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, 5, 374-378

Sequential Chromatin Immunoprecipitation to Detect SUMOylated MeCP2 in Neurons

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Sequential Chromatin Immunoprecipitation to Detect SUMOylated MeCP2 in Neurons

Tao Wu et al. Biochem Biophys Rep.

Abstract

The small ubiquitin-like modifier (SUMO) is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM). Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2) is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP) for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.

Keywords: MeCP2; SUMO; Seq-ChIP; Sequential quantitative chromatin immunoprecipitation; Small ubiquitin-like modifier; chromatin biochemistry; neurons.

Figures

Fig. 1
Fig. 1
Scheme of Sequential-Chromatin immunopreciptation (Seq-ChIP) procedure. Chromatin is prepared from neurons. After chromatin shearing, the first ChIP antibody (α-MeCP2) is immunoprecipitated followed by the addition of the second ChIP antibody (α-SUMO1). The order of the antibody addition can be reversed. The crosslinks are reversed and the target genomic regions are isolated and characterized using PCR for the presence of the SUMOylated MeCP2.
Fig. 2
Fig. 2
SUMO1 and MeCP2 are enriched at the Gamt, Mef2c, and Oprk1 promoters in mouse neurons. Chromatin was harvested from HT22 cells and quantitative chromatin immunoprecipitation (qChIP) was performed with primers flanking the guanidinoacetate methyltransferase (Gamt), the myocyte enhancer factor 2C (Mef2c), and the opioid receptor kappa 1 (Oprk1) promoters. The ChIP antibody was α-SUMO1, α-MeCP2, and the IgG control. Relative enrichment was normalized with input. Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean.
Fig. 3
Fig. 3
Sequential-ChIP reveals the occupancy of SUMOylated MeCP2 at the Gamt promoter. Chromatin was harvested from HT22 cells and sequential-chromatin immunoprecipitation (Seq-ChIP) was performed two different ways with primers flanking the guanidinoacetate methyltransferase (Gamt), the myocyte enhancer factor 2C (Mef2c), and the opioid receptor kappa 1 (Oprk1) promoters. (A) The first ChIP antibody (1) was α-MeCP2 and the second ChIP antibody (2) was either α-SUMO1 or the IgG control. (B) The first ChIP antibody (1) was α-SUMO1 and the second ChIP antibody (2) was either α-MeCP2 or the IgG control. Relative enrichment was normalized with input. Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean. ND=not detected; n.s.=not significant.

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