Abstract
Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes. By utilizing the buoyant density of DNA–protein complexes after cross-linking, we here develop a mass spectrometry-based approach to quantify the chromatin-associated proteome at separate stages of the cell cycle. While epigenetic modifiers that promote transcription are lost from mitotic chromatin, repressive modifiers generally remain associated. Furthermore, while proteins involved in transcriptional elongation are evicted, most identified transcription factors are retained on mitotic chromatin to varying degrees, including core promoter binding proteins. This predicts conservation of the regulatory landscape on mitotic chromosomes, which we confirm by genome-wide measurements of chromatin accessibility. In summary, this work establishes an approach to study chromatin, provides a comprehensive catalog of chromatin changes during the cell cycle, and reveals the degree to which the genomic regulatory landscape is maintained through mitosis.
Highlights
Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes
To enrich for chromatin-bound proteins, we adapted a strategy based on the distinct buoyant density of cross-linked DNA–protein complexes in a cesium chloride (CsCl) gradient[21,22]
We show that binding differences can be analyzed comprehensively using biophysical separation of formaldehyde cross-linked protein–DNA complexes combined with quantitative proteomics
Summary
Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes. While proteins involved in transcriptional elongation are evicted, most identified transcription factors are retained on mitotic chromatin to varying degrees, including core promoter binding proteins. This predicts conservation of the regulatory landscape on mitotic chromosomes, which we confirm by genome-wide measurements of chromatin accessibility. Selected factors are thought to be retained and index particular genomic regions to facilitate transcriptional activation upon mitotic exit This concept is potentially relevant for maintenance of cellular identity and is termed mitotic bookmarking[6]. We have termed the method density-based enrichment for mass spectrometry analysis of chromatin (DEMAC), and utilize it to quantify changes in the chromatin-bound proteome (chromatome) across G1-, S-, and M-phase of the human cell cycle. In addition to providing a rich dataset of chromatin composition during the cell cycle, our results reveal pathwayspecific retention of chromatin modifiers on mitotic chromosomes, including a widespread retention of TFs
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