Epi-Decoder: Decoding the Local Proteome of a Genomic Locus by Massive Parallel Chromatin Immunoprecipitation Combined with DNA-Barcode Sequencing

Breugel, Maria Elize van; Leeuwen, Fred van

doi:10.1007/978-1-0716-2140-0_8

Cited by 3 publications

(1 citation statement)

References 31 publications

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“…An innovative report described a method to determine proteome composition at a specific genomic locus in yeast utilizing protein-based purification and a sequencing readout [144]. This technique, Epi-Decoder, relies on crossing yeast strains in which individual proteins are modified with epitope tags with strains in which the genomic region under study has unique DNA barcodes incorporated (Figure 4e) [145]. The pooled libraries are subjected to conventional ChIP-Seq with an antibody against the common epitope tag.…”

Section: Epi-decodermentioning

confidence: 99%

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome

MacKenzie

Cisneros

Maynard

et al. 2023

Cells

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A phenotypic hallmark of cancer is aberrant transcriptional regulation. Transcriptional regulation is controlled by a complicated array of molecular factors, including the presence of transcription factors, the deposition of histone post-translational modifications, and long‑range DNA interactions. Determining the molecular identity and function of these various factors is necessary to understand specific aspects of cancer biology and reveal potential therapeutic targets. Regulation of the genome by specific factors is typically studied using chromatin immunoprecipitation followed by sequencing (ChIP‑Seq) that identifies genome-wide binding interactions through the use of factor-specific antibodies. A long-standing goal in many laboratories has been the development of a ‘reverse-ChIP’ approach to identify unknown binding partners at loci of interest. A variety of strategies have been employed to enable the selective biochemical purification of sequence-defined chromatin regions, including single-copy loci, and the subsequent analytical detection of associated proteins. This review covers mass spectrometry techniques that enable quantitative proteomics before providing a survey of approaches toward the development of strategies for the purification of sequence-specific chromatin as a ‘reverse-ChIP’ technique. A fully realized reverse-ChIP technique holds great potential for identifying cancer-specific targets and the development of personalized therapeutic regimens.

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Section: Epi-decodermentioning

confidence: 99%

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome

MacKenzie

Cisneros

Maynard

et al. 2023

Cells

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Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly

van Breugel,

van Kruijsbergen,

Mittal

et al. 2023

Molecular Cell

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A chromatin-associated regulator of RNA Polymerase III assembly at tRNA genes revealed by locus-specific proteomics

Breugel

Kruijsbergen

Mittal

et al. 2023

Preprint

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Transcription of tRNA genes by RNA Polymerase III (RNAPIII) is tightly regulated by signaling cascades in response to nutrient availability. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulatory factors have not been described. For that reason, we decoded the proteome of a single native tRNA gene locus in yeast. We observed dynamic reprogramming of the core RNAPIII transcription machinery upon nutrient perturbation. In addition, we identified Fpt1, a protein of unknown function. Fpt1 uniquely occupied tRNA genes but its occupancy varied and correlated with the efficiency of RNAPIII eviction upon nutrient perturbation. Decoding the proteome of a tRNA gene in the absence of Fpt1 revealed that Fpt1 promotes eviction of RNAPIII. Cells without Fpt1 also showed impaired shutdown of ribosome biogenesis genes upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and for tuning an integrated physiological response to changing metabolic demands.

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Epi-Decoder: Decoding the Local Proteome of a Genomic Locus by Massive Parallel Chromatin Immunoprecipitation Combined with DNA-Barcode Sequencing

Cited by 3 publications

References 31 publications

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome

Reverse-ChIP Techniques for Identifying Locus-Specific Proteomes: A Key Tool in Unlocking the Cancer Regulome

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly

A chromatin-associated regulator of RNA Polymerase III assembly at tRNA genes revealed by locus-specific proteomics

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