End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

Satoru Ide,Jerome Dejardin

doi:10.1038/ncomms7674

Abstract

The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals.

Highlights

The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes
As the regulation of ribosomal RNA transcription is deeply linked to the proliferation of cancer cells, we used end-targeting PICh (ePICh) to purify an B1-kb segment of chromatin from the ribosomal RNA gene promoter in order to identify factors that are important for proliferation
We identify more than 100 promoter-associated proteins, including most known ribosomal RNA (rRNA) promoter interactors. ePICh identified zinc finger protein 106 (ZFP106) at the rRNA promoter and we show that this protein plays an important role in pre-rRNA synthesis by recruiting the RNA polymerase I machinery on the promoter region

Summary

Introduction

The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. Defects in chromatin regulation have usually profound consequences on cellular identity or on the proliferative potential How these diverse types of players function at specific genomic regions and how their activities are controlled and integrated to drive biological reactions are critical questions that remain difficult to address experimentally. Recent reports have described the identification of novel components after the affinity purification of specific chromatin proteins[7,8,9] These approaches do not target loci directly but rather have relied on the use of antibodies to immunoprecipitate adaptor or endogenous proteins, and purify protein complexes dissociated from the DNA or multiple loci bound by the target factor. EPICh allows the unbiased identification of new proteins bound in vivo to endogenous mammalian regulatory elements with complex sequences

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Conclusion