HIRA complex deposition of histone H3.3 is driven by histone tetramerization and histone-DNA binding.

The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in the deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.

Structure of the Hir histone chaperone complex.

The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4)2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4)2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.

Interneurons Regulate Locomotion Quiescence via Cyclic Adenosine Monophosphate Signaling During Stress-Induced Sleep in Caenorhabditis elegans.

Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A. C. elegans sleep in response to cellular stress caused by environmental insults [stress-induced sleep (SIS)], a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allowing for cellular dissection of cAMP signaling during sleep. We employed a red-light activated adenylyl cyclase, IlaC22, to identify cells involved in SIS regulation. We found that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein. Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic overexpression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Overexpression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.

Molecular basis for chromatin assembly and modification by multiprotein complexes.

Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post-translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.

EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data.

Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family .