The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification.

The PAR-domain basic leucine zipper (PAR bZip) transcription factors DBP, TEF, and HLF accumulate in a highly circadian manner in several peripheral tissues, including liver and kidney. Mice devoid of all three of these proteins are born at expected Mendelian ratios, but are epilepsy prone, age at an accelerated rate, and die prematurely. In the hope of identifying PAR bZip target genes whose altered expression might contribute to the high morbidity and mortality of PAR bZip triple knockout mice, we compared the liver and kidney transcriptomes of these animals to those of wild-type or heterozygous mutant mice. These experiments revealed that PAR bZip proteins control the expression of many enzymes and regulators involved in detoxification and drug metabolism, such as cytochrome P450 enzymes, carboxylesterases, and constitutive androstane receptor (CAR). Indeed, PAR bZip triple knockout mice are hypersensitive to xenobiotic compounds, and the deficiency in detoxification may contribute to their early aging.

Distinct Roles of Candida albicans Drug Resistance Transcription Factors TAC1, MRR1, and UPC2 in Virulence.

Azoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur in Candida albicans principally by overexpression of multidrug transporter gene CDR1, CDR2, or MDR1 or by overexpression of ERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs) TAC1 (involved in the control of CDR1 and CDR2), MRR1 (involved in the control of MDR1), and UPC2 (involved in the control of ERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial to C. albicans survival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence of C. albicans in the absence of the selective drug pressure. In this work, the effect of GOF mutations on C. albicans virulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neither TAC1 nor MRR1 GOF mutations had a significant effect on C. albicans virulence. In contrast, the presence of two hyperactive UPC2 alleles in C. albicans resulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate with UPC2 hyperactive alleles was observed in competition experiments with the wild type in vivo but not in vitro. Interestingly, UPC2 hyperactivity delayed filamentation of C. albicans after phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining the UPC2 GOF mutation with another hyperactive TF did not compensate for the negative effect of UPC2 on virulence. In conclusion, among the major TFs involved in azole resistance, only UPC2 had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

Using transcription modules to identify expression clusters perturbed in Williams-Beuren syndrome.

The genetic dissection of the phenotypes associated with Williams-Beuren Syndrome (WBS) is advancing thanks to the study of individuals carrying typical or atypical structural rearrangements, as well as in vitro and animal studies. However, little is known about the global dysregulations caused by the WBS deletion. We profiled the transcriptomes of skin fibroblasts from WBS patients and compared them to matched controls. We identified 868 differentially expressed genes that were significantly enriched in extracellular matrix genes, major histocompatibility complex (MHC) genes, as well as genes in which the products localize to the postsynaptic membrane. We then used public expression datasets from human fibroblasts to establish transcription modules, sets of genes coexpressed in this cell type. We identified those sets in which the average gene expression was altered in WBS samples. Dysregulated modules are often interconnected and share multiple common genes, suggesting that intricate regulatory networks connected by a few central genes are disturbed in WBS. This modular approach increases the power to identify pathways dysregulated in WBS patients, thus providing a testable set of additional candidates for genes and their interactions that modulate the WBS phenotypes.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

Estrogen receptor level determines sex-specific in vitro transcription from the Xenopus vitellogenin promoter.

Female-specific expression of the Xenopus laevis vitellogenin gene was reconstituted in vitro by addition of recombinant vaccinia-virus-produced estrogen receptor to nuclear extracts from male livers, in which this gene is silent. Transcription enhancement was at least 30 times and was selectively restricted to vitellogenin templates containing the estrogen-responsive unit. Thus, in male hepatocytes, estrogen receptor is the limiting regulatory factor that in the female liver controls efficient and accurate sex-specific expression of the vitellogenin gene. Furthermore, the Xenopus liver factor B, which is essential in addition to the estrogen receptor for the activation of this gene, was successfully replaced in the Xenopus extract by purified human nuclear factor I, identifying factor B of Xenopus as a functional homolog of this well-characterized human transcription factor.

Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution.

Arising from either retrotransposition or genomic duplication of functional genes, pseudogenes are "genomic fossils" valuable for exploring the dynamics and evolution of genes and genomes. Pseudogene identification is an important problem in computational genomics, and is also critical for obtaining an accurate picture of a genome's structure and function. However, no consensus computational scheme for defining and detecting pseudogenes has been developed thus far. As part of the ENCyclopedia Of DNA Elements (ENCODE) project, we have compared several distinct pseudogene annotation strategies and found that different approaches and parameters often resulted in rather distinct sets of pseudogenes. We subsequently developed a consensus approach for annotating pseudogenes (derived from protein coding genes) in the ENCODE regions, resulting in 201 pseudogenes, two-thirds of which originated from retrotransposition. A survey of orthologs for these pseudogenes in 28 vertebrate genomes showed that a significant fraction ( approximately 80%) of the processed pseudogenes are primate-specific sequences, highlighting the increasing retrotransposition activity in primates. Analysis of sequence conservation and variation also demonstrated that most pseudogenes evolve neutrally, and processed pseudogenes appear to have lost their coding potential immediately or soon after their emergence. In order to explore the functional implication of pseudogene prevalence, we have extensively examined the transcriptional activity of the ENCODE pseudogenes. We performed systematic series of pseudogene-specific RACE analyses. These, together with complementary evidence derived from tiling microarrays and high throughput sequencing, demonstrated that at least a fifth of the 201 pseudogenes are transcribed in one or more cell lines or tissues.

Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes.

In the liver of oviparous vertebrates vitellogenin gene expression is controlled by estrogen. The nucleotide sequence of the 5' flanking region of the Xenopus laevis vitellogenin genes A1, A2, B1 and B2 has been determined. These sequences have been compared to each other and to the equivalent region of the chicken vitellogenin II and apo-VLDLII genes which are also expressed in the liver in response to estrogen. The homology between the 5' flanking region of the Xenopus genes B1 and B2 is higher than between the corresponding regions of the other closely related genes A1 and A2. Four short blocks of sequence homology which are present at equivalent positions in the vitellogenin genes of both Xenopus laevis and chicken are characterized. A short sequence with two-fold rotational symmetry (GGTCANNNTGACC) was found at similar positions upstream of the five vitellogenin genes and is also present in two copies close to the 5' end of the chicken apo-VLDLII gene. The possible functional significance of this sequence, common to liver estrogen-responsive genes, is discussed.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development.

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3-deficient mice show several hallmarks of ciliopathies including left-right asymmetry defects and hydrocephalus. Here we show that Rfx3-deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.

Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

Membrane mu poly(A) signal and 3' flanking sequences function as a transcription terminator for immunoglobulin-encoding genes.

Developmentally regulated mechanisms involving alternative RNA splicing and/or polyadenylation, as well as transcription termination, are implicated in controlling the levels of secreted mu (mu s), membrane mu (mu m) and delta immunoglobulin (Ig) heavy chain mRNAs during B cell differentiation (mu gene encodes the mu heavy chain). Using expression vectors constructed with genomic DNA segments composed of the mu m polyadenylation signal region, we analyzed poly(A) site utilization and termination of transcription in stably transfected myeloma cells and in murine fibroblast L cells. We found that the gene segment containing the mu m poly(A) signals, along with 536 bp of downstream flanking sequence, acted as a transcription terminator in both myeloma cells and L cell fibroblasts. Neither a 141-bp DNA fragment (which directed efficient polyadenylation at the mu m site), nor the 536-bp flanking nucleotide sequence alone, were sufficient to obtain a similar regulation. This shows that the mu m poly(A) region plays a central role in controlling developmentally regulated transcription termination by blocking downstream delta gene expression. Because this gene segment exhibited the same RNA processing and termination activities in fibroblasts, it appears that these processes are not tissue-specific.