PXD040771-1

PXD040771 is an original dataset announced via ProteomeXchange.

Title	SETD2 N-terminus mediates interaction with Lamins
Description	Histone-Lysine N-methyltransferase SETD2 is an evolutionarily conserved histone methyltransferase that associates with RNA Polymerase II (RNAPII) and catalyzes trimethylation of histone H3 lysine 36 (H3K36me3) in transcribed regions. Interestingly, SETD2 is frequently mutated in cancers. Clear cell renal cell carcinomas (ccRCCs) have extensive mutation of SETD2, implicating SETD2 as a tumor suppressor. ccRCCs are known for early driving events where the 3p chromosomal arm is lost, deleting one copy of SETD2, resulting in haploinsufficiency. While the role of SETD2 mediated H3K36me3 and interaction of RNAPII has been the focus of many studies, haploinsufficient SETD2 tumors retain H3K36me3, yet have drastic genome stability defects. The C-terminus of SETD2 is required for H3K36me3 while the N-terminus of SETD2 remains poorly characterized compared to the C-terminus. The N-terminus is highly disordered and has recently been shown to contribute to SETD2 stability, implicating regulatory functions. To better understand how SETD2 contributes to chromatin biology, we first sought to identify SETD2 protein interactors. A stable, immortalized normal human kidney epithelial cell (HKC) line with a dox inducible SETD2-APEX2 construct was used for a proximity-based biotinylation assay. Initial analysis yielded 2083 biotinylated proteins with significant enrichment (log2 fold change > 2 p-value < 0.05) in SETD2-APEX2 compared to control. Gene Ontology analysis of these interactors revealed enrichment of proteins involved in nuclear envelope functions, including lamin A/C, lamin B1, lamin B2 and emerin. These interactions were confirmed via western and reciprocal immunoprecipitation (IP). To determine effect of SETD2 depletion on lamin A/C and lamin B1 phosphorylation, we performed lamin A/C or lamin B1 IP from control or SETD2-knockdown (KD) cells and interrogated via LC-MS/MS. Mass spectrometry analysis revealed a significant decrease in lamin A/C S22 phosphorylation, and modest decrease in S390 phosphorylation, in SETD2 depleted cells compared to controls. While lamin B1 S23 phosphorylation was detected in control cells, we did not detect it in SETD2-KD cells, indicating a significant reduction in S23 phosphorylation upon SETD2 depletion. Next, we sought to identify the effect of SETD2 N-terminal deletion on the SETD2 protein interactome. We compared the protein interactors of dox inducible WT-SETD2 APEX2 fusion to an N-terminal deletion mutant of SETD2 (tSETD2-APEX2). Deletion of the N-terminus resulted in loss of lamin B1 and emerin interaction. Intriguingly, tSETD2 selectively associated with lamin C, whereas WT SETD2 interacted with both lamin A and C. These data together reveal an interaction with SETD2 and nuclear lamins. Further experiments revealed a role for the SETD2 N-terminus in facilitating the association of lamins with CDK1, thereby promoting lamin phosphorylation and depolymerization required for nuclear envelope disassembly during mitosis. Together, the data reveal a non-catalytic role of SETD2 during mitosis.
HostingRepository	PRIDE
AnnounceDate	2025-06-04
AnnouncementXML	Submission_2025-06-04_05:42:25.666.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Christine Mills
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Q Exactive HF; Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-03-10 06:36:33	ID requested
⏵ 1	2025-06-04 05:42:26	announced

Dataset with its publication pending

submitter keyword: SETD2, CDK1, mitosis, lamin

Brian Strahl
contact affiliation	Department of Biochemistry and Biophysics, RNA Discovery Center, and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill
contact email	brian_strahl@med.unc.edu
lab head
Christine Mills
contact affiliation	University of North Carolina - Chapel Hill
contact email	allie.mills@unc.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD040771
     1. Label: PRIDE project
     2. Name: SETD2 N-terminus mediates interaction with Lamins