Subtle Variation: Despite vast phenotypic differences, vertebrates have many readily recognizable specific cell types, like liver hepatocytes. The gene expression that defines specific cells depends on evolutionarily conserved orthologous transcription factors. Schmidt et al. (p. 1036 , published online 8 April) studied the conservation and divergence in the genome-wide binding of two such transcription factors, CEBPA and HNF4A, in livers from human, dog, mouse, short-tailed opossum, and chicken. Although the sequence bound by orthologous transcription factors was similar, the vast majority of binding events were unique to each species.

Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4⁺ 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.

Gaining knowledge on the metabolism of a drug, the enzymes involved and its inhibition or induction potential is a necessary step in pharmaceutical development of new compounds. Primary human hepatocytes are considered a cellular model of reference, as they express the majority of drug-metabolising enzymes, respond to enzyme inducers and are capable of generating invitro a metabolic profile similar to what is found in vivo. However, hepatocytes show phenotypic instability and have a restricted accessibility. Different alternatives have been explored in the past recent years to overcome the limitations of primary hepatocytes. These include immortalisation of adult or fetal human hepatic cells by means of transforming tumour virus genes, oncogenes, conditionally immortalised hepatocytes, and cell fusion. New strategies are currently being used to upregulate the expression of drug-metabolising enzymes in cell lines or to derive hepatocytes from progenitor cells. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism studies as well as the state-of-the-art of the strategies pursued in order to generate metabolically competent hepatic cell lines.

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.

CYP3A4 is the most abundantly expressed drug-metabolizing P450 enzyme in human liver and contributes to the metabolism of a large number of drugs in use today. CYP3A4 is constitutively expressed in adult hepatocytes but it can also be transcriptionally induced by a variety of structurally diverse xenochemicals. CYP3A4 strongly contributes to the important variability in the therapeutic and toxic effects of drugs owing to the major role it plays in xenobiotic metabolism and the large intra- and inter-individual variability to which it is subjected. The functional examination of up to 13 kb of the CYP3A4 5-flanking region has revealed that the regulation of this gene is a complex issue, with numerous transcription factors interacting with multiple promoter/enhancer elements. This also suggests that a high degree of human variability in the hepatic CYP3A4 expression could result from regulatory polymorphisms. Several transcription factors and nuclear receptors contribute to the hepatic-specific expression of CYP3A4, including: C/EBPγ , C/EBPβ , HNF4γ , HNF3γ , CAR and PXR. The induction phenomenon and the down-regulation of CYP3A4 in pathophysiological conditions, such as inflammatory situations, are key processes involved in the toxic vs. therapeutic effects of many drugs. Since CYP3A4 variation may affect the efficacy and toxicity of new drugs, development of reliable hepatic models for the assessment and prediction of the role of CYP3A4 in drug metabolism are important for drug development. Cultured human hepatocytes are the closest model to the human liver as far as CYP3A4 regulation and induction are concerned. However, other hepatic models should be considered in drug development for screening purposes owing to the limited availability of human hepatocytes.