XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast.

Non-coding (nc)RNAs are key players in numerous biological processes such as gene regulation, chromatin domain formation and genome stability. Large ncRNAs interact with histone modifiers and are involved in cancer development, X-chromosome inactivation and autosomal gene imprinting. However, despite recent evidence showing that pervasive transcription is more widespread than previously thought, only a few examples mediating gene regulation in eukaryotes have been described. In Saccharomyces cerevisiae, the bona-fide regulatory ncRNAs are destabilized by the Xrn1 5'-3' RNA exonuclease (also known as Kem1), but the genome-wide characterization of the entire regulatory ncRNA family remains elusive. Here, using strand-specific RNA sequencing (RNA-seq), we identify a novel class of 1,658 Xrn1-sensitive unstable transcripts (XUTs) in which 66% are antisense to open reading frames. These transcripts are polyadenylated and RNA polymerase II (RNAPII)-dependent. The majority of XUTs strongly accumulate in lithium-containing media, indicating that they might have a role in adaptive responses to changes in growth conditions. Notably, RNAPII chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis of Xrn1-deficient strains revealed a significant decrease of RNAPII occupancy over 273 genes with antisense XUTs. These genes show an unusual bias for H3K4me3 marks and require the Set1 histone H3 lysine 4 methyl-transferase for silencing. Furthermore, abolishing H3K4me3 triggers the silencing of other genes with antisense XUTs, supporting a model in which H3K4me3 antagonizes antisense ncRNA repressive activity. Our results demonstrate that antisense ncRNA-mediated regulation is a general regulatory pathway for gene expression in S. cerevisiae.

Linking the DNA strand asymmetry to the spatio-temporal replication program: II. Accounting for neighbor-dependent substitution rates.

In paper I, we addressed the impact of the spatio-temporal program on the DNA composition evolution in the case of time homogeneous and neighbor-independent substitution rates. But substitution rates do depend on the flanking nucleotides as exemplified in vertebrates where CpG sites are hypermutable so that the substitution rate C --> T depends dramatically (ten fold) on whether the cytosine belongs to a CG dinucleotide or not. With the specific goal to account for neighbor-dependence, we revisit our minimal modeling of neutral substitution rates in the human genome. When assuming that r = CpG --> TpG and its reverse complement r(c) = CpG --> CpA are (by far) the main neighbor-dependent substitution rates, we demonstrate, using perturbative analysis, that neighbor-dependence does not affect the decomposition of the compositional asymmetry into a transcription- and a replication-associated components, the former increases in magnitude with transcription rate and changes sign with gene orientation, whereas the latter is proportional to the replication fork polarity. Indeed the neighbor dependence case differs from the neighbor-independent model by an additional source term related to the CG dinucleotide content in both the transcription and replication-associated components. We finally discuss the case of time-dependent substitution rates confirming as a very general result the fact that the skew can still be decomposed into a transcription- and a replication-associated components.

Linking the DNA strand asymmetry to the spatio-temporal replication program. I. About the role of the replication fork polarity in genome evolution.

Two key cellular processes, namely transcription and replication, require the opening of the DNA double helix and act differently on the two DNA strands, generating different mutational patterns (mutational asymmetry) that may result, after long evolutionary time, in different nucleotide compositions on the two DNA strands (compositional asymmetry). We elaborate on the simplest model of neutral substitution rates that takes into account the strand asymmetries generated by the transcription and replication processes. Using perturbation theory, we then solve the time evolution of the DNA composition under strand-asymmetric substitution rates. In our minimal model, the compositional and substitutional asymmetries are predicted to decompose into a transcription- and a replication-associated components. The transcription-associated asymmetry increases in magnitude with transcription rate and changes sign with gene orientation while the replication-associated asymmetry is proportional to the replication fork polarity. These results are confirmed experimentally in the human genome, using substitution rates obtained by aligning the human and chimpanzee genomes using macaca and orangutan as outgroups, and replication fork polarity determined in the HeLa cell line as estimated from the derivative of the mean replication timing. When further investigating the dynamics of compositional skew evolution, we show that it is not at equilibrium yet and that its evolution is an extremely slow process with characteristic time scales of several hundred Myrs.

Thermodynamics of intragenic nucleosome ordering.

The nucleosome ordering observed in vivo along yeast genes is described by a thermodynamical model of nonuniform fluid of 1D hard rods confined by two excluding energy barriers at gene extremities. For interbarrier distances L less than or approximately equal to 1.5 kbp, nucleosomes equilibrate into a crystal-like configuration with a nucleosome repeat length (NRL) L/n approximately 165 bp, where n is the number of regularly positioned nucleosomes. We also observe "bistable" genes with a fuzzy chromatin resulting from a statistical mixing of two crystal states, one with an expanded chromatin (NRL approximately L/n) and the other with a compact one (NRL approximately L/(n+1)). By means of single nucleosome switching, bistable genes may drastically alter their expression level as suggested by their higher transcriptional plasticity. These results enlighten the role of the intragenic chromatin on gene expression regulation.

Bifractality of human DNA strand-asymmetry profiles results from transcription.

We use the wavelet transform modulus maxima method to investigate the multifractal properties of strand-asymmetry DNA walk profiles in the human genome. This study reveals the bifractal nature of these profiles, which involve two competing scale-invariant (up to repeat-masked distances less, or similar 40 kbp) components characterized by Hölder exponents h{1}=0.78 and h{2}=1, respectively. The former corresponds to the long-range-correlated homogeneous fluctuations previously observed in DNA walks generated with structural codings. The latter is associated with the presence of jumps in the original strand-asymmetry noisy signal S. We show that a majority of upward (downward) jumps co-locate with gene transcription start (end) sites. Here 7228 human gene transcription start sites from the refGene database are found within 2 kbp from an upward jump of amplitude DeltaS > or = 0.1 which suggests that about 36% of annotated human genes present significant transcription-induced strand asymmetry and very likely high expression rate.

High levels of tyrosine phosphorylated proto-ret in sporadic phenochromocytomas.

Pheochromocytomas are tumors originating from chromaffin cells, the large majority of which are sporadic neoplasms. The genetic and molecular events determining their tumorigenesis continue to remain unknown. On the other hand, RET germ-line mutations cause the inheritance of familial tumors in multiple endocrine neoplasia (MEN)-2 diseases, which account for a minority of pheochromocytomas. We investigated the expression of the RET gene in 14 sporadic tumors harboring no activating mutations. A subset of highly RET-expressing tumors (50%) could be distinguished. They showed RET transcript, protein amounts as well as Ret-associated phosphotyrosine levels similar to those measured in MEN-2A-associated pheochromocytomas. We also determined the GDNF and GDNF family receptor alpha (GFRalpha)-1 transcript levels in tumors and in normal tissues. Whereas the GFRalpha-1 transcripts were detected at similar levels in normal tissues and in tumors, GDNF was frequently found expressed in sporadic tumors at levels several times higher than in controls. These results led us to propose the existence of an autocrine or paracrine loop leading to chronic stimulation of the Ret signaling pathway, which could participate in the pathogenesis of a number of sporadic pheochromocytomas.

Prediction of rho-independent Escherichia coli transcription terminators. A statistical analysis of their RNA stem-loop structures.

Escherichia coli rho-independent transcription terminators are characterized by an RNA structure having a G+C-rich stem-loop followed by a series of uridine residues, but they can be only partially predicted by the stability of this structure or by its primary sequence. A large number of such terminators have been identified or proposed in the literature, and we have constituted a list of them (148 found in 1021 x 10(3) base-pairs of E. coli DNA sequences) in order to analyze statistically the corresponding RNA hairpins. We show that the size of the loops presents a narrow distribution, that their sequences are not random, and that most loops are closed by a C.G base-pair. In particular, 55% of the loops are tetranucleotides and the most abundant loop sequences are UUCG and GAAA. These loops are abundant in prokaryotic and eukaryotic RNAs, and are known to enhance the stability of RNA hairpins. We propose that these tetraloops play an important role in the nucleation of the nascent RNA structures, as does also the presence of a C.G base-pair closing a hairpin loop. This analysis allows us to propose a model of formation of an RNA hairpin during the termination process and to construct an algorithm of prediction of the terminators in a given DNA sequence. For the E. coli sequences, it clearly distinguishes inter- from intracistronic terminator-like structures, and selects 141 of the 148 rho-independent terminators given in the literature, with a very low background. It also predicts with reasonable accuracy the in vitro termination efficiency of known rho-independent terminators, as well as predicting the existence of 35 as yet uncharacterized terminators.

Transcription-coupled TA and GC strand asymmetries in the human genome.

Analysis of the whole set of human genes reveals that most of them present TA and GC skews, that these biases are correlated to each other and are specific to gene sequences, exhibiting sharp transitions between transcribed and non-transcribed regions. The GC asymmetries cannot be explained solely by a model previously proposed for (G+T) skew based on transitions measured in a small set of human genes. We propose that the GC skew results from additional transcription-coupled mutation process that would include transversions. During evolution, both processes acting on a large majority of genes in germline cells would have produced these transcription-coupled strand asymmetries.

Exon prediction in eucaryotic genomes.

Two independent computer systems, NetPlantGene and AMELIE, dedicated to the identification of splice sites in plant and human genomes, respectively, are introduced here. Both methods were designed in relation to experimental work; they rely on automatically generated rules involving the nucleotide content of sequences regardless of the coding properties of exons. The specificity of plant sequences as considered in NetPlantGene is shown to enhance the quality of detection as opposed to general methods such as GRAIL. A scanning model of the acceptor site recognition is being simulated by AMELIE leading to a relatively accurate selection process of sites.

Influence of the sequence on elastic properties of long DNA chains.

We revisit the results of single-molecule DNA stretching experiments using a rodlike chain (RLC) model that explicitly includes some intrinsic structural disorder induced by the sequence. The investigation of artificial and real genomic sequences shows that the wormlike chain model reproduces quite well the data but with an effective bend stiffness A(eff), which underestimates the true elastic bend stiffness A, independently of the elastic twist stiffness C. Mainly dominated by the amplitude of the structural disorder, this correction seems rather insensitive to the presence of long-range correlations. This RLC model is shown to remarkably fit the experimental data for lambda-DNA when considering A approximately 70+/-10 nm (>A(eff) approximately 50 nm), in good agreement with previous experimental estimates of the "dynamic" persistent length. From the analysis of large human contigs, we speculate about the possible dependence of A(eff) and/or A upon the (G+C) content of the considered sequence.

DNA replication timing data corroborate in silico human replication origin predictions.

We develop a wavelet-based multiscale pattern recognition methodology to disentangle the replication- from the transcription-associated compositional strand asymmetries observed in the human genome. Comparing replication skew profiles to recent high-resolution replication timing data reveals that most of the putative replication origins that border the so-identified replication domains are replicated earlier than their surroundings whereas the central regions replicate late in the S phase. We discuss the implications of this first experimental confirmation of these replication origin predictions that are likely to be early replicating and active in most tissues.

Formation and positioning of nucleosomes: effect of sequence-dependent long-range correlated structural disorder.

The understanding of the long-range correlations (LRC) observed in DNA sequences is still an open and very challenging problem. In this paper, we start reviewing recent results obtained when exploring the scaling properties of eucaryotic, eubacterial and archaeal genomic sequences using the space-scale decomposition provided by the wavelet transform (WT). These results suggest that the existence of LRC up to distances approximately 20-30 kbp is the signature of the nucleosomal structure and dynamics of the chromatin fiber. Actually the LRC are mainly observed in the DNA bending profiles obtained when using some structural coding of the DNA sequences that accounts for the fluctuations of the local double-helix curvature within the nucleosome complex. Because of the approximate planarity of nucleosomal DNA loops, we then study the influence of the LRC structural disorder on the thermodynamical properties of 2D elastic chains submitted locally to mechanical/topological constraint as loops. The equilibrium properties of the one-loop system are derived numerically and analytically in the quite realistic weak-disorder limit. The LRC are shown to favor the spontaneous formation of small loops, the larger the LRC, the smaller the size of the loop. We further investigate the dynamical behavior of such a loop using the mean first passage time (MFPT) formalism. We show that the typical short-time loop dynamics is superdiffusive in the presence of LRC. For displacements larger than the loop size, we use large-deviation theory to derive a LRC-dependent anomalous-diffusion rule that accounts for the lack of disorder self-averaging. Potential biological implications on DNA loops involved in nucleosome positioning and dynamics in eucaryotic chromatin are discussed.

From DNA sequence analysis to modeling replication in the human genome.

We explore the large-scale behavior of nucleotide compositional strand asymmetries along human chromosomes. As we observe for 7 of 9 origins of replication experimentally identified so far, the (TA+GC) skew displays rather sharp upward jumps, with a linear decreasing profile in between two successive jumps. We present a model of replication with well positioned replication origins and random terminations that accounts for the observed characteristic serrated skew profiles. We succeed in identifying 287 pairs of putative adjacent replication origins with an origin spacing approximately 1-2 Mbp that are likely to correspond to replication foci observed in interphase nuclei and recognized as stable structures that persist throughout subsequent cell generations.

T4-induced antipolarity: temporal heterogeneity in response of early transcription units.

When T4 infects Escherichia coli in the absence of protein synthesis, rho-mediated termination takes place on early polycistronic transcription units. During the early period of development, the appearance of delayed early transcripts becomes insensitive to the inhibition of protein synthesis. In the absence of the T4 gene product mot, an inducer for the middle mode of transcription, only the early polycistronic messengers are synthesized. In mot- -infected cells, the synthesis of the distal transcripts still becomes completely insensitive to the polar effect of chloramphenicol. This happens because potential rho-sensitive termination sites are not used in these cells. In this respect, overcoming polarity induced by chloramphenicol can be called a process of antitermination. The mot-independent antitermination can be studied by addition of chloramphenicol during infections with mot- bacteriophage. The effect is stable; it allows a constant percentage of rho-sensitive termination sites in the cell to be traversed by RNA polymerase for at least 10 min at 42 degrees C. By examining six different transcription units on the T4 genome, we find that each transcription unit has a cis-acting component (or components) which determines when its rho-sensitive termination site stops functioning. In extreme cases, rho acts with 100% efficiency in some transcription units, whereas it is almost inactive in others.

In vitro system for induction of delayed early RNA of bacteriophage T4.

Concentrated lysates of Escherichia coli that had been infected with bacteriophage T4 in the presence of chloramphenicol show the same restriction of transcription in vitro as is found in vivo. Restricted lysates can be complemented with lysates from infected cells to induce production of delayed early RNA. Complementation takes place between the RNA polymerase of the restricted lysate and the DNA of the unrestricted lysate. We present evidence that delayed early RNA in these lysates is initiated at quasi-late (middle) promoters, and that such recognition is related to changes in the state of the template DNA.

A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis.

The role of cbiK, a gene found encoded within the Salmonella typhimurium cob operon, has been investigated by studying its in vivo function in Escherichia coli. First, it was found that cbiK is not required for cobalamin biosynthesis in the presence of a genomic cysG gene (encoding siroheme synthase) background. Second, in the absence of a genomic cysG gene, cobalamin biosynthesis in E. coli was found to be dependent upon the presence of cobA(P. denitrificans) (encoding the uroporphyrinogen III methyltransferase from Pseudomonas denitrificans) and cbiK. Third, complementation of the cysteine auxotrophy of the E. coli cysG deletion strain 302delta a could be attained by the combined presence of cobA(P. denitrificans) and the S. typhimurium cbiK gene. Collectively these results suggest that CbiK can function in fashion analogous to that of the N-terminal domain of CysG (CysG(B)), which catalyzes the final two steps in siroheme synthesis, i.e., NAD-dependent dehydrogenation of precorrin-2 to sirohydrochlorin and ferrochelation. Thus, phenotypically CysG(B) and CbiK have very similar properties in vivo, although the two proteins do not have any sequence similarity. In comparison to CysG, CbiK appears to have a greater affinity for Co2+ than for Fe2+, and it is likely that cbiK encodes an enzyme whose primary role is that of a cobalt chelatase in corrin biosynthesis.

Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis.

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-l-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

New species of human tyrosine hydroxylase mRNA are produced in variable amounts in adrenal medulla and are overexpressed in progressive supranuclear palsy.

Alternative splicing of human tyrosine hydroxylase (TH) pre-mRNA produces four mRNAs leading to four different TH isoforms and is thought to have important regulatory functions. We show that the diversity of TH mRNAs is greater than previously described in the autonomous nervous system: New splice junctions corresponding to the skipping of exon 3 were identified by amplification of cDNA synthesized from pheochromocytoma RNA. In all cases the reading frame was maintained. These species were assayed by RNase protection experiments; their abundance (4-6%) was comparable to that of the previously identified human TH-3 and -4 species in normal adrenal medulla. However, higher levels (11-34%) of these species were found in adrenal medullas of patients suffering from progressive supranuclear palsy. Whether such changes are specific to the disease or the consequences of the stress associated with this severe neurodegeneration remains to be established.

Low frequency rhythms in human DNA sequences: a key to the organization of gene location and orientation?

We explore large-scale nucleotide compositional fluctuations of the human genome using multiresolution techniques. Analysis of the GC content and of the AT and GC skews reveals the existence of rhythms with two main periods of 110+/-20 kb and 400+/-50 kb that enlighten a remarkable cooperative gene organization. We show that the observed nonlinear oscillations are likely to display all the characteristic features of chaotic strange attractors which suggests a very attractive deterministic picture: gene orientation and location, in relation with the structure and dynamics of chromatin, might be governed by a low-dimensional nonlinear dynamical system.

Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon.

A 16 kb DNA fragment has been isolated from a Bacillus megaterium genomic library and fully sequenced. The fragment contains 15 open reading frames, 14 of which are thought to constitute a B. megaterium cobalamin biosynthetic (cob) operon. Within the operon, 11 genes display similarity to previously identified Salmonella typhimurium cobalamin biosynthetic genes (cbiH60, -J, -C, -D, -ET, -L, -F, -G, -A, cysGA and btuR), whereas three do not (cbiW, -X and -Y). The genes of the B. megaterium cob operon were compared with the cobalamin biosynthetic genes of Pseudomonas denitrificans, Methanococcus jannaschii and Synechocystis sp. Taking into account the presence of cbiD and cbiG, the absence of a cobF, cobG and cobN, -S and -T, it was concluded that B. megaterium, M. jannaschii and Synechocystis sp., like S. typhimurium, synthesize cobalamin by an anaerobic pathway, in which cobalt is added at an early stage and molecular oxygen is not required.