Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

BackgroundRecognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches.ResultsThe PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA.ConclusionConcerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.

Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

Cells organize their actions partly through tightly controlled protein-protein interactionscollectively 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.

The genomes of all organisms throughout the tree of life are compacted and organized in chromatin by association of chromatin proteins. Eukaryotic genomes encode histones, which are assembled on the genome into octamers, yielding nucleosomes. Post-translational modifications of the histones, which occur mostly on their N-terminal tails, define the functional state of chromatin. Like eukaryotes, most archaeal genomes encode histones, which are believed to be involved in the compaction and organization of their genomes. Instead of discrete multimers, in vivo data suggest assembly of “nucleosomes” of variable size, consisting of multiples of dimers, which are able to induce repression of transcription. Based on these data and a model derived from X-ray crystallography, it was recently proposed that archaeal histones assemble on DNA into “endless” hypernucleosomes. In this review, we discuss the amino acid determinants of hypernucleosome formation and highlight differences with the canonical eukaryotic octamer. We identify archaeal histones differing from the consensus, which are expected to be unable to assemble into hypernucleosomes. Finally, we identify atypical archaeal histones with short N- or C-terminal extensions and C-terminal tails similar to the tails of eukaryotic histones, which are subject to post-translational modification. Based on the expected characteristics of these archaeal histones, we discuss possibilities of involvement of histones in archaeal transcription regulation.

Leukocyte-associated immunoglobulinlike receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagenbinding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagenbinding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis. (Blood.

Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone–DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous end-joining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLFAD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLFAD is intrinsically disordered and binds histone complexes (H3-H4)2 and H2A-H2B specifically and with high affinity. APLFAD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLFAD histone binding domain uses two aromatic side chains to anchor to the α1–α2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLFAD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.