SUZ12 immunoprecipitation followed by protein indentification via mass spectrometry (SUZ12 IP-MS) in mESCs of SUZ12 WT, delta exon 4, KO, and KO rescues with SUZ12-L and SUZ12-S
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ABSTRACT: Suz12 exon 4 encodes 23 amino acids (aa 129–152 in SUZ12-L) that partially overlap with the WD-binding domain 1 (WDB1, 110–145). We reasoned that exon 4 skipping might alter the structure of SUZ12 and, possibly, PRC2 composition. To explore this possibility, we generated ESCs that lack the Suz12 exon 4 via CRISPR-Cas9–induced deletion. In addition, to rule out any biases due to the expression levels and/or SUZ12 epitope masking, we generated Suz12 knockout (KO) ESCs (herein, KO) in which Suz12 expression was subsequently rescued by re-introducing either the Suz12-L or Suz12-S mouse isoform fused to a triple-Flag tag under the regulation of a CAG promoter (KO+L/S; Figures S2H–S2K). We performed SUZ12 immunoprecipitation coupled with mass spectrometry (IP-MS) in the WT and ∆ex4 clones to compare their interactomes and with a flag antibody in the KO and rescue cell lines. As expected, no peptides corresponding to exon 4 were retrieved in ∆ex4 samples, while the rest of the sequence displayed similar coverage. Comparison of interactors in the two conditions revealed that SUZ12 binding to AEBP2 and JARID2 was strongly reduced in ∆ex4 cells with respect to WT cells, whereas SUZ12 binding to most core components or to PRC2.1-specific factors was unchanged or only slightly increased . These observations were confirmed by SUZ12 IP followed by Western blot (WB). Flag IP-MS in rescue cells confirmed that, while the long isoform was able to correctly form comparable amounts of both PRC2.1 and PRC2.2 subtypes, interaction of the SUZ12-S with PRC2.2-specific factors was drastically reduced.
INSTRUMENT(S): LTQ Orbitrap Velos Pro
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Stem Cell
SUBMITTER: Niccolò Arecco
LAB HEAD: Niccolò Arecco
PROVIDER: PXD046660 | Pride | 2024-03-18
REPOSITORIES: Pride
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