Dataset Information

SUZ12 immunoprecipitation followed by PRM of SUZ12 peptides to identify alternatively spliced isoforms of SUZ12 at the protein level.

ABSTRACT: Alternative splicing of SUZ12 exon 4 is predicted to generate two alternative isoforms differing by the inclusion or exclusion of 69 nucleotides in exon 4, which we named SUZ12-long (SUZ12-L) and SUZ12-short (SUZ12-S) respectively. At the protein level 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). To detect the existance of SUZ12-S at the protein levels we performed SUZ12 immunoprecipitation coupled with mass spectrometry (IP-MS) in the WT and ∆ex4 clones to identify SUZ12-S unique peptides. We identified a SUZ12-S specific peptide in both WT #2 and ∆ex4 #1. These observations were confirmed by SUZ12 IP followed by Western blot (WB) (size shift).

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Stem Cell

SUBMITTER: Niccolò Arecco

LAB HEAD: Niccolò Arecco

PROVIDER: PXD046742 | Pride | 2024-03-18

REPOSITORIES: Pride

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Publications

Alternative splicing decouples local from global PRC2 activity.

Arecco Niccolò N Mocavini Ivano I Blanco Enrique E Ballaré Cecilia C Libman Elina E Bonnal Sophie S Irimia Manuel M Di Croce Luciano L

Molecular cell 20240306 6

The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the c ...[more]

PMID: 38452766

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Project description:Whole genome profiling of 4 histone modifications (H3K27me1, H3K27me3,H3K36me1 and H3K36me3) using ChIP-on-chip. It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons ("exon-intron marking"), linking chromatin structure and function to co- transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing.

			Action	DRS
	PRM_Target_Proteomics_SkylineOutput.xlsx	Xlsx
	SUZ121_IP_PRM_dEx4_1.raw	Raw
	SUZ12_IP_PRM_WT_2.raw	Raw
	checksum.txt	Txt