DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network.

A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.

A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates.

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

Phosphorylation of Schizosaccharomyces pombe Dss1 mediates direct binding to the ubiquitin-ligase Dma1 in vitro.

Intrinsically disordered proteins (IDPs) are often multifunctional and frequently posttranslationally modified. Deleted in split hand/split foot 1 (Dss1-Sem1 in budding yeast) is a highly multifunctional IDP associated with a range of protein complexes. However, it remains unknown if the different functions relate to different modified states. In this work, we show that Schizosaccharomyces pombe Dss1 is a substrate for casein kinase 2 in vitro, and we identify three phosphorylated threonines in its linker region separating two known disordered ubiquitin-binding motifs. Phosphorylations of the threonines had no effect on ubiquitin-binding but caused a slight destabilization of the C-terminal α-helix and mediated a direct interaction with the forkhead-associated (FHA) domain of the RING-FHA E3-ubiquitin ligase defective in mitosis 1 (Dma1). The phosphorylation sites are not conserved and are absent in human Dss1. Sequence analyses revealed that the Txx(E/D) motif, which is important for phosphorylation and Dma1 binding, is not linked to certain branches of the evolutionary tree. Instead, we find that the motif appears randomly, supporting the mechanism of ex nihilo evolution of novel motifs. In support of this, other threonine-based motifs, although frequent, are nonconserved in the linker, pointing to additional functions connected to this region. We suggest that Dss1 acts as an adaptor protein that docks to Dma1 via the phosphorylated FHA-binding motifs, while the C-terminal α-helix is free to bind mitotic septins, thereby stabilizing the complex. The presence of Txx(D/E) motifs in the disordered regions of certain septin subunits may be of further relevance to the formation and stabilization of these complexes.

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome.

Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.