Conserved long-range base pairings are associated with pre-mRNA processing of human genes.

The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3'-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation.

Conservation and innovation: Plastome evolution during rapid radiation of Rhodiola on the Qinghai-Tibetan Plateau.

The amount of plastome sequence data available has soared in the last decade, but the nature of plastome evolution during rapid radiations is largely unknown. Moreover, although there is increasing evidence showing that plastomes may have undergone adaptive evolution in order to allow adaptation to various environments, few studies have systematically investigated the role of the plastome in alpine adaptation. To address these questions, we sequenced and analyzed 12 representative species of Rhodiola, a genus which includes ca. 70 perennial herbs mainly growing in alpine habitats in the Qinghai-Tibet Plateau and the Hengduan Mountains. Rapid radiation in this genus was triggered by the uplift of the Qinghai-Tibet Plateau. We also included nine species of Crassulaceae as the outgroups. All plastomes were conserved with respect to size, structure, and gene content and order, with few variations: each contained 134 genes, including 85 protein-coding genes, 37 tRNAs, 8 rRNAs, and 4 potential pseudogenes. Four types of repeat sequence were detected. Slight contraction and expansion of the inverted repeats were also revealed. Both the genome-wide alignment and sequence polymorphism analyses showed that the inverted repeats and coding regions were more conserved than the single-copy regions and the non-coding regions. Positive selection analyses identified three genes containing sites of positive selection (rpl16, ndhA, ndhH), and one gene with a faster than average rate of evolution (psaA). The products of these genes may be involved in the adaptation of Rhodiola to alpine environments such as low CO2 concentration and high-intensity light.

TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis.

DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A-green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A-GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast-ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Evolution of Pentameric Ligand-Gated Ion Channels: Pro-Loop Receptors.

Pentameric ligand-gated ion channels (pLGICs) are ubiquitous neurotransmitter receptors in Bilateria, with a small number of known prokaryotic homologues. Here we describe a new inventory and phylogenetic analysis of pLGIC genes across all kingdoms of life. Our main finding is a set of pLGIC genes in unicellular eukaryotes, some of which are metazoan-like Cys-loop receptors, and others devoid of Cys-loop cysteines, like their prokaryotic relatives. A number of such "Cys-less" receptors also appears in invertebrate metazoans. Together, those findings draw a new distribution of pLGICs in eukaryotes. A broader distribution of prokaryotic channels also emerges, including a major new archaeal taxon, Thaumarchaeota. More generally, pLGICs now appear nearly ubiquitous in major taxonomic groups except multicellular plants and fungi. However, pLGICs are sparsely present in unicellular taxa, suggesting a high rate of gene loss and a non-essential character, contrasting with their essential role as synaptic receptors of the bilaterian nervous system. Multiple alignments of these highly divergent sequences reveal a small number of conserved residues clustered at the interface between the extracellular and transmembrane domains. Only the "Cys-loop" proline is absolutely conserved, suggesting the more fitting name "Pro loop" for that motif, and "Pro-loop receptors" for the superfamily. The infered molecular phylogeny shows a Cys-loop and a Cys-less clade in eukaryotes, both containing metazoans and unicellular members. This suggests new hypotheses on the evolutionary history of the superfamily, such as a possible origin of the Cys-loop cysteines in an ancient unicellular eukaryote. Deeper phylogenetic relationships remain uncertain, particularly around the split between bacteria, archaea, and eukaryotes.

The role of evolutionarily conserved germ-line DH sequence in B-1 cell development and natural antibody production.

Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire.

Molecular cloning and potential function prediction of homologous SOC1 genes in tree peony.

KEY MESSAGE: The central flower integrator PsSOC1 was isolated and its expression profiles were analyzed; then the potential function of PsSOC1 in tree peony was postulated. The six flowering genes PrSOC1, PdSOC1, PsSOC1, PsSOC1-1, PsSOC1-2, and PsSOC1-3 were isolated from Paeonia rockii, Paeonia delavayi, and Paeonia suffruticosa, respectively. Sequence comparison analysis showed that the six genes were highly conserved and shared 99.41% nucleotide identity. Further investigation suggested PsSOC1 was highly homologous to the floral integrators, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), from Arabidopsis. Phylogenetic analysis showed that the SOC1 protein clustering has family specificity and PsSOC1 has a close relationship with homologous SOC1 from Asteraceae species. The studies of PsSOC1's expression patterns in different buds and flower buds, and vegetative organs indicated that PsSOC1 could express in both vegetative and reproductive organs. While the expression of PsSOC1 in different developmental stages of buds was different; high expression levels of PsSOC1 occurred in the bud at the bud sprouting stage and the type I aborted the flower bud. PsSOC1 expression was also shown to be affected by gibberellins (GA), low temperature, and photoperiod. One of the pathways that regulates tree peony flowering may be the GA-inductive pathway. Ectopic expression of PsSOC1 in tobacco demonstrated that greater PsSOC1 expression in the transgenic tobacco plants not only promoted plant growth, but also advanced the flowering time. Finally, the potential function of PsSOC1 in tree peony was postulated.

Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit.

Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.

Conservation of a triple-helix-forming RNA stability element in noncoding and genomic RNAs of diverse viruses.

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold.

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.

The Hippo pathway and apico-basal cell polarity.

The establishment and maintenance of apico-basal cell polarity is a pre-requisite for the formation of a functioning epithelial tissue. Many lines of evidence suggest that cell polarity perturbations favour cancer formation, even though the mechanistic basis for this link remains unclear. Studies in Drosophila have uncovered complex interactions between the conserved Hpo (Hippo) tumour suppressor pathway and apico-basal polarity determinants. The Hpo pathway is a crucial growth regulatory network whose inactivation in Drosophila epithelial tissues induces massive overproliferation. Its core consists of a phosphorylation cascade (comprising the kinases Hpo and Warts) that mediates the inactivation of the pro-growth transcriptional co-activator Yki [Yorkie; YAP (Yes-associated protein) in mammals]. Several apically located proteins, such as Merlin, Expanded or Kibra, have been identified as upstream regulators of the Hpo pathway, leading to the notion that an apical multi-molecular complex modulates core kinase activity and promotes Yki/YAP inactivation. In the present review, we explore the links between apico-basal polarity and Hpo signalling. We focus on the regulation of Yki/YAP by apical proteins, but also on how the Hpo pathway might in turn influence apical domain size as part of a regulatory feedback loop.

Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.

BACKGROUND: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. METHODOLOGY/PRINCIPAL FINDINGS: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. CONCLUSIONS/SIGNIFICANCE: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

Conserved regulatory motifs in osteogenic gene promoters integrate cooperative effects of canonical Wnt and BMP pathways.

Osteoblast differentiation depends on the coordinated network of evolutionary conserved transcription factors during bone formation and homeostasis. Evidence indicates that bone morphogenetic protein (BMP) and Wnt proteins regulate several steps of skeletal development. Here, we provide a molecular description of the cooperative effects of BMP and Wnt canonical pathway on the expression of the early osteogenic genes Dlx5, Msx2, and Runx2 in C2C12 cells, primary cultures of bone marrow-mesenchymal stem cells, and organotypic calvarial cultures. Coordinated regulation of these genes leads to the cooperative activation of their downstream osteogenic target gene osterix. Induction of these genes is mediated through enhancer regions with an evolutionary conserved structure encompassing both Smad and TCF/LEF1 DNA-binding sites. Formation of a cooperative complex is mediated through DNA binding of Smads and TCF4/ß-catenin to their cognate sequences, as well as protein-protein interactions between them. The formation of these cooperative transcriptional complexes results in a more efficient recruitment of coactivators such as p300. We propose that evolutionary conserved regulatory regions in specific osteogenic master genes are key integrative modules during osteogenesis.

Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.

A cis-regulatory signature in ascidians and flies, independent of transcription factor binding sites.

BACKGROUND: Transcription initiation is controlled by cis-regulatory modules. Although these modules are usually made of clusters of short transcription factor binding sites, a small minority of such clusters in the genome have cis-regulatory activity. This paradox is currently unsolved. RESULTS: To identify what discriminates active from inactive clusters, we focused our attention on short topologically unconstrained clusters of two ETS and two GATA binding sites, similar to the early neural enhancer of Ciona intestinalis Otx. We first computationally identified 55 such clusters, conserved between the two Ciona genomes. In vivo assay of the activity of 19 hits identified three novel early neural enhancers, all located next to genes coexpressed with Otx. Optimization of ETS and GATA binding sites was not always sufficient to confer activity to inactive clusters. Rather, a dinucleotide sequence code associated to nucleosome depletion showed a robust correlation with enhancer potential. Identification of a large collection of Ciona regulatory regions revealed that predicted nucleosome depletion constitutes a general signature of Ciona enhancers, which is conserved between orthologous loci in the two Ciona genomes and which partitions conserved noncoding sequences into a major nucleosome-bound fraction and a minor nucleosome-free fraction with higher cis-regulatory potential. We also found this signature in a large fraction of short Drosophila cis-regulatory modules. CONCLUSION: This study indicates that a sequence-based dinucleotide signature, previously associated with nucleosome depletion and independent of transcription factor binding sites, contributes to the definition of a local cis-regulatory potential in two metazoa, Ciona intestinalis and Drosophila melanogaster.

Ultradeep sequencing of a human ultraconserved region reveals somatic and constitutional genomic instability.

Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.

A conserved lysine in the thyroid hormone receptor-alpha1 DNA-binding domain, mutated in hepatocellular carcinoma, serves as a sensor for transcriptional regulation.

Nuclear receptors are hormone-regulated transcription factors that play key roles in normal physiology and development; conversely, mutant nuclear receptors are associated with a wide variety of neoplastic and endocrine disorders. Typically, these receptor mutants function as dominant negatives and can interfere with wild-type receptor activity. Dominant-negative thyroid hormone receptor (TR) mutations have been identified in over 60% of the human hepatocellular carcinomas analyzed. Most of these mutant TRs are defective for corepressor release or coactivator binding in vitro, accounting for their transcriptional defects in vivo. However, two HCC-TR mutants that function as dominant-negative receptors in cells display near-normal properties in vitro, raising questions about the molecular basis behind their transcriptional defects. We report here that a single amino acid substitution, located at the same position in the DNA-binding domain of both mutants, is responsible for their impaired transcriptional activation and dominant-negative properties. Significantly, this amino acid, K74 in TRalpha, is highly conserved in all known nuclear receptors and seems to function as an allosteric sensor that regulates the transcriptional activity of these receptors in response to binding to their DNA recognition sequences. We provide evidence that these two human hepatocellular carcinoma mutants have acquired dominant-negative function as a result of disruption of this allosteric sensing. Our results suggest a novel mechanism by which nuclear receptors can acquire transcriptional defects and contribute to neoplastic disease.