cyclicAMP and glucocorticoid responsiveness of the rat carbamoylphosphate synthetase gene requires the interplay of upstream regulatory units.

Many genes involved in metabolic processes are regulated by glucocorticoids and/or cyclicAMP. The hepatic expression of the urea cycle enzyme carbamoylphosphate-synthetase-I gene (CPS) is regulated at the transcriptional level by both factors. Here, we report that the 5' half of the distal enhancer is necessary and sufficient for full cyclicAMP responsiveness. The cyclicAMP-responsive element (CRE), and FoxA- and C/EBP-binding sites are indispensible for cyclicAMP responsiveness, indicating that these elements make up a cyclicAMP-responsive unit (CRU). In addition to this CRU, the CPS regulatory regions contain two glucocorticoid-response elements (GRE): one in the 3' region of the distal enhancer and one in the proximal enhancer. In presence of the cyclicAMP-responsive region in the distal enhancer, only one of the GREs is required for glucocorticoid-inducible CPS expression, with both GREs acting in an additive fashion to fully confer the inducing effect of glucocorticoids. In contrast, the simultaneous presence of both GREs is required in the absence of the cyclicAMP-responsive region. In this configuration, the distal GRE fully depends on its neighbouring FoxA and C/EBP REs for activity and is, therefore, a glucocorticoid-responsive unit. In conclusion, we show here that the CPS CRU is a bifunctional unit that elicits the cyclicAMP response and, in addition, functions as a glucocorticoid accessory unit to establish a glucocorticoid response from otherwise silent proximal or distal GRUs. Therefore, cyclicAMP and glucocorticoid pathways can induce CPS transcription via overlapping sets of response elements.

Hepatocyte-specific interplay of transcription factors at the far-upstream enhancer of the carbamoylphosphate synthetase gene upon glucocorticoid induction.

Carbamoylphosphate synthetase-I is the flux-determining enzyme of the ornithine cycle, and neutralizes toxic ammonia by converting it to urea. An 80 bp glucocorticoid response unit located 6.3 kb upstream of the transcription start site mediates hormone responsiveness and liver-specific expression of carbamoylphosphate synthetase-I. The glucocorticoid response unit consists of response elements for the glucocorticoid receptor, forkhead box A, CCAAT/enhancer-binding protein, and an unidentified protein. With only four transcription factor response elements, the carbamoylphosphate synthetase-I glucocorticoid response unit is a relatively simple unit. The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined by comparing a carbamoylphosphate synthetase-I-expressing hepatoma cell line with a carbamoylphosphate synthetase-I-negative fibroblast cell line. DNaseI hypersensitivity assays revealed an open chromatin configuration of the carbamoylphosphate synthetase-I enhancer in hepatoma cells only. In vivo footprinting assays showed that the accessory transcription factors of the glucocorticoid response unit bound to their response elements in carbamoylphosphate synthetase-I-positive cells, irrespective of whether carbamoylphosphate synthetase-I expression was induced with hormones. In contrast, the binding of glucocorticoid receptor to the carbamoylphosphate synthetase-I glucocorticoid response unit was dependent on treatment of the cells with glucocorticoids. Only forkhead box A was exclusively present in hepatoma cells, and therefore appears to be an important determinant of the observed tissue specificity of carbamoylphosphate synthetase-I expression. As the glucocorticoid receptor is the only DNA-binding protein specifically recruited to the glucocorticoid response unit upon stimulation by glucocorticoids, it is likely to be directly responsible for the transcriptional activation mediated by the glucocorticoid response unit.

Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology.

BACKGROUND: In experimental biology, including retrovirology and molecular biology, replicate measurement sessions very often show similar proportional differences between experimental conditions, but different absolute values, even though the measurements were presumably carried out under identical circumstances. Although statistical programs enable the analysis of condition effects despite this replication error, this approach is hardly ever used for this purpose. On the contrary, most researchers deal with such between-session variation by normalisation or standardisation of the data. In normalisation all values in a session are divided by the observed value of the 'control' condition, whereas in standardisation, the sessions' means and standard deviations are used to correct the data. Normalisation, however, adds variation because the control value is not without error, while standardisation is biased if the data set is incomplete. RESULTS: In most cases, between-session variation is multiplicative and can, therefore, be removed by division of the data in each session with a session-specific correction factor. Assuming one level of multiplicative between-session error, unbiased session factors can be calculated from all available data through the generation of a between-session ratio matrix. Alternatively, these factors can be estimated with a maximum likelihood approach. The effectiveness of this correction method, dubbed "factor correction", is demonstrated with examples from the field of molecular biology and retrovirology. Especially when not all conditions are included in every measurement session, factor correction results in smaller residual error than normalisation and standardisation and therefore allows the detection of smaller treatment differences. Factor correction was implemented into an easy-to-use computer program that is available on request at: biolab-services@amc.uva.nl?subject=factor. CONCLUSION: Factor correction is an effective and efficient way to deal with between-session variation in multi-session experiments.

The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit.

As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When challenged with glucocorticoids, the glucocorticoid-responsive unit (GRU) in the distal enhancer of the carbamoylphosphate-synthetase gene can only activate gene expression if, in addition to the minimal promoter, the proximal enhancer is present. Here, we identify and characterise two elements in the proximal CPS enhancer that are involved in glucocorticoid-dependent gene activation mediated by the GRU. A purine-rich stretch forming a so-called GAGA-box and a glucocorticoid-response element (GRE) are both crucial for the efficacy of the GRU and appear to constitute a promoter-proximal response unit that activates the promoter. The glucocorticoid response of the CPS gene is, therefore, dependent on the combined action of a distal and a promoter-proximal response unit.

Mechanisms of glucocorticoid signalling.

It has become increasingly clear that glucocorticoid signalling not only comprises the binding of the glucocorticoid receptor (GR) to its response element (GRE), but also involves indirect regulation glucocorticoid-responsive genes by regulating or interacting with other transcription factors. In addition, they can directly regulate gene expression by binding to negative glucocorticoid response elements (nGREs), to simple GREs, to GREs, or to GREs and GRE half sites (GRE1/2s) that are part of a regulatory unit. A response unit allows a higher level of glucocorticoid induction than simple GREs and, in addition, allows the integration of tissue-specific information with the glucocorticoid response. Presumably, the complexity of such a glucocorticoid response unit (GRU) depends on the number of pathways that integrate at this unit. Because GRUs are often located at distant sites relative to the transcription-start site, the GRU has to find a way to communicate with the basal-transcription machinery. We propose that the activating signal of a distal enhancer can be relayed onto the transcription-initiation complex by coupling elements located proximal to the promoter.

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence. With spacers of < or =12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP-FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.