Martina Rath

Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53.

The transcription factor c-Jun mediates several cellular processes, including proliferation and survival, and is upregulated in many carcinomas. Liver-specific inactivation of c-Jun at different stages of tumor development was used to study its role in chemically induced hepatocellular carcinomas (HCCs) in mice. The requirement for c-jun was restricted to early stages of tumor development, and the number and size of hepatic tumors was dramatically reduced when c-jun was inactivated after the tumor had initiated. The impaired tumor development correlated with increased levels of p53 and its target gene noxa, resulting in the induction of apoptosis without affecting cell proliferation. Primary hepatocytes lacking c-Jun showed increased sensitivity to TNF-alpha-induced apoptosis, which was abrogated in the absence of p53. These data indicate that c-Jun prevents apoptosis by antagonizing p53 activity, illustrating a mechanism that might contribute to the early stages of human HCC development.

Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq

The Regulatory Genome Multicellular organisms contain a variety of cell types that are morphologically and functionally distinct even though they typically contain the same genomic DNA. Differences stem from differential gene expression. Gene regulatory genomic regions (enhancers) are well studied, yet despite major efforts, such as Encode and modEncode, the number of enhancers in animal genomes and their genomic positions, cell-type specificity, and strengths are largely unknown. Arnold et al. (p. 1074, published online 17 January) report a method, termed STARR-seq, that measures the strength of enhancers genome-wide, giving insight into the organization of the regulatory genome. A map of thousands of Drosophila cell type–specific enhancers is revealed using a new method. Genomic enhancers are important regulators of gene expression, but their identification is a challenge, and methods depend on indirect measures of activity. We developed a method termed STARR-seq to directly and quantitatively assess enhancer activity for millions of candidates from arbitrary sources of DNA, which enables screens across entire genomes. When applied to the Drosophila genome, STARR-seq identifies thousands of cell type–specific enhancers across a broad continuum of strengths, links differential gene expression to differences in enhancer activity, and creates a genome-wide quantitative enhancer map. This map reveals the highly complex regulation of transcription, with several independent enhancers for both developmental regulators and ubiquitously expressed genes. STARR-seq can be used to identify and quantify enhancer activity in other eukaryotes, including humans.

The Fos-related antigen Fra-1 is an activator of bone matrix formation

Ectopic expression of the transcription factor Fra‐1 in transgenic mice leads to osteosclerosis, a bone disorder characterized by increased bone mass. The molecular basis for this phenotype is unknown and Fra‐1 functions cannot be studied by a conventional loss‐of‐function approach, since fra‐1‐knockout mice die in utero likely due to placental defects. Here we show that the lethality of fra‐1‐knockout mice can be rescued by specific deletion of Fra‐1 only in the mouse embryo and not in the placenta. Mice lacking Fra‐1 (fra‐1Δ/Δ) are viable and develop osteopenia, a low bone mass disease. Long bones of fra‐1Δ/Δ mice appear to have normal osteoclasts but express reduced amounts of bone matrix components produced by osteoblasts and chondrocytes such as osteocalcin, collagen1a2 and matrix Gla protein. The gene for matrix Gla protein seems to be a specific target of Fra‐1 since its expression was markedly increased in the long bones of fra‐1‐transgenic mice. These results uncover a novel function of Fra‐1 in regulating bone mass through bone matrix production by osteoblasts and chondrocytes.

Enhancer–core-promoter specificity separates developmental and housekeeping gene regulation

Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters—one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor—and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors—Dref and Trl—that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer–core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.

Development of pulmonary fibrosis through a pathway involving the transcription factor Fra-2/AP-1

Studies using genetically modified mice have revealed fundamental functions of the transcription factor Fos/AP-1 in bone biology, inflammation, and cancer. However, the biological role of the Fos-related protein Fra-2 is not well defined in vivo. Here we report an unexpected profibrogenic function of Fra-2 in transgenic mice, in which ectopic expression of Fra-2 in various organs resulted in generalized fibrosis with predominant manifestation in the lung. The pulmonary phenotype was characterized by vascular remodeling and obliteration of pulmonary arteries, which coincided with expression of osteopontin, an AP-1 target gene involved in vascular remodeling and fibrogenesis. These alterations were followed by inflammation; release of profibrogenic factors, such as IL-4, insulin-like growth factor 1, and CXCL5; progressive fibrosis; and premature mortality. Genetic experiments and bone marrow reconstitutions suggested that fibrosis developed independently of B and T cells and was not mediated by autoimmunity despite the marked inflammation observed in transgenic lungs. Importantly, strong expression of Fra-2 was also observed in human samples of idiopathic and autoimmune-mediated pulmonary fibrosis. These findings indicate that Fra-2 expression is sufficient to cause pulmonary fibrosis in mice, possibly by linking vascular remodeling and fibrogenesis, and suggest that Fra-2 has to be considered a contributing pathogenic factor of pulmonary fibrosis in humans.

Hepatocyte survival in acute hepatitis is due to c-Jun/AP-1-dependent expression of inducible nitric oxide synthase

Analysis of the molecular factors determining hepatocyte survival or death in response to inflammatory stimuli is essential for understanding the pathogenesis of inflammatory liver disease and for identifying novel therapeutic approaches. c-Jun N-terminal kinase (JNK) is a major mediator of cytokine-induced cell death during hepatitis, but the signaling pathways downstream of JNK remain less well defined. Here we show that the transcription factor c-Jun/AP-1, a prototypic target of JNK, is strongly expressed in the liver of patients with acute liver injury. The molecular function of c-Jun in inflammatory liver disease was analyzed in mice by using the Con A model of T cell-mediated hepatitis. Mice lacking c-Jun in hepatocytes display increased liver cell death and mortality upon Con A injection. This phenotype is caused by impaired expression of inducible nitric oxide synthase (nos2), a direct transcriptional target of c-Jun, and reduced production of hepatoprotective nitric oxide (NO). Moreover, increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be rescued pharmacologically by liver-specific NO delivery. These findings demonstrate that c-Jun/AP-1 is hepatoprotective during acute hepatitis by regulating nos2/NO expression and thus functionally antagonizes the cell death-promoting functions of JNK.

Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution

Gene expression is controlled by enhancers that activate transcription from the core promoters of their target genes. Although a key function of core promoters is to convert enhancer activities into gene transcription, whether and how strongly they activate transcription in response to enhancers has not been systematically assessed on a genome-wide level. Here we describe self-transcribing active core promoter sequencing (STAP-seq), a method to determine the responsiveness of genomic sequences to enhancers, and apply it to the Drosophila melanogaster genome. We cloned candidate fragments at the position of the core promoter (also called minimal promoter) in reporter plasmids with or without a strong enhancer, transfected the resulting library into cells, and quantified the transcripts that initiated from each candidate for each setup by deep sequencing. In the presence of a single strong enhancer, the enhancer responsiveness of different sequences differs by several orders of magnitude, and different levels of responsiveness are associated with genes of different functions. We also identify sequence features that predict enhancer responsiveness and discuss how different core promoters are employed for the regulation of gene expression.

Resolving systematic errors in widely used enhancer activity assays in human cells

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.

A high‐throughput method to identify trans‐activation domains within transcription factor sequences

Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans‐activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high‐throughput approach tAD‐seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix–loop–helix domains that are typically annotated as DNA‐binding. We also identified tADs in the non‐native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which—particularly for TFs of different transcription activating functionalities—is still poorly understood.

Resolving systematic errors in widely-used enhancer activity assays in human cells enables genome-wide functional enhancer characterization

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliable: (1) the function of the bacterial plasmid origin-of-replication (ORI) as a conflicting core-promoter and (2) the activation of a type I interferon (IFN-I) response. These problems cause strongly confounding false-positives and -negatives in luciferase assays and genome-wide STARR-seq screens. We overcome both problems by directly employing the ORI as a core-promoter and by inhibiting two kinases central to IFN-I induction. This corrects luciferase assays and enables genome-wide STARR-seq screens in human cells. Comprehensive enhancer activity profiles in HeLa-S3 cells uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells, and are key to the characterization of human enhancers.