The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases1 and have been implicated in the regulation of transcription, genome stability, metabolism, and lifespan2, 3. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human Sirt6 efficiently removes long chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of Sirt6 reveals a large hydrophobic pocket that can accommodate long chain fatty acyl groups. We demonstrate further that Sirt6 promotes the secretion of tumor necrosis factor α (TNFα) by removing the fatty acyl modification on K19 and K20 of TNFα. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data suggest that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of Sirt6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of the previously ignored protein posttranslational modification.

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Many studies have revealed pathways of epigenetic gene silencing by Polycomb Repressive Complex 2 (PRC2) in vivo, but understanding molecular mechanisms requires biochemistry. Here we analyze reconstituted human PRC2-nucleosome complexes. Histone modifications, the H3K27M cancer mutation, and inclusion of JARID2 or EZH1 in the PRC2 complex have unexpectedly minor effects on PRC2-nucleosome binding. Instead, protein-free linker DNA dominates the PRC2-nucleosome interaction. Specificity for CG-rich sequences is consistent with PRC2 occupying CG-rich DNA in vivo. Intriguingly, PRC2 preferentially binds methylated DNA via AEBP2, suggesting how DNA- and histone-methylation collaborate to repress chromatin. RNA is known to inhibit PRC2 activity. We find that RNA is not a methyltransferase inhibitor per se, but instead sequesters PRC2 from nucleosome substrates; this occurs because PRC2 binding requires linker DNA, and RNA and DNA binding are mutually exclusive. Together, we provide a model for PRC2 recruitment and a straightforward explanation of how actively transcribed portions of the genome bind PRC2 but escape silencing.

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag® fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.