The ADP-ribosylation of proteins is an important posttranslational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.

Poly-ADP-ribosylation is a post-translational modification catalyzed by PARP enzymes with roles in transcription and chromatin biology. Here we show that distinct macrodomains, including those of histone macroH2A1.1, are recruited to sites of PARP1 activation induced by laser-generated DNA damage. Chemical PARP1 inhibitors, PARP1 knockdown and mutation of ADP-ribose-binding residues in macroH2A1.1 abrogate macrodomain recruitment. Notably, histone macroH2A1.1 senses PARP1 activation, transiently compacts chromatin, reduces the recruitment of DNA damage factor Ku70-Ku80 and alters gamma-H2AX patterns, whereas the splice variant macroH2A1.2, which is deficient in poly-ADP-ribose binding, does not mediate chromatin rearrangements upon PARP1 activation. The structure of the macroH2A1.1 macrodomain in complex with ADP-ribose establishes a poly-ADP-ribose cap-binding function and reveals conformational changes in the macrodomain upon ligand binding. We thus identify macrodomains as modules that directly sense PARP activation in vivo and establish macroH2A histones as dynamic regulators of chromatin plasticity.

To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance.

Histone macroH2A is a hallmark of mammalian heterochromatin. Here we show that human macroH2A1.1 binds the SirT1-metabolite O-acetyl-ADP-ribose (OAADPR) through its macro domain. The 1.6-A crystal structure and mutants reveal how the metabolite is recognized. Mutually exclusive exon use in the gene H2AFY produces macroH2A1.2, whose tissue distribution differs. MacroH2A1.2 shows only subtle structural changes but cannot bind nucleotides. Alternative splicing may thus regulate the binding of nicotinamide adenine dinucleotide (NAD) metabolites to chromatin.

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Assigning functions to the vast array of proteins present in eukaryotic cells remains challenging. To identify relationships between proteins, and thereby enable functional annotations of proteins, we determined changes of abundance of 10,323 human proteins in response to 294 biological perturbations using isotope-labelling mass spectrometry. We applied the machine learning algorithm treeClust to reveal functional associations between co-regulated human proteins from ProteomeHD, a compilation of our own data and datasets from the Proteomics Identifications (PRIDE) database. This produced a co-regulation map of the human proteome. Co-regulation was able to capture relationships between proteins that do not physically interact or co-localize. For example, co-regulation of the peroxisomal membrane protein PEX11β with mitochondrial respiration factors led us to discover an organelle interface between peroxisomes and mitochondria ✉

Genes are not randomly distributed in the genome. In humans, 10% of protein‐coding genes are transcribed from bidirectional promoters and many more are organised in larger clusters. Intriguingly, neighbouring genes are frequently coexpressed but rarely functionally related. Here we show that coexpression of bidirectional gene pairs, and closeby genes in general, is buffered at the protein level. Taking into account the 3D architecture of the genome, we find that co‐regulation of spatially close, functionally unrelated genes is pervasive at the transcriptome level, but does not extend to the proteome. We present evidence that non‐functional mRNA coexpression in human cells arises from stochastic chromatin fluctuations and direct regulatory interference between spatially close genes. Protein‐level buffering likely reflects a lack of coordination of post‐transcriptional regulation of functionally unrelated genes. Grouping human genes together along the genome sequence, or through long‐range chromosome folding, is associated with reduced expression noise. Our results support the hypothesis that the selection for noise reduction is a major driver of the evolution of genome organisation.

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.