Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. © 2009 Corcoran et al

Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i-->i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, alpha-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable alpha-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ~9 kcal/mol, but this was compensated by increased conformational entropy (TDS ≤ 7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by alpha-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases

Intragenic DNA methylation: implications of this epigenetic mechanism for cancer research

Epigenetics is the study of all mechanisms that regulate gene transcription and genome stability that are maintained throughout the cell division, but do not include the DNA sequence itself. The best-studied epigenetic mechanism to date is DNA methylation, where methyl groups are added to the cytosine base within cytosine–guanine dinucleotides (CpG sites). CpGs are frequently clustered in high density (CpG islands (CGIs)) at the promoter of over half of all genes. Current knowledge of transcriptional regulation by DNA methylation centres on its role at the promoter where unmethylated CGIs are present at most actively transcribed genes, whereas hypermethylation of the promoter results in gene repression. Over the last 5 years, research has gradually incorporated a broader understanding that methylation patterns across the gene (so-called intragenic or gene body methylation) may have a role in transcriptional regulation and efficiency. Numerous genome-wide DNA methylation profiling studies now support this notion, although whether DNA methylation patterns are a cause or consequence of other regulatory mechanisms is not yet clear. This review will examine the evidence for the function of intragenic methylation in gene transcription, and discuss the significance of this in carcinogenesis and for the future use of therapies targeted against DNA methylation

Vaccination with DNA plasmids expressing Gn coupled to C3d or alphavirus replicons expressing Gn protects mice against rift valley fever virus

Background: Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent. Methodology/Principal Findings: We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12. Conclusion/Significance: These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use. © 2010 Bhardwaj et al

TOPOLOGICAL DEFECTS AND HIGHEST ENERGY COSMIC AND GAMMA RAYS

In this paper we review the hypothesis that a considerable part of the cosmic ray flux observed above about 10^{19}\eV may be produced by decaying or annihilating topological defects left over from phase transitions in the early universe at grand unification energy scales (\approx10^{16}\GeV). Possible signatures of cosmic ray producing defect models are discussed which could be tested experimentally in the near future. We thereby focus on model independent universal spectral properties of the predicted particle fluxes.Comment: 11 pages of uuencoded compressed postscript, including 3 figures, to be published in Space Science Reviews

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

DNA Methylation of the First Exon Is Tightly Linked to Transcriptional Silencing

Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude

Ancient Ancestry of KFDV and AHFV Revealed by Complete Genome Analyses of Viruses Isolated from Ticks and Mammalian Hosts

Alkhurma hemorrhagic fever (AHF) and Kyasanur Forest disease (KFD) viruses both cause serious and sometimes fatal human disease in their respective ranges, Saudi Arabia and India. AHFV was first identified in the mid-1990s and due to its strong genetic similarity to KFDV it has since been considered the result of a recent introduction of KFDV into Saudi Arabia. To gain a better understanding of the evolutionary history of AHFV and KFDV, we sequenced the full-length genomes of 3 KFDV and 16 AHFV. Sequence analyses show a greater genetic diversity within AHFV than previously thought, particularly within the tick population. The phylogeny constructed with these 19 full-length sequences and two AHFV sequences from GenBank indicates AHFV diverged from KFDV almost 700 years ago. Given the presence of competent tick vectors in the regions between and surrounding Saudi Arabia and India and the recent identification of AHFV in Egypt, these results suggest a broader geographic range of AHFV and KFDV, and raise the possibility of other AHFV/KFDV–like viruses circulating in these regions

Mice have a transcribed L-threonine aldolase/GLY1 gene, but the human GLY1 gene is a non-processed pseudogene

BACKGROUND: There are three pathways of L-threonine catabolism. The enzyme L-threonine aldolase (TA) has been shown to catalyse the conversion of L-threonine to yield glycine and acetaldehyde in bacteria, fungi and plants. Low levels of TA enzymatic activity have been found in vertebrates. It has been suggested that any detectable activity is due to serine hydroxymethyltransferase and that mammals lack a genuine threonine aldolase. RESULTS: The 7-exon murine L-threonine aldolase gene (GLY1) is located on chromosome 11, spanning 5.6 kb. The cDNA encodes a 400-residue protein. The protein has 81% similarity with the bacterium Thermotoga maritima TA. Almost all known functional residues are conserved between the two proteins including Lys242 that forms a Schiff-base with the cofactor, pyridoxal-5'-phosphate. The human TA gene is located at 17q25. It contains two single nucleotide deletions, in exons 4 and 7, which cause frame-shifts and a premature in-frame stop codon towards the carboxy-terminal. Expression of human TA mRNA was undetectable by RT-PCR. In mice, TA mRNA was found at low levels in a range of adult tissues, being highest in prostate, heart and liver. In contrast, serine/threonine dehydratase, another enzyme that catabolises L-threonine, is expressed very highly only in the liver. Serine dehydratase-like 1, also was most abundant in the liver. In whole mouse embryos TA mRNA expression was low prior to E-15 increasing more than four-fold by E-17. CONCLUSION: Mice, the western-clawed frog and the zebrafish have transcribed threonine aldolase/GLY1 genes, but the human homolog is a non-transcribed pseudogene. Serine dehydratase-like 1 is a putative L-threonine catabolising enzyme