HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae

Guillen-Ahlers, Hector; Rao, Prahlad K.; Levenstein, Mark E.; Kennedy‐Darling, Julia; Perumalla, Danu S.; Jadhav, Avinash Y.L.; Glenn, Jeremy P.; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, María; Göring, Harald H.H.; Anderson, Corianna D.; Scalf, Mark; Gildersleeve, Heidi; Cole, Regina; Greene, A; Oduro, Akua K.; Lazarova, Katarina; Cesnik, Anthony J.; Barfknecht, Jared; Cirillo, Lisa Ann; Gasch, Audrey P.; Shortreed, Michael R.; Smith, Lloyd M.; Olivier, Michael

doi:10.1016/j.ygeno.2016.05.002

Cited by 12 publications

(16 citation statements)

References 25 publications

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“…Important progress has been made for multi-copy DNA loci such as repetitive DNA elements [7][8] . Recently, oligo-capture 44 and CRISPR-Cas9 technology 45 have been applied to identify proteins at single copy genomic loci. However, the problem of assessing a unique locus in a comprehensive and quantitative manner by mass spectrometry has not been solved and many challenges remain, a major one being the high degree of purification of the locus of interest while obtaining sufficient material for good coverage in MS analysis 6 .…”

Section: Discussionmentioning

confidence: 99%

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Korthout

Poramba‐Liyanage

Verzijlbergen

et al. 2017

Preprint

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Transcription, replication and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-ChIPBarcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

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Section: Discussionmentioning

confidence: 99%

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Korthout

Poramba‐Liyanage

Verzijlbergen

et al. 2017

Preprint

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“…Novel findings on Pab1 functions are continuously emerging, indicating that the role of this protein in cell physiology could be larger than expected. For example, chromatin immunoprecipitation assays revealed that Pab1 binds DNA at the ENO2 and GAL1 promoter and that the carbon source can alter Pab1 enrichment at the promoter site (Guillen‐Ahlers et al, ). This result suggests a possible role of Pab1 in the regulation of transcription and/or in the epigenetic control of chromatin function (Byrum, Raman, Taverna, & Tackett, ; Guillen‐Ahlers et al, ).…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology

Brambilla

Martani

Bertacchi

et al. 2018

Yeast

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Pab1, the major poly (A) binding protein of the yeast Saccharomyces cerevisiae, is involved in many intracellular functions associated with mRNA metabolism, such as mRNA nuclear export, deadenylation, translation initiation and termination. Pab1 consists of four RNA recognition motifs (RRM), a proline‐rich domain (P) and a carboxy‐terminal (C) domain. Due to its modular structure, Pab1 can simultaneously interact with poly (A) tails and different proteins that regulate mRNA turnover and translation. Furthermore, Pab1 also influences cell physiology under stressful conditions by affecting the formation of quinary assemblies and stress granules, as well as by stabilizing specific mRNAs to allow translation re‐initiation after stress. The main goal of this review is to correlate the structural complexity of this protein with the multiplicity of its functions.

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“…We have used an approach known as HyCCAPP, or hybridization capture of chromatin-associated proteins for proteomics. 23,24 Briefly, human K562 cells are cross-linked using formaldehyde to fix protein-DNA interactions, then cells are lysed and chromatin is sheared. The lysate is incubated with biotinylated capture oligonucleotides, whose sequences are designed to complement the human alpha satellite consensus sequence, 25 and hybridized complexes are isolated using streptavidin-coated magnetic beads.…”

Section: Introductionmentioning

confidence: 99%

“…HyCCAPP has proven to be a useful tool, revealing both known as well as novel protein-DNA interactions in yeast. 23,24 Here, we apply HyCCAPP to the human genome for the first time, providing a DNA-centric lens through which to view protein-alphoid DNA interactions. Using this approach, 90 proteins were identified as enriched at the alpha satellite repeats, including many known centromere-binding proteins in addition to a number of novel alpha satellite-binding proteins, which may provide new insights into centromere structure or function.…”

Section: Introductionmentioning

confidence: 99%

Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

Buxton¹,

Kennedy‐Darling²,

Shortreed³

et al. 2017

J. Proteome Res.

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The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.

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HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae

Cited by 12 publications

References 25 publications

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology

Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

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