A multi-tissue analysis identifies HLA complex group 9 gene methylation differences in bipolar disorder.

Epigenetic studies of DNA and histone modifications represent a new and important activity in molecular investigations of human disease. Our previous epigenome-wide scan identified numerous DNA methylation differences in post-mortem brain samples from individuals affected with major psychosis. In this article, we present the results of fine mapping DNA methylation differences at the human leukocyte antigen (HLA) complex group 9 gene (HCG9) in bipolar disorder (BPD). Sodium bisulfite conversion coupled with pyrosequencing was used to interrogate 28 CpGs spanning ∼700 bp region of HCG9 in 1402 DNA samples from post-mortem brains, peripheral blood cells and germline (sperm) of bipolar disease patients and controls. The analysis of nearly 40 000 CpGs revealed complex relationships between DNA methylation and age, medication as well as DNA sequence variation (rs1128306). Two brain tissue cohorts exhibited lower DNA methylation in bipolar disease patients compared with controls at an extended HCG9 region (P=0.026). Logistic regression modeling of BPD as a function of rs1128306 genotype, age and DNA methylation uncovered an independent effect of DNA methylation in white blood cells (odds ratio (OR)=1.08, P=0.0077) and the overall sample (OR=1.24, P=0.0011). Receiver operating characteristic curve A prime statistics estimated a 69-72% probability of correct BPD prediction from a case vs control pool. Finally, sperm DNA demonstrated a significant association (P=0.018) with BPD at one of the regions demonstrating epigenetic changes in the post-mortem brain and peripheral blood samples. The consistent multi-tissue epigenetic differences at HCG9 argue for a causal association with BPD.

A high-resolution map of human chromosome 12.

Our sequence-tagged site-content map of chromosome 12 is now integrated with the whole-genome fingerprinting effort. It provides accurate and nearly complete bacterial clone coverage of chromosome 12. We propose that this integrated mapping protocol serves as a model for constructing physical maps for entire genomes.

Large-scale human promoter mapping using CpG islands.

Vertebrate genomic DNA is generally CpG depleted, possibly because methylation of cytosines at 80% of CpG dinucleotides results in their frequent mutation to thymine, and thus CpG to TpG dinucleotides. There are, however, genomic regions of high G+C content (CpG islands), where the occurrence of CpGs is significantly higher, close to the expected frequency, whereas the methylation concentration is significantly lower than the overall genome. CpG islands are longer than 200 bp and have over 50% of G+C content and CpG frequency, at least 0.6 of that statistically expected. Approximately 50% of mammalian gene promoters are associated with one or more CpG islands. Although biologists often intuitively use CpG islands for 5' gene identification, this has not been rigorously quantified. We have determined the features that discriminate the promoter-associated and non-associated CpG islands. This led to an effective algorithm for large-scale promoter mapping (with 2-kb resolution) with a concentration of false-positive predictions of promoters much lower than previously obtained. Using this algorithm, we correctly discriminated approximately 85% of the CpG islands within an interval (-500 to +1500) around a transcriptional start site (TSS) from those that lie further away from TSSs. We also correctly mapped approximately 93% of the promoters containing CpG islands.

Periodical distribution of transcription factor sites in promoter regions and connection with chromatin structure.

Nucleosomes regulate transcriptional initiation when positioned in the promoter area. This may require the transcription factor (TF) sites to be correlated with the nucleosome positions and phased on the nucleosome surface. If this is the case, one would expect a periodical distribution of TF sites in the vicinity of promoters, with the nucleosomal period of 10.1-10.5 bp. We examined the distributions of putative binding sites of 323 different TFs along 1, 057 sequences of the Eukaryotic Promoter Database (release 50) [Cavin Perier, R., Junier, T. & Bucher, P. (1998) Nucleic Acids Res. 26, 353-357] and of 218 TFs on 673 sequences of the Lead Exon Database of human promoter sequences. We obtained a statistically significant overrepresentation of TF sites distributed with the main period of 10.1-10.5 bp in the region -50 to +120 around the transcription start site and in few locations nearby. Correlation of the positioning of the TF sites with the nucleosomes is further reinforced by sequence-directed mapping of the nucleosomes, a method previously developed.

Interdependence between DNA template secondary structure and priming efficiencies of short primers.

Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.

Enhancement of the nucleosomal pattern in sequences of lower complexity.

Intuitively, the complexity of a given DNA sequence is related to the number of various superimposed biological messages it contains. Here we assess the expectation that in nucleosome DNA sequences of lower linguistic complexity, the nucleosome DNA positioning pattern would be more pronounced than in those of higher linguistic complexity. The nucleosome DNA positioning pattern is one of the weakest (highly degenerate) sequence patterns. It has been extracted recently by specially designed multiple alignment procedures. We applied the most sensitive of these procedures to nearly equal subsets of a nucleosome database separated according to linguistic complexity. The pattern extracted from the subset of the simpler nucleosome sequences not only possesses all major attributes of the known nucleosomal pattern, but is substantially stronger with respect to amplitude in comparison with the total database. This result constitutes the first demonstration that a weak pattern can be significantly enhanced by selective treatment of a lower complexity subset of the sequence ensemble under consideration.

Applicability of the multiple alignment algorithm for detection of weak patterns: periodically distributed DNA pattern as a study case.

MOTIVATION: A nucleosome DNA positioning pattern is known to be one of the weakest (highly degenerated) patterns. The alignment procedure that has been developed recently for the extraction of such a pattern is based on a statistical matching of the sequences, and its success depends on the pattern/background ratio in the individual sequences and in the generated pattern. The heuristic nature of the method and distinctive properties of the pattern bring up the question of efficiency and sensitivity in the procedure. This paper presents a method of verification for this multiple sequence alignment algorithm. RESULTS: To verify the applicability of the multiple alignment approach, we constructed a set of sequences carrying the hidden pattern. The pattern was presented by weak ('signal') oscillations of occurrences of AA and TT dinucleotides along otherwise random sequences. Only a few dinucleotides of any given 145 base long sequence would correspond to the signal, appearing in about the same phase within the simulated periodic pattern. The novelty of our simulation approach is that we simulated a database as a whole, as opposed to simulating each sequence separately. The correlation between the hidden pattern and a sequence from the database is negligible on average, but our statistical multicycle alignment procedure produced the pattern with attributes very close to the simulated ones. The accuracy of the procedure was tested and calibrated. The presence in a typical sequence of as little as three dinucleotides corresponding to the signal is sufficient to generate (detect) the pattern hidden in a collection of 204 sequences.

Nucleosome DNA sequence pattern revealed by multiple alignment of experimentally mapped sequences.

Five different algorithms have been applied for detecting DNA sequence pattern hidden in 204 DNA sequences collected from the literature which are experimentally found to be involved in nucleosome formation. Each algorithm was used to perform a multiple alignment of the nucleosome DNA sequences within the window 145 nt, the size of a nucleosome core DNA. From these alignments five pairs of AA and TT dinucleotide positional frequency distributions have been computed. The frequency profiles calculated by different algorithms are rather different due to substantial noise. They, however, share several important features. Both AA and TT dinucleotide positional frequencies display periodicity with the period of 10.3(+/- 0.2) bases. TT dinucleotides appear to be distributed symmetrically relative to AA dinucleotides of the same DNA strand, with the center of symmetry at the midpoint of the nucleosome core DNA. The phase shift between the AA and TT patterns is about 6 bp. Superposition of the five pairs of the AA (TT) positional frequency profiles has produced the refined pattern, with the above features well pronounced. An interesting novel feature of the pattern is an absence of central peaks in the periodical AA and TT distributions. This may indicate that the central section of nucleosome DNA, 15 bp around the dyad axis of the nucleosome, is not bent. Positional distributions of other dinucleotides were not found in this study to be as informative as the ones for AA and TT.

Preferred positions of AA and TT dinucleotides in aligned nucleosomal DNA sequences.

Multiple alignment of 118 nucleosomal DNA sequences by maximizing simultaneously match of AA dinucleotides and match of TT dinucleotides results in a pattern of the dinucleotide distributions which is characteristic of the nucleosomal DNA sequences. The AA dinucleotides are found to be distributed symmetrically relative to the TT dinucleotide distribution, around the middle point of the nucleosomal DNA sequence. The distances between major peaks of the distributions are multiples of about 10.4 bases. The peaks of the TT distribution are shifted by 6 bases downstream from the peaks of the AA distribution.