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A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β -hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr′s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones. [Display omitted] • SiTomics uses genetic code expansion combined with proteomic and genomic profiling • In situ and multi-dimensional identification of chromatinized interactome • Linkage between the interacting proteome and genome via dynamic H3K56 acylations • A role of super-enhancers along the "metabolites-modification-regulation" axis A genetic code expansion approach enables functional characterization of site-specific changes and interactome of histone lysine modifications in response to short chain fatty acid stimulations, linking the location of the modified nucleosomes with associated proteins on the physiological chromatin. [ABSTRACT FROM AUTHOR]

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis. • Phosphorylation of pro-caspase-8 at Thr265 releases the blockade of necroptosis • RSK phosphorylates pro-caspase-8 at Thr265 in the necrosome • PDK1 activates RSK by an ERK-independent mechanism to promote necroptosis • RSK inhibition protects mice from TNF-induced cecum injury and lethality An intrinsic mechanism—RSK, activated by TNF via PDK1—for overcoming necroptosis blockade is revealed by Yang et al. that phosphorylates pro-caspase-8 at Thr265 within the necrosome to inactivate pro-caspase-8. [ABSTRACT FROM AUTHOR]