Maximum Caliber Can Characterize Genetic Switches with Multiple Hidden Species.

Gene networks with feedback often involve interactions between multiple species of biomolecules, much more than experiments can actually monitor. Coupled with this is the challenge that experiments often measure gene expression in noisy fluorescence instead of protein numbers. How do we infer biophysical information and characterize the underlying circuits from this limited and convoluted data? We address this by building stochastic models using the principle of Maximum Caliber (MaxCal). MaxCal uses the basic information on synthesis, degradation, and feedback-without invoking any other auxiliary species and ad hoc reactions-to generate stochastic trajectories similar to those typically measured in experiments. MaxCal in conjunction with Maximum Likelihood (ML) can infer parameters of the model using fluctuating trajectories of protein expression over time. We demonstrate the success of the MaxCal + ML methodology using synthetic data generated from known circuits of different genetic switches: (i) a single-gene autoactivating circuit involving five species (including mRNA), (ii) a mutually repressing two-gene circuit (toggle switch) with seven species (including mRNA) considering stochastic time traces of two proteins, and (iii) the same toggle switch circuit considering stochastic time traces of only one of the two proteins. To further challenge the MaxCal + ML inference scheme, we repeat our analysis for the second and third scenario with traces expressed in noisy fluorescence instead of protein number to closely mimic typical experiments. We show that, for all of these models with increasing complexity and obfuscation, the minimal model of MaxCal is still able to capture the fluctuations of the trajectory and infer basic underlying rate parameters when benchmarked against the known values used to generate the synthetic data. Importantly, the model also yields an effective feedback parameter that can be used to quantify interactions within these circuits. These applications show the promise of MaxCal's ability to characterize circuits with limited data, and its utility to better understand evolution and advance design strategies for specific functions.

Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Activation of the beta globin locus by transcription factors and chromatin modifiers.

Locus control regions (LCRs) alleviate chromatin-mediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is influenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Krüppel-like factor (EKLF) also influence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF influences the expression of the gamma- more than the beta-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position.

Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

The dynamics of globin gene expression and position effects.

We have used gene competition to study the regulation of the human beta-globin locus in transgenic mice as a model system of a multigene locus. The locus is regulated by the locus control region (LCR), which is required for the expression of all the genes. Analysis of the locus at the single-cell level shows that the LCR appears to interact directly with the genes via a looping mechanism. This interaction is monogenic, and the level of transcription is determined by the frequency and stability of LCR/gene complex formation. These parameters are dependent both on the distance between the LCR and gene(s), and the concentration of transcription factors in the nucleus. Disturbance of complex formation leads to position effects, particularly when the locus is integrated in a heterochromatic environment.

Sequence analysis and tissue expression of a non-Bohr beta-globin cDNA from Atlantic salmon.

The presence of a haemoglobin protein which does not exhibit a Bohr effect has been found only in fish living in fast flowing waters. We report the cloning of the first non-Bohr effect beta-globin cDNA from an adult Atlantic salmon kidney bank. Nucleotide sequence analysis of this cDNA shows that the predicted beta-globin peptide comprises 147 amino acids with a calculated molecular mass of 15 975 Da and an overall amino acid homology of 40 to 50% to higher vertebrates and 60-90% to fish sequences. This sequence confirms the important amino acid residues which are changed thus causing loss of the Bohr effect [Powers, D.A. and Edmunson, A.B. (1972) Multiple hemoglobins of catostomid fish. J. Biol. Chem. 247, 6686-6693; Brunori, M. (1975) Molecular adaptation to physiological requirements: the hemoglobin system of trout. Curr. Topics Cell. Regul. 9, 1-39]. This loss allows the haemoglobin protein to have a higher oxygen affinity, as it does not release oxygen when the pH of the surrounding environment decreases, which is an important ability for the fish in times of stress.

Structural organization and sequence analysis of the globin locus in Atlantic salmon.

Preliminary analysis of Atlantic salmon alpha- and beta-globin genes indicated that these genes are linked in a 3' to 3' orientation, with the RNA-coding sequences located on opposite strands. In this report, we show that two different alpha-globin genes have the same orientation and are encoded on the same strand whereas two different beta-globin genes are encoded on the opposite strand and also have the same orientation. This cluster of globin genes is divided into two subclusters: one for the Bohr globin genes and one for the non-Bohr globin genes. This is the first evidence for this type of arrangement found for globin genes. DNase I footprint analysis of two of the globin promoters show erythroid-specific transcription factor binding sites that have also been found in human and other mammalian globin genes.

Salmon HNF1: cDNA sequence, evolution, tissue specificity and binding to the salmon serum albumin promoter.

cDNA clones coding for the transcription factor HNF1 have been isolated from Atlantic salmon (Salmo salar L.). The 559 amino acid residue long encoded protein shows high conservation, with respect to other species, of the domains necessary for DNA-binding: the HNF1 atypical homeodomain, the POU related sequence and the dimerisation domain. Alignment with rat HNF1 protein reveals that the transcription activation domains ADI and ADIII are relatively conserved in the fish sequence whereas ADII is not. Phylogenetic analysis indicates that higher vertebrate HNF1s and the related variant HNF1s (vHNF1s) are more closely related to each other than any of them is to Salmon HNF1, suggesting that the duplication event from which HNF1 and vHNF1 genes arose occurred after the divergence of the tetrapod and teleost ancestors. Northern blot analysis show a single transcript, of about 2.6 kb, which is not exclusive to liver but is also present in intestine, kidney and spleen. Using polymerase chain reaction (PCR) we have isolated the salmon albumin gene promoter which contains, upstream of the TATA box, a potential binding site for HNF1. The salmon HNF1 protein synthesized by in vitro transcription-translation of the full-length cDNA is able to bind specifically with equivalent affinities to either the rat or salmon albumin promoter.

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes.

We report the cloning of a cDNA and two corresponding beta-globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for alpha-globins. Nucleotide sequence analysis of the cDNA shows that the predicted beta-globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40-50% to higher vertebrates and 60-90% to fish sequences. The study of the genomic organization of alpha- and beta-globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked alpha- and beta-globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the alpha- and beta-globin genes are oriented 3' to 3' relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes.