Regulation of histone acetylation is fundamental to the utilization of eukaryotic genomes in chromatin. Aberrant acetylation contributes to disease and can be clinically combated by inhibiting the responsible enzymes. Our knowledge of the histone acetylation system is patchy because we so far lacked the methodology to describe acetylation patterns and their genesis by integrated enzyme activities. We devised a generally applicable, mass spectrometry-based strategy to precisely and accurately quantify combinatorial modification motifs. This was applied to generate a comprehensive inventory of acetylation motifs on histones H3 and H4 in Drosophila cells. Systematic depletion of known or suspected acetyltransferases and deacetylases revealed specific alterations of histone acetylation signatures, established enzyme-substrate relationships, and unveiled an extensive crosstalk between neighboring modifications. Unexpectedly, overall histone acetylation levels remained remarkably constant upon depletion of individual acetyltransferases. Conceivably, the acetylation level is adjusted to maintain the global charge neutralization of chromatin and the stability of nuclei.

Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. Here, we assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, we find that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, we decreased the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau. We find that these targeted interventions both alleviate the observed aging-associated changes and promote longevity. Our findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span.

Transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila males is brought about by a ribonucleoprotein assembly called Male-Specific-Lethal or Dosage Compensation Complex (MSL-DCC). This machinery is formed in male flies and specifically associates with active genes on the X chromosome. After assembly at dedicated high-affinity ''entry'' sites (HAS) on the X chromosome, the complex distributes to the nearby active chromatin. High-resolution, genome-wide mapping of the MSL-DCC subunits by chromatin immunoprecipitation (ChIP) on oligonucleotide tiling arrays suggests a rather homogenous spreading of the intact complex onto transcribed chromatin. Coupling ChIP to deep sequencing (ChIP-seq) promises to map the chromosomal interactions of the DCC with improved resolution. We present ChIP-seq binding profiles for all complex subunits, including the first description of the RNA helicase MLE binding pattern. Exploiting the preferential representation of direct chromatin contacts upon highenergy shearing, we report a surprising functional and topological separation of MSL protein contacts at three classes of chromosomal binding sites. Furthermore, precise determination of DNA fragment lengths by paired-end ChIP-seq allows decrypting of the local complex architecture. Primary contacts of MSL-2 and MLE define HAS for the DCC. In contrast, association of the DCC with actively transcribed gene bodies is mediated by MSL-3 binding to nucleosomes. We identify robust MSL-1/MOF binding at a fraction of active promoters genome-wide. Correlation analyses suggest that this association reflects a function outside dosage compensation. Our comprehensive analysis provides a new level of information on different interaction modes of a multiprotein complex at distinct regions within the genome.

Loss of cellular homeostasis during aging results in altered tissue functions and leads to a general decline in fitness and, ultimately, death. As animals age, the control of gene expression, which is orchestrated by multiple epigenetic factors, degenerates. In parallel, metabolic activity and mitochondrial protein acetylation levels also change. These two hallmarks of aging are effectively linked through the accumulating evidence that histone acetylation patterns are susceptible to alterations in key metabolites such as acetyl-CoA and NAD(+), allowing chromatin to function as a sensor of cellular metabolism. In this review we discuss experimental data supporting these connections and provide a context for the possible medical and physiological relevance.

SUMMARY The functionality of stem cells declines during aging thereby contributing to aging-associated impairments in tissue regeneration and function1. Alterations in developmental pathways have been associated with declines in stem cell function during aging2–6 but the nature of this process remains poorly defined. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in aging7,8. This study identifies an altered epigenetic stress response in muscle stem cells (also known as satellite cells = SCs) of aged compared to young mice. This includes aberrant global and site-specific induction of active chromatin marks in activated SCs from aged mice resulting in the specific induction of Hoxa9 among all Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor separating gene expression of SCs from aged compared to young mice. This includes most of the currently known inhibitors of SC function in aging muscle such as Wnt-, TGFß-, JAK/STAT- and senescence signaling2–4,6. Inhibition of aberrant chromatin activation or deletion of Hoxa9 suffices to improve SC function and muscle regeneration in aged mice, while overexpression of Hoxa9 mimics aging-associated defects in SCs from young mice, which can be rescued by inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated SCs from aged mice, which limits SC function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

The H4K16 acetyltransferase MOF plays a crucial role in dosage compensation in Drosophila but has additional, global functions. We compared the molecular context and effect of MOF in male and female flies, combining chromosome-wide mapping and transcriptome studies with analyses of defined reporter loci in transgenic flies. MOF distributes dynamically between two complexes, the dosage compensation complex and a complex containing MBD-R2, a global facilitator of transcription. These different targeting principles define the distribution of MOF between the X chromosome and autosomes and at transcription units with 5' or 3' enrichment. The male X chromosome differs from all other chromosomes in that H4K16 acetylation levels do not correlate with transcription output. The reconstitution of this phenomenon at a model locus revealed that the activation potential of MOF is constrained in male cells in the context of the DCC to arrive at the 2-fold activation of transcription characteristic of dosage compensation.

The MOF (males absent on the first)-containing NSL (non-specific lethal) complex binds to a subset of active promoters in Drosophila melanogaster and is thought to contribute to proper gene expression. The determinants that target NSL to specific promoters and the circumstances in which the complex engages in regulating transcription are currently unknown. Here, we show that the NSL complex primarily targets active promoters and in particular housekeeping genes, at which it colocalizes with the chromatin remodeler NURF (nucleosome remodeling factor) and the histone methyltransferase Trithorax. However, only a subset of housekeeping genes associated with NSL are actually activated by it. Our analyses reveal that these NSL-activated promoters are depleted of certain insulator binding proteins and are enriched for the core promoter motif ‘Ohler 5’. Based on these results, it is possible to predict whether the NSL complex is likely to regulate a particular promoter. We conclude that the regulatory capacity of the NSL complex is highly context-dependent. Activation by the NSL complex requires a particular promoter architecture defined by combinations of chromatin regulators and core promoter motifs.

Chromatin modifications regulate genome function by recruiting protein factors to the genome. However, the protein composition at distinct chromatin modifications remains to be fully characterized. Here, we use natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 a H3K27 residues. We first demonstrate their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localisation, genomic distribution and histone modification–binding preference. By fusing eCRs to the biotin ligase BASU, we establish ChromID, a method for identifying the chromatin-dependent protein interactome based on proximity biotinylation, and apply it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncover the protein composition at bivalent promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.