DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication

Mattiroli, Francesca; Gu, Yajie; Yadav, Tejas; Balsbaugh, Jeremy L.; Harris, Michael; Findlay, Eileen S; Liu, Yang; Radebaugh, Catherine A.; Stargell, Laurie A.; Ahn, Natalie G.; Whitehouse, Iestyn; Luger, Karolin

doi:10.7554/elife.22799

Cited by 83 publications

(134 citation statements)

References 62 publications

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“…3c). This was confirmed when we measured nucleosome assembly activity of the tCAF-1 subcomplexes in a quantitative nucleosome assembly (NAQ) assay37 (Fig. 3d).…”

Section: Resultssupporting

confidence: 65%

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

Mattiroli

Balsbaugh

et al. 2017

Sci Rep

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Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

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“…3c). This was confirmed when we measured nucleosome assembly activity of the tCAF-1 subcomplexes in a quantitative nucleosome assembly (NAQ) assay37 (Fig. 3d).…”

Section: Resultssupporting

confidence: 65%

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

Mattiroli

Balsbaugh

et al. 2017

Sci Rep

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“…CAF-1 is a heterotrimeric chaperone composed of p150, p60 and RbAp48 (also known as p48) subunits, also known as Cac1, Cac2, and Cac3 in yeast, respectively. Early studies suggested that human CAF-1 binds to and deposits H3.1/H4 dimers in vivo [23], however more recent evidence suggests that yeast CAF-1 forms tetramers of two H3/H4 dimers and deposits the tetrameric form onto DNA [154,155], though the mechanisms of tetramerization in the human protein remains an active area of research. The Cac1/p150 subunit is largely disordered and contains a large acidic stretch (the glutamate/aspartate or ED domain) in the middle of the protein sequence (Figure 3).…”

Section: Introductionmentioning

confidence: 99%

Fly Fishing for Histones: Catch and Release by Histone Chaperone Intrinsically Disordered Regions and Acidic Stretches

Warren

Shechter

2017

Journal of Molecular Biology

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Chromatin is the complex of eukaryotic DNA and proteins required for the efficient compaction of the nearly two-meter long human genome into a roughly ten-micron diameter cell nucleus. The fundamental repeating unit of chromatin is the nucleosome: 147bp of DNA wrapped about an octamer of histone proteins. Nucleosomes are stable enough to organize the genome yet must be dynamically displaced and reassembled to allow access to the underlying DNA for transcription, replication, and DNA damage repair. Histone chaperones are a non-catalytic group of proteins that are central to the processes of nucleosome assembly and disassembly, and thus the fluidity of the ever-changing chromatin landscape. Histone chaperones are responsible for binding the highly basic histone proteins, shielding them from non-specific interactions, facilitating their deposition onto DNA, and aiding in their eviction from DNA. Though most histone chaperones perform these common functions, recent structural studies of many different histone chaperones reveal that there are few commonalities in their folds. Importantly, sequence-based predictions show that histone chaperones are highly enriched in intrinsically disordered regions (IDRs) and acidic stretches. In this review, we focus on the molecular mechanisms underpinning histone binding, selectivity, and regulation of these highly dynamic protein regions. We highlight new evidence suggesting that IDRs are often critical for histone chaperone function and play key roles in chromatin assembly and disassembly pathways.

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“…Data normalization to a loading control DNA allows us to compare and accurately quantify the amount of nucleosomes formed in different samples. The NAQ assay has been successfully used to measure the activity of the chromatin assembly factor 1 (CAF-1) in vitro (Mattiroli et al ., 2017a and 2017b), and has the potential to reveal the differential contribution of histone chaperones to nucleosome assembly in cells. This will pave the way for the complete understanding of their functional roles in nucleosome dynamics.…”

Section: Introductionmentioning

confidence: 99%

Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay

Mattiroli

Luger

2018

BIO-PROTOCOL

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Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack of quantitative assays. Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation in vitro. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of ~150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins in vivo.

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DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication

Cited by 83 publications

References 62 publications

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

Fly Fishing for Histones: Catch and Release by Histone Chaperone Intrinsically Disordered Regions and Acidic Stretches

Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay

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