SummaryThe mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.

SynopsisTranscription factor binding differences can contribute to organismal evolution by altering downstream gene expression programmes. Recent genome-wide studies in Drosophila and mammals have revealed common quantitative and combinatorial properties of in vivo DNAbinding, as well as significant differences in the rate and mechanisms of metazoan transcription factor binding evolution. Here, we review the recently-discovered, rapid re-wiring of in vivo transcription factor binding between related metazoan species and summarize general principles underlying the observed patterns of evolution. We then consider what might explain genome evolution differences between metazoan phyla, and outline the conceptual and technological challenges facing the field.Complex, multicellular organisms require a means to create hundreds of distinct tissue types from a single genome. Most (if not all) of these tissues are shared among all known vertebrates 1, 2 ; for instance, two tissues with distinctive morphologies and evolutionarily conserved functions are the heart, controlling blood flow, and the liver, controlling blood detoxification and circulating lipids. Vertebrate tissues have broadly conserved transcriptional programmes 3,4 , and are often known to be controlled by a highly conserved set of tissue-specific DNA binding transcription factors 5 . Such tissue-specific master regulators include the transcription factors MYOD1 in muscle, HNF4A in liver or NKX2-5 in heart, which have functional roles both in development to establish tissue identity and in adulthood to maintain tissue-specific functions.It would be reasonable to suppose that the protein-DNA contacts that connect conserved transcription factors and (downstream) conserved tissue-specific gene expression programs are under strong constraint--a paradigm which has prompted the use of many diverse model organisms to model human regulatory and developmental processes. On the other hand, differences in gene regulation have long been recognized as major contributors to phenotypic diversity [6][7][8] , especially between closely related species 9 . Only recently, and mainly due to the advent of functional genomics approaches such as ChIP-Seq, have we been able to experimentally test how widely TF binding differs between species and how Correspondence to: Paul Flicek; Duncan T Odom. Competing interests statement None Europe PMC Funders GroupAuthor Manuscript Nat Rev Genet. Author manuscript; available in PMC 2014 October 01. Published in final edited form as:Nat Rev Genet. 2014 April ; 15(4): 221-233. doi:10.1038/nrg3481. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts rapidly these differences accumulate, thus fundamentally reshaping our understanding of how transcription factor binding evolves and the potential consequences for how complex eukaryotic tissues are created.Here, we will review recent advances in evolutionary analysis of transcription factor binding, with a focus on genome-wide studies in metazoans. We start by briefly discussi...

The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Hence, the complete identification of HIF targets is essential for understanding the cellular responses to hypoxia. Herein we describe a computational strategy based on the combination of phylogenetic footprinting and transcription profiling meta-analysis for the identification of HIF-target genes. Comparison of the resulting candidates with published HIF1a genome-wide chromatin immunoprecipitation indicates a high sensitivity (78%) and specificity (97.8%). To validate our strategy, we performed HIF1a chromatin immunoprecipitation on a set of putative targets. Our results confirm the robustness of the computational strategy in predicting HIF-binding sites and reveal several novel HIF targets, including RE1-silencing transcription factor co-repressor (RCOR2). In addition, mapping of described polymorphisms to the predicted HIF-binding sites identified several single-nucleotide polymorphisms (SNPs) that could alter HIF binding. As a proof of principle, we demonstrate that SNP rs17004038, mapping to a functional hypoxia response element in the macrophage migration inhibitory factor (MIF) locus, prevents induction of this gene by hypoxia. Altogether, our results show that the proposed strategy is a powerful tool for the identification of HIF direct targets that expands our knowledge of the cellular adaptation to hypoxia and provides cues on the inter-individual variation in this response.

When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1α or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-α glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.

To gain insight into how mammalian gene expression is controlled by rapidly evolving regulatory elements, we jointly analysed promoter and enhancer activity with downstream transcription levels in liver samples from fifteen species. Genes associated with complex regulatory landscapes generally exhibit high expression levels that remain evolutionarily stable. While the number of regulatory elements is the key driver of transcriptional output and resilience, regulatory conservation matters: elements active across mammals most effectively stabilise gene expression. In contrast, recently-evolved enhancers typically contribute weakly, consistent with their high evolutionary plasticity. These effects are observed across the entire mammalian clade and robust to potential confounders, such as gene expression level. Using liver as a representative somatic tissue, our results illuminate how the evolutionary stability of gene expression is profoundly entwined with both the number and conservation of surrounding promoters and enhancers.

Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species - a notion that was long-debated without the benefit of this new and compelling evidence.

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.