TTS mapping: integrative WEB tool for analysis of triplex formation target DNA sequences, G-quadruplets and non-protein coding regulatory DNA elements in the human genome

Abstract

Background: DNA triplexes can naturally occur, co-localize and interact with many other regulatory DNA elements (e.g. G-quadruplex (G4) DNA motifs), specific DNA-binding proteins (e.g. transcription factors (TFs)), and micro-RNA (miRNA) precursors. Specific genome localizations of triplex target DNA sites (TTSs) may cause abnormalities in a double-helix DNA structure and can be directly involved in some human diseases. However, genome localization of specific TTSs, their interconnection with regulatory DNA elements and physiological roles in a cell are poor defined. Therefore, it is important to identify comprehensive and reliable catalogue of specific potential TTSs (pTTSs) and their co-localization patterns with other regulatory DNA elements in the human genome.

Results: "TTS mapping" database is a web-based search engine developed here, which is aimed to find and annotate pTTSs within a region of interest of the human genome. The engine provides descriptive statistics of pTTSs in a given region and its sequence context. Different annotation tracks of TTS-overlapping gene region(s), G4 motifs, CpG Island, miRNA precursors, miRNA targets, transcription factor binding sites (TFBSs), Single Nucleotide Polymorphisms (SNPs), small nucleolar RNAs (snoRNA), and repeat elements are also mapped based onto a sequence location provided by UCSC genome browser, G4 database http://www.quadruplex.org and several other datasets. The results pages provide links to UCSC genome browser annotation tracks and relative DBs. BLASTN program was included to check the uniqueness of a given pTTS in the human genome. Recombination- and mutation-prone genes (e.g. EVI-1, MYC) were found to be significantly enriched by TTSs and multiple co-occurring with our regulatory DNA elements. TTS mapping reveals that a high-complementary and evolutionarily conserved polypurine and polypyrimidine DNA sequence pair linked by a non-conserved short DNA sequence can form miR-483 transcribed from intron 2 of IGF2 gene and bound double-strand nucleic acid TTSs forming natural triplex structures.

Conclusion: TTS mapping provides comprehensive visual and analytical tools to help users to find pTTSs, G-quadruplets and other regulatory DNA elements in various genome regions. TTS Mapping not only provides sequence visualization and statistical information, but also integrates knowledge about co-localization TTS with various DNA elements and facilitates that data analysis. In particular, TTS Mapping reveals complex structural-functional regulatory module of gene IGF2 including TF MZF1 binding site and ncRNA precursor mir-483 formed by the high-complementary and evolutionarily conserved polypurine- and polypyrimidine-rich DNA pair. Such ncRNAs capable of forming helical triplex structures with a polypurine strand of a nucleic acid duplexes (DNA or RNA) via Hoogsteen or reverse Hoogsteen hydrogen bonds. Our web tool could be used to discover biologically meaningful genome modules and to optimize experimental design of anti-gene treatment.

Figure 1

Schema of three triplex motifs bind to TTS by different orientation. This schema shows three triplex motifs of third sequence strand which can form triplex structure with polypurine of duplex (or TTS) via Hoogsteen or reverse Hoogsteen hydrogen bond. In CT motif, the third strand is parallel with TTS by forming Hoogsteen hydrogen bond. In the GT motif, the third strand can be either anti-parallel to the third strand by forming reverse Hoogsteen or parallel by forming Hoogsteen hydrogen bond. In GA motif, the third strand is anti-parallel with TTS by forming.

Figure 2

Schema of data flow in TTS mapping. A chromosome coordinate is used to initiate a search of pTTSs in a given region and map pTTSs to several annotation tracks. The pTTSs search parameters should be provided by user or by default. The results of the search are reported in a summary table and also forward to map the given pTTS to selected annotation tracks. After mapping all pTTSs to available annotation tracks, the number of found annotation tracks, the number of the annotation tracks overlapped with these pTTSs, and number of the pTTSs overlapped with the annotation tracks are reported to summary table.

Figure 3

The interface page of TTS mapping tools. To run the program, user has to define chromosome region and parameters for TTS search. "TTS density" option could be used to show TTS density in the running window with a given span and window moving step.

Figure 4

The summary tables of pTTSs search and mapping in a human genome region. The results display summary tables of: chromosome position, TTS search parameters, statistics of pTTSs found, the gene appearing in the given chromosome position, and the data of selected annotation tracks found in the chromosome region.

Figure 5

The list of pTTSs and integrative annotation information. The list of to TTS ID which describe the descriptive information of each pTTSs and integrate annotation tracks overlapped with pTTSs. The link of TTS ID enable the user to view information about pTTSs in context of the chromosome sequence, tandem repeat inside the pTTSs, the known repeat element overlapping with pTTSs, other annotation tracks overlapped, and the BLAST genome link. The BLAST link is use to view the uniqueness of pTTSs on the human genome.

Figure 6

Result Tables on the properties of pTTS and neighboring annotation information. Descriptive information about pTTS, neighboring gene region which overlapped with the pTTS and annotation tracks which overlapped with the given pTTS.

Figure 7

Mapping of pTTSs and other regulatory elements on MYC gene region. Bottom plot shows detail map of pTTSs and maps other regulatory elements on MYC gene region and up plot shows zoomed region and regulatory sequences in the vicinity of start position of MYC gene. Red oval in the zoomed region: The uniquely mapped TTS (TTS15008128817597) located in the first exon of MYC gene is overlapped with the TFBS V$E2F_02 and with the CpG island. The next downstream TTS (TTS15008128817681) in the fist exon of MYC gene is also overlapped with other TFBS V$P300_01. However, for the last pTTS multiple locations in the human genome were found. Two G4 sequences (Quad-8-CCC-5773 and Quad-8-CC-5774) are also shown in a vicinity of start position of MYC gene One of this G4 (Quad-8-CCC-5773) is overlapped with pTTS TTS15008128817345 and include other pTTS TTS15008128817365.

Figure 8

Mapping of pTTSs and other regulatory elements on EVI-1 gene region. Red oval: the unique pTTS is overlapped with transcription factor binding site.

Figure 9

Mapping of pTTSs and miRNA included into IGF2 gene region. A: Mapping on primary miRNA (hsa-mir-483) and pTFO on the evolutionally conserve intronic region of IGF2 gene. B: secondary structure of primary miRNA (hsa-mir-483). Pink color of nucleotide symbol: mature siRNA, blue color of nucleotide symbol: polypurine sequence, red color of nucleotide symbol: polypyrimidine sequence located on the some strand as double helix DNA. One substitution on Y in the R starch is allowed. C: pTTS (TTS15011002112005) includes binding site (TTCCCCTCTCCC) of transcription factor MZF1.