography and elution using 3xFLAG peptide. We identified all four proteins in the elution fraction (Fig. 3a) suggesting protein complex formation.
To reconstitute the REST/NRSF–SIN3 transcriptional corepressor complex (Fig. 1b, fourth complex), two expres- sion cassettes that encoded for the subcomplexes SIN3A- HDAC1-RBAP46/RBBP7-REST/NRSF and SIN3A-SAP30-SDS3, respectively, were expressed independently in insect cells. In both constructs, SIN3A is tagged with a 3xFLAG. Nuclear extracts containing both subcomplexes were then mixed and subjected to anti-FLAG immunoaffinity purifica- tion. All proteins co-eluted using 3xFLAG peptide as com- petitor as shown by SDS-PAGE (Fig. 3b) as well as western blotting (Fig. 3c) and functional analyses (see below). The protein yield was about 1.5–2.0 mg per Liter of cell culture volume. To address whether the proteins indeed assembled into the REST/NRSF–SIN3 transcriptional corepressor com- plex upon mixing of the SIN3A-HDAC1-RBAP46/RBBP7- REST/NRSF and SIN3A-SAP30-SDS3 nuclear extracts

Fig. 2 Expression of the REST/NRSF–SIN3A/B complex. a SDS- PAGE of the REST/NRSF–SIN3A and the b REST/NRSF–SIN3B complex during purification (top). Proteins were separated on an SDS-PAGE and Coomassie-stained. M marker, CE cytoplasmic extract, NE nuclear extract, FT flow through, W and W1-W2 wash fractions, E and E1-E5 elution fractions, R resin. REST/NRSF, SIN3A and SIN3B protein bands are indicated on right. Protein iden- tities were confirmed by western blotting (bottom) remained as smaller SIN3A-associated subcomplexes, we further separated the anti-FLAG eluates by a glycerol gra- dient ultracentrifugation. SDS-PAGE analysis of the gradi- ent fractions demonstrated that all proteins co-migrated in a clear peak in fractions 4–5 (Fig. 3d). We conclude that the REST/NRSF–SIN3 transcriptional corepressor complex was efficiently expressed in the baculovirus/insect cell expression system and isolated from the nuclear extracts by anti-FLAG affinity selection.
REST/NRSF–SIN3 Complexes Bind to RE1/NRSE
Next, we tested whether or not the complexes are function- ally active using EMSA and histone deacetylation assays. First, we examined whether or not the REST/NRSF–SIN3 complexes interact with RE1/NRSE sites. EMSA experi- ments were performed for the REST/NRSF–SIN3B complex (Fig. 1b, second complex) and the REST/NRSF–SIN3 tran- scriptional corepressor complex (Fig. 1b, fourth complex) using a 37 bp DNA fragment with a canonical RE1/NRSE site (Fig. 4a). Cy5-labeled RE1 alone did not shift (Fig. 4b, lane 1). There was a band-shift using increasing concen- trations of the REST/NRSF–SIN3B complex incubated with the Cy5-labeled probe (Fig. 4b, lanes 2–5). Unlabeled competitor RE1 DNA prevented the occurrence of the band- shift (Fig. 4b, lane 6). Similarly, we observed a band-shift, when the Cy5-labeled probe was incubated with the REST/ NRSF–SIN3 transcriptional corepressor complex (Fig. 4c, lanes 2 and 3; Cy5-labeled probe alone is shown in lane 1). Again, unlabeled competitor RE1 DNA interfered with the occurrence of the band-shift (Fig. 4c, lane 4). Thus, the REST/NRSF complexes bind to the RE1/NRSE site when they are in complex with SIN3A and SIN3B.
Histone Deacetylase Activity of the REST/NRSF–SIN3 Transcriptional Corepressor Complex Finally, we tested whether the REST/NRSF–SIN3 tran- scriptional corepressor complex is active in deacetylation and whether the activity is inhibited by small-molecule drugs. To this end, we incubated the REST/NRSF–SIN3 transcriptional corepressor complex with DMSO or

Fig. 3 Expression of the REST/NRSF-SIN3A-HDAC1-RBAP46/ RBBP7 and the REST/NRSF–SIN3 transcriptional corepressor com- plex. a Purification of the REST/NRSF-SIN3A-HDAC1-RBAP46/ RBBP7 complex and b the REST/NRSF–SIN3 transcriptional core- pressor complex. c Western blot analysis of the REST/NRSF–SIN3 transcriptional corepressor complex. d Fractions of the REST/NRSF–SIN3 transcriptional corepressor complex following ultracentrifuga- tion in a 10–30% glycerol gradient (1, bottom fraction; 11, top frac- tion of the gradient). Shown are Coomassie-stained SDS-PAGEs (a, b, d), and western blot signals (c), respectively. Protein bands are labeled on the right increasing concentrations of the HDAC inhibitors SAHA, romidepsin and mocetinostat. While the DMSO-exposed samples were active in the HDAC assay, the addition of the HDAC inhibitors significantly interfered with the HDAC activity in a concentration-dependent manner (Fig. 5). We thus conclude that the recombinantly expressed REST/ NRSF–SIN3 transcriptional corepressor complex is func- tionally active and targetable by HDAC inhibitors.

Fig. 4 EMSA of REST/NRSF–SIN3B, and the REST/NRSF–SIN3 transcriptional corepressor complex using a canonical RE1/ NRSE site. a Schematic representation of the DNA fragment used. b The REST/NRSF–SIN3B complex binds to canonical RE1/NRSE. Cy5-labeled probe only in lane 1; lanes 2–5 show increasing amounts (1x, 2x, 4x, and 8x) of protein samples along with Cy5-labeled probe; unlabeled competitor probe, Cy5-labeled probe and protein sample (8x) in lane 6. (c) The REST/NRSF–SIN3 transcriptional corepres- sor complex (NRC) binds to canonical RE1/NRSE. Cy5-labeled probe in lane 1; increasing amounts (1x and 4x) of protein samples and Cy5-labeled probe in lane 2 and 3; unlabeled competitor probe, Cy5-labeled probe and protein sample (4x) in lane 4. Band-shifts are indicated by lines, and asterisks indicate non-shifted bands [14, 36]. Molecular studies on these complexes are chal- lenging as multiple large proteins (up to > 100 kDa) have to be expressed and isolated. In this study, we took advan- tage of a recombinant expression toolbox [28] that was successfully applied to assemble functional C-terminal REST/NRSF complexes comprising REST/NRSF, CoR- EST, LSD1 and HDAC1 [29]. Previous structural data and pull-down assays indicated that PAH2 and PAH1 domains of Sin3a and Sin3b bind to the N terminus of Rest/Nrsf [10, 37]. Here, we pulled down a stable complex of REST/ NRSF–SIN3A and REST/NRSF–SIN3B and thus provided further support for a SIN3-REST/NRSF interaction.
Moreover, we reconstituted the fully assembled REST/ NRSF–SIN3 transcriptional corepressor complex by com- bining two subcomplexes, REST/NRSF-SIN3A-HDAC1- RBAP46/RBBP7 and SIN3A-SDS3-SAP30. All proteins co-purified using SIN3A as bait, and co-migrated in glycerol gradients indicating complex formation. We noted that the band intensities of the individual proteins of the REST/ NRSF–SIN3 transcriptional corepressor complex differed taking the molecular weight of the proteins into account. For example, REST/NRSF, which apparently migrated in multiple bands in the gel, appeared to be underrepresented with respect to SIN3A. In this respect, it has been shown that an Sds3 fragment is able to dimerize a Sin3a fragment [19] suggesting that at least SDS3 and SIN3A might be pre- sent in two copies in the REST/NRSF–SIN3 transcriptional corepressor complex. In a recent report, the pre-assembled REST/NRSF- CoREST complex was shown to bind to the RE1/NRSE site as did isolated REST/NRSF alone [29]. In the present study, we demonstrated that both, the REST/NRSF–SIN3B complex and the REST/NRSF–SIN3 transcriptional core- pressor complex, interacts with a canonical RE1/NRSE

Fig. 5 Histone deacetylase activity of the REST/NRSF–SIN3 tran- scriptional corepressor complex. DMSO was used as control; SAHA, romidepsin and mocetinostat were added to the REST/NRSF–SIN3 transcriptional corepressor complex at different concentrations as indicated. The histogram depicts the mean relative fluorescence units (RFU) ± SEM of three replicates (n = 3). **p < 0.01, and ***p < 0.001 site at a salt concentration of about 172 mM during com- plex formation. REST/NRSF acts as a site-specific DNA- binding protein [38] and is thus expected to drive the RE1/ NRSE DNA–protein complex interaction. Besides REST/ NRSF, however, additional proteins of REST/NRSF–SIN3 transcriptional corepressor complex have been shown to interact with DNA. Among these proteins, SDS3 has been shown to interact with DNA under low-salt conditions (0.2 × Tris/Borate/EDTA (TBE) buffer) [19], and SAP30 binds to DNA at a salt concentration of 125 mM inde- pendent of the DNA sequence [39]. Furthermore, a com- plex comprising the Sin3a PAH3 and Sap30 SID binds to DNA [40]. The presence of these additional DNA-binding proteins may thus further stabilize the interaction of the REST/NRSF–SIN3 transcriptional corepressor complex with DNA.

Discussion

HDAC1 and HDAC2 contribute to the repression of gene transcription by the deacetylation of nucleosomal histone proteins [34]. HDAC1/2 typically forms multi-protein complexes [35] such as, for example, the REST/NRSF complexes that assemble on genomic RE1/NRSE sites
Finally, we demonstrated the deacetylation activity of the REST/NRSF–SIN3 transcriptional corepressor complex indicating that the isolates are not only active in binding to DNA but also support the functional deacetylation activity of the HDAC1 protein. Using the HDAC inhibitors SAHA, mocetinostat, and romidepsin, the activity of the complex could be blocked in an inhibitor concentration-dependent manner. While the REST/NRSF–SIN3 transcriptional core- pressor complex was functionally active at a very low con- centration of the inhibitors, increasing concentrations of the small-molecule drugs significantly inhibited the histone deacetylase activity. In conclusion, we present a functional molecular repressor system for studying SIN3 complexes as targets of HDAC inhibition.

Acknowledgements We are grateful to Golshah Ayoubi and Susanne
N. Stubbe for excellent technical assistance, and wish to thank Zong- pei Zhao for technical support. We acknowledge access to laboratory facilities at the Danish Neuroscience Center House. This work has been supported by the Lundbeck Foundation’s Fellowship program, the Sapere Aude Program of the Danish Council for Independent Research, the Danish Cancer Society, the Carlsberg Foundation, the A.P. Møller Foundation for the Advancement of Medical Sciences, the Fabrikant Einar Willumsens Mindelegat and the Helga og Peter Kornings Fond to M.M.G.

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