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PLAMseq enables the simultaneous proteo-genomic characterization of chromatin-associated proteins and protein interactions in a single workflow.

ABSTRACT: Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP) assays are the most common approaches to characterize the genomic localization and protein interactors, respectively for a protein of interest. However, these approaches require the use of specific antibodies, which are costly reagents that often face sensitivity and specificity issues. Based on TurboID, we developed PLAMseq (Proximity Labelled Affinity-purified Mass spectrometry plus sequencing), after a short biotin pulse, DNA-protein crosslinks are induced by formaldehyde and the interactors of a protein of interest together with their associated DNA sequences are purified simultaneously and identified by mass spectrometry-based proteomics and Next Generation Sequencing, respectively. To validate PLAMseq, we performed the proteo-genomic characterization of two proteins which genomic loci are very well characterized, namely, RNA polymerase II and CTCF with excellent robustness and reproducibility. Next, we applied PLAMseq to characterize Histone H1 SUMOylation, a histone post-translational modification which study has remained elusive due to the lack of specific reagents. SETDB1 binds to SUMOylated histone H1 and, accordingly, SUMOylated histone H1 colocalize with H3K9me3 at repetitive regions of the genome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE294161 | GEO | 2025/11/07

REPOSITORIES: GEO

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Similar Datasets

PLAMseq enables the proteo-genomic characterization of chromatin-associated proteins and protein interactions in a single workflow.

Project description:Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP) assays are the most common approaches to characterize the genomic localization and protein interactors, respectively, for a protein of interest. However, these approaches require the use of specific antibodies, which are costly reagents that often face sensitivity and specificity issues. Based on TurboID, we developed PLAMseq (Proximity Labelled Affinity-purified Mass spectrometry plus sequencing), which enables, in the same workflow, to identify the genomic loci and the interacting proteome of a protein of interest. Moreover, PLAMseq can also be applied to specifically map protein interactions across the genome or to specifically detect proteins which are ubiquitin(-like) modified. After a short biotin pulse, DNA-protein crosslinks are induced by formaldehyde and the interactors of a protein of interest, together with their associated DNA sequences, are purified simultaneously and identified by mass spectrometry-based proteomics and Next Generation Sequencing, respectively.To validate PLAMseq, we performed the proteo-genomic characterization of two proteins which genomic loci are very well characterized, namely, RNA polymerase II and CTCF, with excellent robustness and reproducibility. Next, we applied PLAMseq to characterize Histone H1 SUMOylation, a histone post-translational modification which study has remained elusive due to the lack of specific reagents. We found that SETDB1 binds to SUMOylated histone H1.2 and H1.4 and, accordingly, SUMOylated histone H1 colocalizes with H3K9me3 at repetitive regions of the genome.

2025-10-24 | PXD062203 | Pride

Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

Project description:The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest.

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The interactome of site-specifically acetylated linker histone H1

Project description:Linker histone H1 plays a key role in chromatin organization and maintenance, however, our knowledge of the regulation of H1 functions by its posttranslational modifications (PTMs) is very limited. In this study, we report on the generation of homogeneously and site-specifically mono- and di-acetylated H1 (H1 Ac) using genetic code expansion. We used these modified histones to identify and comprehensively characterize the acetylation-dependent cellular interactome for linker histone H1 and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Intriguingly, H1 acetylation-specific interactors comprise translational initiation factors and are involved in transcriptional regulation, suggesting that acetylation of H1 may indeed act as a regulator of the linker histone H1 by modulation of protein-protein interactions.

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The dynamic interactome and genomic targets of Polycomb complexes during stem cell differentiation

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2025-09-11 | PXD068298 |

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A novel proteomic approach reveals GREB1 as an Estrogen Receptor co-factor

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