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We previously developed sans spike-in quantitative chromatin immunoprecipitation sequencing (siQ-ChIP), a technique that introduces an absolute quantitative scale to ChIP-seq data without reliance on spike-in normalization approaches. The physical model of siQ-ChIP predicted that the IP step of ChIP would produce a classical binding isotherm when antibody or epitope was titrated. Here, we define experimental conditions in which this titration is observable for antibodies that recognize modified states of histone proteins. We show that minimally sequenced points along an isotherm can reveal differential binding specificities that are associated with on- and off-target epitope interactions. This work demonstrates that the interpretation of histone post-translational modification distribution from ChIP-seq data has a dependence on antibody concentration. Collectively, these studies introduce a simplified and reproducible experimental method to generate quantitative ChIP-seq data without spike-in normalization and demonstrate that histone antibody specificity can be analyzed directly in ChIP-seq experiments.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) – including β-hydroxybutyrate (βOHB) and acetoacetate (AcAc) – as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and βOHB directly increases CD8+ Teff cell IFN-γ production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that βOHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was determined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating ’tracks’ for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the widespread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.

Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis.We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not.We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.

Chromatin functions are influenced by the addition, removal, and recognition of histone post-translational modifications (PTMs). Ubiquitin and ubiquitin-like (UBL) PTMs on histone proteins can function as signaling molecules by mediating protein–protein interactions. Fueled by the identification of novel ubiquitin and UBL sites and the characterization of the writers, erasers, and readers, the breadth of chromatin functions associated with ubiquitin signaling is emerging. Here, we highlight recently appreciated roles for histone ubiquitination in DNA methylation control, PTM crosstalk, nucleosome structure, and phase separation. We also discuss the expanding diversity and functions associated with histone UBL modifications. We conclude with a look toward the future and pose key questions that will drive continued discovery at the interface of epigenetics and ubiquitin signaling.

Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) “reader” proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.