Thierry Grange

Glucocorticoid-induced DNA demethylation and gene memory during development.

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver‐specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2–3 days following rapid (<1 h) chromatin remodeling and recruitment of a first transcription factor, HNF‐3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine‐tuning of the enhancer and to the memorization of a regulatory event during development.

In vivo footprinting of rat TAT gene: Dynamic interplay between the glucocorticoid receptor and a liver-specific factor

HNF5, a liver-specific DNA-binding protein, interacts with DNA in a manner that allows DNAase I cleavage in the middle of its recognition sequence. Using this property we have identified in vivo HNF5 bound to its sites within two glucocorticoid-responsive units of the rat tyrosine aminotransferase (TAT) gene. One HNF5-binding site is also a glucocorticoid receptor-binding site; glucocorticoid-dependent HNF5 binding could be detected at this site even though it is incompatible with glucocorticoid receptor binding. HNF5 binds within 10 min of hormone addition, indicating that it participates in transcriptional activation. In the TAT gene glucocorticoid-dependent HNF5 binding occurs where there is glucocorticoid-dependent disruption of nucleosomal structure; constitutive binding occurs in constitutively disrupted regions. These results suggest a hit-and-run mechanism of transcriptional activation by glucocorticoid receptor: the activated receptor binds its target sequence, modifies local chromatin structure, then leaves its site accessible to another factor.

Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor

The structures of two remote glucocorticoid responsive units (GRUs) that cooperatively interact to promote cell-type specific glucocorticoid induction of rat tyrosine aminotransferase gene expression have been analyzed. DNAase I footprinting and gel mobility shift analyses reveal a complex array of contiguous and overlapping sites for cell type-specific DNA binding proteins. Apart from the glucocorticoid receptor, two liver-specific nuclear factors possess multiple binding sites in each of these GRUs: C/EBP and a newly identified liver-specific factor: HNF5. C/EBP possesses four binding sites in each GRU; a DNA-binding protein with similar binding specificity has been identified in fibroblasts; this protein could be related to AP-3. HNF5 possesses two binding sites in one GRU and four in the other. There are also HNF5 binding sites in numerous regulatory regions of other liver-specific genes. The interaction of HNF5 with DNA gives a characteristic DNAase I footprint with hypersensitive sites in the middle of the recognition sequence. Some of the C/EBP and HNF5 binding sites overlap in a conserved arrangement.

Freshly excavated fossil bones are best for amplification of ancient DNA.

Despite the enormous potential of analyses of ancient DNA for phylogeographic studies of past populations, the impact these analyses, most of which are performed with fossil samples from natural history museum collections, has been limited to some extent by the inefficient recovery of ancient genetic material. Here we show that the standard storage conditions and/or treatments of fossil bones in these collections can be detrimental to DNA survival. Using a quantitative paleogenetic analysis of 247 herbivore fossil bones up to 50,000 years old and originating from 60 different archeological and paleontological contexts, we demonstrate that freshly excavated and nontreated unwashed bones contain six times more DNA and yield twice as many authentic DNA sequences as bones treated with standard procedures. This effect was even more pronounced with bones from one Neolithic site, where only freshly excavated bones yielded results. Finally, we compared the DNA content in the fossil bones of one animal, a ≈3,200-year-old aurochs, excavated in two separate seasons 57 years apart. Whereas the washed museum-stored fossil bones did not permit any DNA amplification, all recently excavated bones yielded authentic aurochs sequences. We established that during the 57 years when the aurochs bones were stored in a collection, at least as much amplifiable DNA was lost as during the previous 3,200 years of burial. This result calls for a revision of the postexcavation treatment of fossil bones to better preserve the genetic heritage of past life forms.

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Background PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Methodology/Principal Findings Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. Conclusions/Significance There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.

An Antisense RNA Regulates the Bidirectional Silencing Property of the Kcnq1 Imprinting Control Region

ABSTRACT The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

DNA physical properties determine nucleosome occupancy from yeast to fly

Nucleosome positioning plays an essential role in cellular processes by modulating accessibility of DNA to proteins. Here, using only sequence-dependent DNA flexibility and intrinsic curvature, we predict the nucleosome occupancy along the genomes of Saccharomyces cerevisiae and Drosophila melanogaster and demonstrate the predictive power and universality of our model through its correlation with experimentally determined nucleosome occupancy data. In yeast promoter regions, the computed average nucleosome occupancy closely superimposes with experimental data, exhibiting a <200 bp region unfavourable for nucleosome formation bordered by regions that facilitate nucleosome formation. In the fly, our model faithfully predicts promoter strength as encoded in distinct chromatin architectures characteristic of strongly and weakly expressed genes. We also predict that nucleosomes are repositioned by active mechanisms at the majority of fly promoters. Our model uses only basic physical properties to describe the wrapping of DNA around the histone core, yet it captures a substantial part of chromatins structural complexity, thus leading to a much better prediction of nucleosome occupancy than methods based merely on periodic curved DNA motifs. Our results indicate that the physical properties of the DNA chain, and not just the regulatory factors and chromatin-modifying enzymes, play key roles in eukaryotic transcription.

Local DNA demethylation in vertebrates: how could it be performed and targeted?

In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site‐specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid‐dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.

Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000–12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern‐East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000–67 000 ya using an average mammalian microsatellite mutation rate and at 4700–44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70–110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.