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Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

ABSTRACT: PPIs play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spec-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic acitivty a,d expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleavage mutant to allow the use of the POI in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we build a GOlden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A split-link design, we applied it to identify PPIs of GR, NCAP, and NSP7 by TurboID proximity labeling proteomics. Our results demisntrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Louis Delhaye

LAB HEAD: Sven Eyckerman

PROVIDER: PXD046412 | Pride | 2024-06-10

REPOSITORIES: Pride

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			Action	DRS
	B102794_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-183-1.raw	Raw
	B102794_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-183-2.raw	Raw
	B102796_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-184-1.raw	Raw
	B102796_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-184-2.raw	Raw
	B102798_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-187-1.raw	Raw

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Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

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Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

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