Celia Pilar Martinez‐Jimenez

Transcription factors (TFs) direct gene expression by binding to DNA regulatory regions. To explore the evolution of gene regulation, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine experimentally the genome-wide occupancy of two TFs, CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha, in the livers of five vertebrates. Although each TF displays highly conserved DNA binding preferences, most binding is species-specific, and aligned binding events present in all five species are rare. Regions near genes with expression levels that are dependent on a TF are often bound by the TF in multiple species yet show no enhanced DNA sequence constraint. Binding divergence between species can be largely explained by sequence changes to the bound motifs. Among the binding events lost in one lineage, only half are recovered by another binding event within 10 kilobases. Our results reveal large interspecies differences in transcriptional regulation and provide insight into regulatory evolution.

T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.

Male gametes are generated through a specialised differentiation pathway involving a series of developmental transitions that are poorly characterised at the molecular level. Here, we use droplet-based single-cell RNA-Sequencing to profile spermatogenesis in adult animals and at multiple stages during juvenile development. By exploiting the first wave of spermatogenesis, we both precisely stage germ cell development and enrich for rare somatic cell-types and spermatogonia. To capture the full complexity of spermatogenesis including cells that have low transcriptional activity, we apply a statistical tool that identifies previously uncharacterised populations of leptotene and zygotene spermatocytes. Focusing on post-meiotic events, we characterise the temporal dynamics of X chromosome re-activation and profile the associated chromatin state using CUT&RUN. This identifies a set of genes strongly repressed by H3K9me3 in spermatocytes, which then undergo extensive chromatin remodelling post-meiosis, thus acquiring an active chromatin state and spermatid-specific expression.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Adaptation of liver to nutritional signals is regulated by several transcription factors that are modulated by intracellular metabolites. Here, we demonstrate a transcription factor network under the control of hepatocyte nuclear factor 4alpha (HNF4alpha) that coordinates the reciprocal expression of fatty acid transport and metabolizing enzymes during fasting and feeding conditions. Hes6 is identified as a novel HNF4alpha target, which in normally fed animals, together with HNF4alpha, maintains PPARgamma expression at low levels and represses several PPARalpha-regulated genes. During fasting, Hes6 expression is diminished, and peroxisome proliferator-activated receptor alpha (PPARalpha) replaces the HNF4alpha/Hes6 complex on regulatory regions of target genes to activate transcription. Gene expression and promoter occupancy analyses confirmed that HNF4alpha is a direct activator of the Pparalpha gene in vivo and that its expression is subject to feedback regulation by PPARalpha and Hes6 proteins. These results establish the fundamental role of dynamic regulatory interactions between HNF4alpha, Hes6, PPARalpha, and PPARgamma in the coordinated expression of genes involved in fatty acid transport and metabolism.