ChIP-less analysis of chromatin states

BACKGROUND: Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. RESULTS: Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. CONCLUSIONS: Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms

Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity

Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer

SummaryChromatin factors have emerged as the most frequently dysregulated family of proteins in cancer. We have previously identified the histone deacetylase SIRT6 as a key tumor suppressor, yet whether point mutations are selected for in cancer remains unclear. In this manuscript, we characterized naturally occurring patient-derived SIRT6 mutations. Strikingly, all the mutations significantly affected either stability or catalytic activity of SIRT6, indicating that these mutations were selected for in these tumors. Further, the mutant proteins failed to rescue sirt6 knockout (SIRT6 KO) cells, as measured by the levels of histone acetylation at glycolytic genes and their inability to rescue the tumorigenic potential of these cells. Notably, the main activity affected in the mutants was histone deacetylation rather than demyristoylation, pointing to the former as the main tumor-suppressive function for SIRT6. Our results identified cancer-associated point mutations in SIRT6, cementing its function as a tumor suppressor in human cancer

Racial and Socioeconomic Disparities Are More Pronounced in Inflammatory Breast Cancer Than Other Breast Cancers

Inflammatory breast cancer (IBC) is a rare yet aggressive form of breast cancer. We examined differences in patient demographics and outcomes in IBC compared to locally advanced breast cancer (LABC) and all other breast cancer patients from the Breast and Prostate Cancer Data Quality and Patterns of Care Study (POC-BP), containing information from cancer registries in seven states. Out of 7,624 cases of invasive carcinoma, IBC and LABC accounted for 2.2% (N = 170) and 4.9% (N = 375), respectively. IBC patients were more likely to have a higher number (P = 0.03) and severity (P = 0.01) of comorbidities than other breast cancer patients. Among IBC patients, a higher percentage of patients with metastatic disease versus nonmetastatic disease were black, on Medicaid, and from areas of higher poverty and more urban areas. Black and Hispanic IBC patients had worse overall and breast cancer-specific survival than white patients; moreover, IBC patients with Medicaid, patients from urban areas, and patients from areas of higher poverty and lower education had worse outcomes. These data highlight the effects of disparities in race and socioeconomic status on the incidence of IBC as well as IBC outcomes. Further work is needed to reveal the causes behind these disparities and methods to improve IBC outcomes

SIRT3 Mediates Multi-Tissue Coupling for Metabolic Fuel Switching

SummarySIRT3 is a member of the Sirtuin family of NAD+-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations; however, the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild-type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis

Metabolite of SIR2 reaction modulates TRPM2 Ion channel

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gatin

Gut microbiome variation modulates the effects of dietary fiber on host metabolism

Background: There is general consensus that consumption of dietary fermentable fiber improves cardiometabolic health, in part by promoting mutualistic microbes and by increasing production of beneficial metabolites in the distal gut. However, human studies have reported variations in the observed benefits among individuals consuming the same fiber. Several factors likely contribute to this variation, including host genetic and gut microbial differences. We hypothesized that gut microbial metabolism of dietary fiber represents an important and differential factor that modulates how dietary fiber impacts the host. Results: We examined genetically identical gnotobiotic mice harboring two distinct complex gut microbial communities and exposed to four isocaloric diets, each containing different fibers: (i) cellulose, (ii) inulin, (iii) pectin, (iv) a mix of 5 fermentable fibers (assorted fiber). Gut microbiome analysis showed that each transplanted community preserved a core of common taxa across diets that differentiated it from the other community, but there were variations in richness and bacterial taxa abundance within each community among the different diet treatments. Host epigenetic, transcriptional, and metabolomic analyses revealed diet-directed differences between animals colonized with the two communities, including variation in amino acids and lipid pathways that were associated with divergent health outcomes. Conclusion: This study demonstrates that interindividual variation in the gut microbiome is causally linked to differential effects of dietary fiber on host metabolic phenotypes and suggests that a one-fits-all fiber supplementation approach to promote health is unlikely to elicit consistent effects across individuals. Overall, the presented results underscore the importance of microbe-diet interactions on host metabolism and suggest that gut microbes modulate dietary fiber efficacy. [MediaObject not available: see fulltext.]Fil: Murga Garrido, Sofia M.. Universidad Nacional Autónoma de México; México. University of Wisconsin; Estados UnidosFil: Hong, Qilin. University of Wisconsin; Estados UnidosFil: Cross, Tzu Wen L.. University of Wisconsin; Estados Unidos. Purdue University; Estados UnidosFil: Hutchison, Evan R.. University of Wisconsin; Estados UnidosFil: Han, Jessica. Wisconsin Institute for Discovery; Estados UnidosFil: Thomas, Sydney P.. Wisconsin Institute for Discovery; Estados UnidosFil: Vivas, Eugenio I.. University of Wisconsin; Estados UnidosFil: Denu, John. Wisconsin Institute for Discovery; Estados UnidosFil: Ceschin, Danilo Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; ArgentinaFil: Tang, Zheng Zheng. University of Wisconsin; Estados Unidos. Wisconsin Institute for Discovery; Estados UnidosFil: Rey, Federico E.. University of Wisconsin; Estados Unido

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress

Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease

Genetic mutations that severely diminish the activity of aspartoacylase (ASPA) result in the fatal brain dysmyelinating disorder, Canavan disease. There is no effective treatment. ASPA produces free acetate from the concentrated brain metabolite, N-acetylaspartate (NAA). Because acetyl coenzyme A is a key building block for lipid synthesis, we postulated that the inability to catabolize NAA leads to a brain acetate deficiency during a critical period of CNS development, impairing myelination and possibly other aspects of brain development. We tested the hypothesis that acetate supplementation during postnatal myelination would ameliorate the severe phenotype associated with ASPA deficiency using the tremor rat model of Canavan disease. Glyceryltriacetate (GTA) was administered orally to tremor rats starting 7 days after birth, and was continued in food and water after weaning. Motor function, myelin lipids, and brain vacuolation were analyzed in GTA-treated and untreated tremor rats. Significant improvements were observed in motor performance and myelin galactocerebroside content in tremor rats treated with GTA. Further, brain vacuolation was modestly reduced, and these reductions were positively correlated with improved motor performance. We also examined the expression of the acetyl coenzyme A synthesizing enzyme acetyl coenzyme A synthase 1 and found upregulation of expression in tremor rats, with a return to near normal expression levels in GTA-treated tremor rats. These results confirm the critical role played by NAA-derived acetate in brain myelination and development, and demonstrate the potential usefulness of acetate therapy for the treatment of Canavan disease

NUP98-PHF23 Is a Chromatin-Modifying Oncoprotein That Causes a Wide Array of Leukemias Sensitive to Inhibition of PHD Histone Reader Function

In this report, we show that expression of a NUP98-PHF23 (NP23) fusion, associated with acute myeloid leukemia (AML) in humans, leads to myeloid, erythroid, T-cell, and B-cell leukemia in mice. The leukemic and pre-leukemic tissues display a stem cell-like expression signature including Hoxa, Hoxb, and Meis1 genes. The PHF23 PHD domain is known to bind H3K4me3 residues, and chromatin immunoprecipitation experiments demonstrated that the NP23 protein bound chromatin at a specific subset of H3K4me3 sites, including Hoxa, Hoxb, and Meis1. Treatment of NP23 cells with disulfiram, which inhibits the binding of PHD domains to H3K4me3 residues, rapidly and selectively killed NP23 myeloblasts; cell death was preceded by decreased expression of Hoxa, Hoxb, and Meis1. Furthermore, AML driven by a related fusion gene, NUP98-JARID1A (NJL), was also sensitive to disulfiram. Thus, the NP23 mouse provides a platform to evaluate compounds that disrupt binding of oncogenic PHD proteins to H3K4me3