The role of histone modifications in transcription remains incompletely understood. Here we used experimental perturbations combined with sensitive machine learning tools that infer the distribution of histone marks using maps of nascent transcription. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3 consistent with a role for RNA in recruiting PRC2. A subset of DNase-I hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than a regulatory, role in transcription.

Cytosine methylation patterns have not yet been thoroughly studied in horses. Here, we profile n = 333 samples from 42 horse tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). Using the blood and liver tissues from horses, we develop five epigenetic aging clocks: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. In addition, using blood methylation data from three additional equid species (plains zebra, Grevy’s zebras and Somali asses), we develop another clock that applies across all equid species. Castration does not significantly impact the epigenetic aging rate of blood or liver samples from horses. Methylation and RNA data from the same tissues define the relationship between methylation and RNA expression across horse tissues. We expect that the multi-tissue atlas will become a valuable resource.

Human DNA methylation profiles have been used successfully to develop highly accurate biomarkers of aging ("epigenetic clocks"). Here, we describe epigenetic clocks for horses, based on methylation profiles of CpGs with flanking DNA sequences that are highly conserved between multiple mammalian species. Methylation levels of these CpGs were measured using a custom-designed Infinium array (HorvathMammalMethylChip40). We generated 336 DNA methylation profiles from 42 different horse tissues and body parts, which we used to develop five epigenetic clocks for horses: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. Epigenetic age measured by these clocks show that while castration affects the basal methylation levels of individual cytosines, it does not exert a significant impact on the epigenetic aging rate of the horse. We observed that most age-related CpGs are adjacent to developmental genes. Consistently, these CpGs reside in bivalent chromatin domains and polycomb repressive targets, which are elements that control expression of developmental genes. The availability of an RNA expression atlas of these tissues allowed us to correlate CpG methylation, their corresponding contextual chromatin features and gene expression. This analysis revealed that while increased methylation of CpGs in enhancers is likely to repress gene expression, methylation of CpGs in bivalent chromatin domains on the other hand is likely to stimulate expression of the corresponding downstream loci, which are often developmental genes. This supports the notion that aging may be accompanied by increased expression of developmental genes. It is expected that the epigenetic clocks will be useful for identifying and validating anti-aging interventions for horses.

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The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.