Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF AD) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF AD-histone octamer complex shows that APLF AD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF AD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted

Histone interaction landscapes visualized by crosslinking mass spectrometry in intact cell nuclei

Cells organize their actions partly through tightly controlled protein-protein interactions - collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry

Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry

Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation

Histone interaction landscapes visualized by crosslinking mass spectrometry in intact cell nuclei

Cells organize their actions partly through tightly controlled protein-protein interactions - collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF$^{AD}$) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF$^{AD}$-histone octamer complex shows that APLF$^{AD}$ tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF$^{AD}$ affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted