Identification of Modified Histones as Binding Substrates of Human Spindlin Family Member 4 (SPIN4) by Peptide Arrays and Native Nucleosome Pulldown

Summary

Genetic variants in human Spindlin Family Member 4 (SPIN4) have recently been identified in patients with bone overgrowth, a novel developmental disorder. A protocol and new results describing the biochemical identification of modified histones in the context of nucleosomes as SPIN4 binding substrates are presented.

Abstract

Human Spindlin Family Member 4 (SPIN4) has just emerged as a novel regulator of bone growth. Loss-of-function variants in SPIN4 were identified in patients exhibiting a pre- and post-natal overgrowth syndrome, which is inherited in an X-linked dominant fashion. SPIN4 contains three tandem repeats of Tudor domains, each forming an individual aromatic cage. These Tudor domains are considered epigenetic reader domains for methylated arginine and lysine residues of histones based on sequence homology. However, the exact binding substrates of SPIN4 remain unclear. Human SPIN4, in both its wild-type and disease-causing mutant forms, was successfully generated via an insect cell expression system. By a histone modification peptide array, the differential binding affinities between wild-type and mutant SPIN4 were determined. Functional validations further demonstrated that SPIN4 is a bona fide reader of histone H3 Lysine 4 methylation (H3K4me), as evidenced by biochemical pulldown of native mononucleosomes. This integrated approach facilitates a rapid binding target identification of epigenetic reader proteins.

Introduction

Spindlin Family Member 4 (SPIN4) is an epigenetic reader and has been recently implicated in overgrowth syndrome, presenting with a constellation of pre- and postnatal overgrowth and tall stature^1,2. Spin4 knockout (KO) mice recapitulated the exact human phenotype, supporting the causality. To date, SPIN4 is the only identified exclusive epigenetic reader that negatively regulates bone growth. However, the biochemical properties of SPIN4, a critical epigenetic reader for growth, have not been characterized, leaving a critical knowledge gap in understanding the fundamental, disease-causing mechanisms i....

Protocol

All experimental procedures involving HEK293FT cells were conducted in compliance with the Institutional Biosafety Committee (IBC) guidelines of Virginia Commonwealth University.

1. Purification of recombinant human SPIN4

1. Clone full-length wild-type SPIN4 or SPIN4^D82H with an N-terminal 1× FLAG epitope tag into a baculoviral vector^6,7.
2. Prepare baculovirus by transfecting Sf9 cells with baculoviral vectors, harvest viruses when cells burst, aliquot, and store the virus at −80 °C^6,

Results

To investigate the binding substrates of human SPIN4, wild-type SPIN4 and a patient-derived mutant associated with overgrowth syndrome were reconstituted using a Sf9 insect cell expression system. Specifically, this is an aspartic acid (D) to histidine (H) missense mutation at residue 82 (D82H), which resides within the first Tudor domain of SPIN4, implying its role in regulating the substrate binding of SPIN4. A baculoviral vector encoding a single FLAG-tagged SPIN4 was used for protein expression in Sf9 cells, followed.......

Discussion

A time- and cost-effective method for biochemically pinpointing specific histone PTMs as targets of a poorly characterized protein, and for assessing how its disease-associated mutation affects the target's binding affinity, was presented. The advantage of this method is that it requires no expensive or time-consuming reconstitution of synthetic nucleosomes. This approach leveraged the convenience of commercialized histone PTM peptide arrays while ensuring that SPIN4 is a bona fide H3K4me reader by using native monon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes	Sarstedt	72.690.001	General experiments
15 cm Tissue Culture Dish	Sarstedt	83.3903.300	General experiments
15 mL Conical Centrifuge Tubes	Sarstedt	62.554.101	General experiments
50 mL Conical Centrifuge Tubes	Sarstedt	62.548.004	General experiments
500 mL Erlenmeyer Flask	Stellar Scientific	CT229809	Sf9 cell culture
Amersham ImageQuant 800 CCD Imager	Cytiva	IMQ800	CCD imaging system
Anti-FLAG M2 Affinity Gel	Sigma-Aldrich	A2220	Biochemical pulldown
Anti-FLAG M2 antibody	Sigma-Aldrich	F1804	Western blot
Aprotinin	MedChemExpress	HY-P0017	Protease inhibitor
Array Analyze Sofrware	Active Motif	https://www.activemotif.com/documents/Array_Analyze_Software_v16.1.zip	MODified peptide array data analysis
ASI Agarose LE	Alkali Scientific	A7700	DNA gel
Bac-to-Bac Baculovirus Expression System	Thermo Scientific	10359016	Baculoviral expression
Benzamidine hydrochloride	MedChemExpress	HY-W018781	Protease inhibitor
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A9647	Protein quantification standard
Bromophenol Blue	Thermo Scientific	A18469.08	Lammeli buffer
Coomassie Brilliant Blue R-250	Thermo Scientific	20278	Protien gel staining
Dithiothreitol (DTT)	Sigma-Aldrich	D5545	Lysis buffer
EDTA	RPI research products	E58100	Lysis buffer
Enhanced Chemiluminescence (ECL) substrate	Cytiva	RPN2232	Western blot
ESF 921 Insect Cell Culture Medium	Expression Systems	96-001	Sf9 cell culture
Ethanol (200 proof)	Spectrum Chemical	ET107	DNA precipitation
EVOS M5000 Imaging System	Invitrogen	M5000	General imaging
FLAG peptide	MedChemExpress	HY-P7006	Pulldown elution
Glycerol	Sigma-Aldrich	G77893	Storage buffer
H3K4me1 antibody	Active Motif	39300	Western blot
H3K4me2 antibody	Cell Signaling	9725T	Western blot
H3K4me3 antibody	Cell Signaling	9751T	Western blot
HEK293FT cells	Thermo Scientific	R70007	Human embryonic kidney fibroblastcell line
HEPES	Sigma-Aldrich	H3375	Lysis buffer
ImageJ	National Institutes of Health	https://imagej.net/ij/download.html	Java-based image processing program
Leupeptin hemisulfate	MedChemExpress	HY-18234A	Protease inhibitor
Micrococcal Nuclease (MNase)	NEB	M0247S	Endonuclease for chromatin digestion
Model 120 Sonic Dismembrator	Fisher Scientific	FB120110	Sonication
MODified Histone Peptide Array	Active Motif	13005	Protein binding assay
Pepstatin A	MedChemExpress	HY-P0018	Protease inhibitor
Peroxidase IgG Fraction Monoclonal Goat Anti-Mouse IgG, light chain specific	Jackson ImmunoResearch	115-035-174	Western blot
pFastBac1-Flag-SPIN4	Thermo Scientific	10360014	Baculoviral expression vector
pFastBac1-Flag-SPIN4-D82H	Thermo Scientific	10360014	Baculoviral expression vector
Phenol:Chloroform:Isoamyl Alcohol (25:24:1)	Sigma-Aldrich	P2069	DNA purification
Pierce Rapid Gold BCA Protein Assay Kit	Thermo Scientific	PIA53226	Protein quantificaiton
Pierce Silver Stain Kit	Thermo Scientific	24612	Protein detection
PMSF Protease Inhibitor	Thermo Scientific	36978	Protease inhibitor
Polyacrylamide Gel	Made in-house	N/A	Protein gel
Sf9 insect cells	ATCC	CRL-1711	Insect cell line for baculovirus expression
Slide-A-Lyzer MINI Dialysis Device, 10K MWCO	Thermo Scientific	69572	Protein dialysis
Sodium acetate	Sigma-Aldrich	S8750	DNA precipitation
Sodium Chloride	Fischer	BP358-1	General buffers
Sodium Dodecyl Sulfate (SDS)	Sigma-Aldrich	L3771	General buffers
Trypan Blue Solution (0.4%)	Thermo Scientific	15250061	Nuclei staining