The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease. This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Objective: Fibrotic organ responses have recently been identified as long-term complications in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date. Methods: Based on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single-nuclei RNA sequencing. Results: Hepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice fed a lipogenic MCD diet. Single-nuclei RNA sequencing revealed a distinct TSC22D4-dependent gene signature identifying an upregulation of mitochondrial-related processes in hepatocytes upon loss of TSC22D4. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models of hepatocyte-selective TSC22D4 loss-of-function. Conclusions: Together, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Hepatocyte-specific reduction of TSC22D4 improves hepatic steatosis and promotes hepatocyte survival via mitochondrial-related mechanisms thus paving the way for targeted therapies.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.

Cellular heterogeneity is revolutionizing the way to study, monitor and dissect complex diseases. This has been possible with the technological and computational advances associated to single-cell genomics and epigenomics. Deeper understanding of cell-to-cell variation and its impact on tissue function will open new avenues for early disease detection, accurate diagnosis and personalized treatments, all together leading to the next generation of health care. This review focuses on the recent discoveries that single-cell genomics and epigenomics have facilitated in the context of human health. It highlights the potential of single-cell omics to further advance the development of personalized treatments and precision medicine in cancer, diabetes and chronic age-related diseases. The promise of single-cell technologies to generate new insights about the differences in function between individual cells is just emerging, and it is paving the way for identifying biomarkers and novel therapeutic targets to tackle age, complex diseases and understand the effect of life style interventions and environmental factors.

How the age-associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the gamma delta T-cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell-intrinsic effects on function and global gene expression of gamma delta T cells, and gamma delta TCR diversity remains stable. However, ageing alters TCR delta chain usage and clonal structure of gamma delta T-cell subsets. Importantly, IL-17-producing gamma delta 17 T cells dominate the gamma delta T-cell pool of aged mice-mainly due to the selective expansion of V gamma 6(+) gamma delta 17 T cells and augmented gamma delta 17 polarisation of V gamma 4(+) T cells. Expansion of the gamma delta 17 T-cell compartment is mediated by increased IL-7 expression in the T-cell zone of old mice. In a Lewis lung cancer model, pro-tumourigenic V gamma 6(+) gamma delta 17 T cells are exclusively activated in the tumour-draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in gamma delta T-cell pool in the pLN lead to an unbalanced gamma delta T-cell response in the tumour that is associated with accelerated tumour growth.

How age-associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterized the cellular composition of the γδ T cell pool in peripheral lymph nodes (pLNs) upon ageing. We found that ageing has minimal cell-intrinsic effects on function and global gene expression of γδ T cells, and TCRγδ diversity remained stable. However, ageing altered TCRδ chain usage and clonal structure of γδ T cell subsets. Importantly, IL-17-producing γδ17 T cells dominated the γδ T cell pool of aged mice - mainly due to the selective expansion of Vγ6+ γδ17 T cells and augmented γδ17-polarisation of Vγ4+ T cells. Expansion of the γδ17 T cell compartment was supported by increased Interleukin-7 expression in the T cell zone of old mice. In a Lewis lung cancer model, pro-tumourigenic Vγ6+ γδ17 T cells were exclusively activated in the tumour-draining LN and their infiltration into the tumour correlated with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes of γδ T cell pool in a dysregulated pLN microenvironment promote tumour growth.

Understanding male fertility requires an in-depth characterisation of spermatogenesis, the developmental process by which male gametes are generated. Spermatogenesis occurs continuously throughout a male's reproductive window and involves a complex sequence of developmental steps, both of which make this process difficult to decipher at the molecular level. To overcome this, we transcriptionally profiled single cells from multiple distinct stages during the first wave of spermatogenesis, where the most mature germ cell type is known. This naturally enriches for spermatogonia and somatic cell types present at very low frequencies in adult testes. Our atlas, available as a shiny app (https://marionilab.cruk.cam.ac.uk/SpermatoShiny), allowed us to reconstruct the three main processes of spermatogenesis: spermatogonial differentiation, meiosis, and spermiogenesis. Additionally, we profiled the chromatin changes associated with meiotic silencing of the X chromosome, revealing a set of genes specifically and strongly repressed by H3K9me3 in the spermatocyte stage, but which escape post-meiotic silencing in spermatids.

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.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan member of the nuclear receptor family of transcription factors whose activities are modulated upon binding of small molecules into an hydrophobic ligand-binding pocket (LBP). Although the LBP of COUP-TFII is filled with aromatic amino-acid side chains, alternative modes of ligand binding could potentially lead to regulation of the orphan receptor. Here, we screened a synthetic and natural compound library in a yeast one-hybrid assay and identified 4-methoxynaphthol as an inhibitor of COUP-TFII. This synthetic inhibitor was able to counteract processes either positively or negatively regulated by COUP-TFII in different mammalian cell systems. Hence, we demonstrate that the true orphan receptor COUP-TFII can be targeted by small chemicals which could be used to study the physiological functions of COUP-TFII or to counteract detrimental COUP-TFII activities in various pathological conditions.

INTRODUCTION: For the optimal management of children with cystic fibrosis, there are currently no efficient tools for the precise adjustment of pancreatic enzyme replacement therapy, either for advice on appropriate dietary intake or for achieving an optimal nutrition status. Therefore, we aim to develop a mobile application that ensures a successful nutritional therapy in children with cystic fibrosis. METHODS AND ANALYSIS: A multidisciplinary team of 12 partners coordinate their efforts in 9 work packages that cover the entire so-called 'from laboratory to market' approach by means of an original and innovative co-design process. A cohort of 200 patients with cystic fibrosis aged 1-17 years are enrolled. We will develop an innovative, clinically tested mobile health application for patients and health professionals involved in cystic fibrosis management. The mobile application integrates the research knowledge and innovative tools for maximising self-management with the aim of leading to a better nutritional status, quality of life and disease prognosis. Bringing together different and complementary areas of knowledge is fundamental for tackling complex challenges in disease treatment, such as optimal nutrition and pancreatic enzyme replacement therapy in cystic fibrosis. Patients are expected to benefit the most from the outcomes of this innovative project. ETHICS AND DISSEMINATION: The project is approved by the Ethics Committee of the coordinating organisation, Hospital Universitari La Fe (Ref: 2014/0484). Scientific findings will be disseminated via journals and conferences addressed to clinicians, food scientists, information and communications technology experts and patients. The specific dissemination working group within the project will address the wide audience communication through the website (http://www.mycyfapp.eu), the social networks and the newsletter.

Recent technological and computational advances in understanding the transcriptional and chromatin features of single cells have begun answering longstanding questions in the extent and impact of biological heterogeneity. Here, we outline the intrinsic and extrinsic mechanisms that underlie the transcriptional and functional diversity within superficially homogeneous populations, and we discuss how fascinating new studies have afforded novel insight into each mechanism. The studies are chosen in part to include initial reports of novel functional genomics tools where the eventual applications will clearly have profound impact on our understanding the dynamics of cell-to-cell transcriptional variation-from individual cells to whole organisms.

Technological breakthroughs are emphasizing the impact of epigenetic mechanisms in human health highlighting the importance of a fine-tune orchestration of DNA methylation, micro RNAs, histone modifications, and chromatin structure. Transcriptional regulators sense the concentration of intermediary metabolites associated to a wide variety of biological processes including the long-term imprinting and heritable DNA methylation. Recent epigenetic mechanisms associated with cholesterol and lipid homeostasis have a critical impact in the susceptibility, development and progression of complex diseases such as type 2 diabetes mellitus, non-alcoholic fatty liver, obesity and metabolic syndrome. The heritability of epigenetic states emerge as an additional level of complexity where the extension of somatic as well as inherited epigenetic modifications may require a thoughtful reconsideration in many human diseases related with metabolic disorders.

Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5'-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.

Chromatin structure complexity requires the interaction and coordinated work of a multiplicity of factors at different transcriptional regulation stages. Transcription control comprises a set of processes that ensures proper balance in the gene expression under different conditions, such as signals, metabolic states, or development. We could frame those steps from epigenetic marks to mRNA stability to support the holistic view of a fine-tune balance of final mRNA levels through mRNA transcription, export, stability, translation, and degradation. Transport of mRNA from the nucleus to the cytoplasm is a key process in regulated gene expression. Transcriptional elongation and mRNA export are coregulated steps that determine the mature mRNA levels in the cytoplasm. In this paper, recent insights into the coordination of these processes in eukaryotes will be summarised.

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.

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.

To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes, we generated mice with liver-specific disruption of the TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the downregulation of most hepatocyte-specific genes during embryonic life and a defect in liver organogenesis. Unexpectedly, however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Furthermore, TFIID dissociation from promoters leads to the re-expression of several postnatally silenced hepatic genes. Promoter occupancy analyses, combined with expression profiling, demonstrate that TFIID is required for the initial activation or postnatal repression of genes, while it is dispensable for maintaining ongoing transcription.

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/EBPalpha, C/EBPbeta, HNF4alpha, HNF3gamma, 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.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a key transcription factor for the constitutive expression of cytochromes P450 (P450s) in the liver. However, human hepatoma HepG2 cells show a high level of HNF4alpha but express only marginal P450 levels. We found that the HNF4alpha-mediated P450 transcription in HepG2 is impaired by the low level of coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC1alpha) and steroid receptor coactivator 1 (SRC1). Reporter assays with a chimeric CYP2C9-LUC construct demonstrated that the sole transfection of coactivators induced luciferase activity in HepG2 cells. In HeLa cells however, CYP2C9-LUC activity only significantly increased when coactivators were cotransfected with HNF4alpha. A deletion mutant lacking the two proximal HNF4alpha binding sites in the CYP2C9 promoter did not respond to PGC1alpha or SRC1, demonstrating that coactivators were acting through HNF4alpha response elements. Adenovirus-mediated transfection of PGC1alpha in human hepatoma cells caused a significant dose-dependent increase in CYP2C9, CYP1A1, and CYP1A2 and in the positive control CYP7A1. PGC1alpha also showed a moderate activating effect on CYP3A4, CYP3A5, and CYP2D6. Adenoviral transfection of SRC1 had a lessened effect on P450 genes. Chromatin immunoprecipitation assay demonstrated in vivo binding of HNF4alpha and PGC1alpha to HNF4alpha response sequences in the CYP2C9 promoter and to three new regulatory regions in the common 23.3 kilobase spacer sequence of the CYP1A1/2 cluster. Insulin treatment of HepG2 and human hepatocytes caused repression of PGC1alpha and a concomitant down-regulation of P450s. Our results establish the importance of coactivators PGC1alpha and SRC1 for the hepatic expression of human P450s and uncover a new HNF4alpha-dependent regulatory mechanism to constitutively control the CYP1A1/2 cluster.

Hepatocyte nuclear factor 4alpha (HNF4alpha) plays critical roles during liver development and in the transcriptional regulation of many hepatic genes in adult liver. Here we have demonstrated that in human hepatoma HepG2 cells, HNF4alpha is expressed at levels as high as in human liver but its activity on target genes is very low or absent. We have discovered that the low expression of key coactivators (PGC1alpha, SRC1, SRC2, and PCAF) might account for the lack of function of HNF4alpha in HepG2 cells. Among them, PGC1alpha and SRC1 are the two most important HNF4alpha coactivators as revealed by reporter assays with an Apo-CIII promoter construct. Moreover, the expression of these two coactivators was found to be down-regulated in all human hepatomas investigated. Overexpression of SRC1 and PGC1alpha by recombinant adenoviruses led to a significant up-regulation of well characterized HNF4alpha-dependent genes (ApoCIII, ApoAV, PEPCK, AldoB, OTC, and CYP7A1) and forced HepG2 cells toward a more differentiated phenotype as demonstrated by increased ureogenic rate. The positive effect of PGC1alpha was seen to be dependent on HNF4alpha. Finally, insulin treatment of human hepatocytes and HepG2 cells caused repression of PGC1alpha and a concomitant down-regulation of ApoCIII, PEPCK, AldoB, and OTC. Altogether, our results suggest that SRC1, and notably PGC1alpha, are key coactivators for the proper function of HNF4alpha in human liver and for an integrative control of multiple hepatic genes involved in metabolism and homeostasis. The down-regulation of key HNF4alpha coactivators could be a determinant factor for the dedifferentiation of human hepatomas.

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 in vitro 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.

Because of the intrinsic variability and limited accessibility of primary cultured human hepatocytes, other cell models (e.g. cell lines) have been considered in drug metabolism studies. Unfortunately, hepatic cell lines express only marginal levels of drug-metabolising cytochrome P450s (CYPs) and are not a real alternative. Lack of CYP expression in hepatomas appears to be the consequence of an altered expression of liver-enriched transcription factors and co-regulators. We have attempted to up-regulate CYP expression by transfecting hepatoma cells with selected factors. Re-expression of one or several of these factors proved to be effective in re-activating different CYPs, and supports the notion that this can be an appropriate strategy to achieve metabolically competent cell lines.

CCAAT/enhancer-binding proteins (C/EBPs) are key transcription factors involved in the constitutive expression of several cytochrome P450 genes in the liver. Their concentration and activity change in several pathophysiological conditions. For instance, during inflammation, released cytokines induce repressive C/EBPbeta-liver inhibitory protein (LIP), which antagonizes constitutive C/EBP transactivators [C/EBPalpha and C/EBPbeta-liver activating protein (LAP)], down-regulating genes such as CYP3A4. However, the mechanism by which hepatic C/EBP factors modulate transcription of the CYP3A4 gene is not known. To elucidate the mechanism of action, we cotransfected luciferase reporter vectors, containing 5'-flanking deletions of the CYP3A4 gene, along with expression vectors for C/EBPbeta-LAP, C/EBPbeta-LIP, and C/EBPalpha, in hepatic (HepG2) and nonhepatic (HeLa) cells. Analysis of the -3557 to -6954 base pair (bp) region demonstrated the existence of a 288-bp sequence at -5.95 kilobases (kb), which showed maximal response to C/EBPbeta-LAP ( approximately 30-fold increase in HepG2 cells). Coexpression of LAP with increasing amounts of LIP reduced the activating effect by approximately 70%. Site-directed mutagenesis of predicted C/EBPbeta binding sites demonstrated the presence of four functional C/EBPbeta-responsive motifs within this distal flanking region. Further experiments using chromatin immunoprecipitation proved the binding of endogenous C/EBPbeta to the -5.95-kilobase enhancer of the CYP3A4 gene in human hepatocytes. Expression of recombinant LAP and LIP by means of adenoviral vectors resulted in their binding to this region, which was followed by activation/repression of CYP3A4. Together, our results uncover a new distal enhancer site in the CYP3A4 gene where C/EBPbeta-LAP binds and activates transcription, whereas the truncated form, C/EBPbeta-LIP, antagonizes LAP activity and causes gene repression.

The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated in order to control intrahepatic cholesterol and bile acid levels. Ligands of the xenobiotic-sensing pregnane X receptor inhibit CYP7A1 expression. To retrace the evolution of the molecular mechanisms underlying CYP7A1 inhibition, we used a chicken hepatoma cell system that retains the ability to be induced by phenobarbital and other drugs. Whereas bile acids regulate CYP7A1 via small heterodimer partner and liver receptor homolog-1, mRNA expression of these nuclear receptors is unchanged by xenobiotics. Instead, drugs repress chicken hepatic nuclear factor 4alpha (HNF4alpha) transcript levels concomitant with a reduction in CYP7A1 expression. Importantly, no reduction of HNF4alpha levels is found in mouse liver in vivo and in human primary hepatocyte cultures, respectively. Thus, besides the importance of HNF4alpha in CYP7A1 regulation in all species, birds and mammals use different signaling pathways to adjust CYP7A1 levels after exposure to xenobiotics.

Knowledge of metabolism, enzymes so far involved, and potential enzyme-inhibiting or enzyme-inducing properties of new compounds is a key issue in drug development. Primary cultured hepatocytes, cytochrome P450 (CYP)-engineered cells and hepatoma cell lines are currently being used for this purpose, but only primary cultures can produce a metabolic profile of a drug similar to that found in vivo and can respond to inducers. Because of their limited accessibility, alternatives to replace human hepatocytes are currently being explored, including the immortalisation of hepatocytes by using different strategies (i.e. SV40 T-large antigen, conditionally immortalised hepatocytes, transfection with c-myc, cH-ras, N-ras oncogenes, transgenic animals over-expressing growth factors or oncogenes and cre-lox recombination/excision). However, none of the resulting cells has the desirable phenotypic characteristics to replace primary cultures in drug metabolisms studies. We investigated why these differentiated human hepatomas do not express CYP genes and found that the levels of certain key transcription factors clearly differ from those found in hepatocytes. It was then conceivable that re-expression of one (or more) of these transcription factors could lead to an efficient transcription of CYP genes. The feasibility of this hypothesis was demonstrated by genetic engineering of Hep G2 cells with liver-enriched transcription factors followed by the analysis of the expression of the most relevant human CYPs.