Chromatin enrichment for proteomics

Abstract

During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a 'snapshot' of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses.

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Figure 1: Outline of the ChEP procedure.
Figure 2: Comparison of protein enrichment after isolation of ChEP-based gelatinous chromatin and the 'classical' chromatin pellet isolated according to Shiio et al.35.
Figure 3: Typical results from ChEP-based chromatin proteomics.

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Acknowledgements

We thank F. de Lima Alves and L. Peil for their assistance with mass spectrometric analyses and N. Hegarat and H. Hochegger for testing this protocol on chicken DT40 cells. The Wellcome Trust generously supported this work through a Senior Research Fellowship to J.R. (084229), two Wellcome Trust Centre Core Grants (077707 and 092076) and an instrument grant (091020). G.K. was supported by a Federation of European Biochemical Societies (FEBS) long-term fellowship.

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Authors

Contributions

G.K. and J.R. conceived the chromatin enrichment procedure. G.K., K.L.H.W. and C.F. performed the experiments. G.K. and J.R. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Juri Rappsilber.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The effect of RNase treatment on ChEP-isolated chromatin fractions.

(a) Boxplot showing the impact of RNase digestion on various functional categories of proteins. Proteins with functions related to RNA processing (purple) are depleted by RNase treatment, presumably because they are cross-linked to chromatin indirectly via RNA. Proteins without expected chromatin function (i.e. contaminants, blue) are also somewhat reduced by RNase treatment, but proteins with canonical chromatin functions are not (red). (b) Boxplot showing that RNase treatment reduces the co-purification of ribosomes, which are typical contaminants of chromatin fractions. Note that the family of serine / arginine (SR)-rich splicing factors is not affected by RNase treatment, as these proteins generally act co-transcriptionally and are thus likely to be cross-linked directly to DNA or other chromatin factors.

Supplementary information

Supplementary Figure 1

The effect of RNase treatment on ChEP-isolated chromatin fractions. (PDF 152 kb)

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Kustatscher, G., Wills, K., Furlan, C. et al. Chromatin enrichment for proteomics. Nat Protoc 9, 2090–2099 (2014). https://doi.org/10.1038/nprot.2014.142

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