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A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

ABSTRACT: Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Schneider-2 Cell

SUBMITTER: Richard Imre

LAB HEAD: Alexander Stark

PROVIDER: PXD048161 | Pride | 2024-11-13

REPOSITORIES: Pride

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			Action	DRS
	20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep1.raw	Raw
	20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep2.raw	Raw
	20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_sh_rep3.raw	Raw
	20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_wt_rep1.raw	Raw
	20201012_Lumos_RSLC10_Reiter_Stark_IMP_onbead_10per_DLM3_wt_rep2.raw	Raw

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Project description:The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.

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A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

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