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

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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 deacetylase (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.

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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

RBAP48:

Retinoblastoma-associated protein 48

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

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Acknowledgements

We are grateful to Golshah Ayoubi and Susanne N. Stubbe for excellent technical assistance, and wish to thank Zongpei 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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Author notes

  1. Bjoern Sander

    Present address: Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, 8000, Aarhus C, Denmark

    Sakthidasan Jayaprakash & Monika M. Golas

  2. Stereology and EM Laboratory, Department of Clinical Medicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, 8000, Aarhus C, Denmark

    Le T. M. Le & Bjoern Sander

  3. Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, 8000, Aarhus C, Denmark

    Bjoern Sander

  4. Department of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Monika M. Golas

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Jayaprakash, S., Le, L.T.M., Sander, B. et al. Expression of the Neural REST/NRSF–SIN3 Transcriptional Corepressor Complex as a Target for Small-Molecule Inhibitors. Mol Biotechnol 63, 53–62 (2021). https://doi.org/10.1007/s12033-020-00283-7

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