Project description:Increasing numbers of senseâantisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural senseâantisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-proteinâcoding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of senseâantisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (1) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of senseâantisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome. Keywords: Expression profile of mouse and human sense-antisense transcript The RNA samples used for the mouse oligo-array experiments came from the brain, NIH3T3 cells (fibroblast cell line), liver, heart, and testis. RNA from mouse tissue (C57BL/6J, 8 to 10 weeks, male and female mixed) and mouse fibroblast line NIH3T3 was isolated by using Trizol reagent (Invitrogen). The mouse custom oligo DNA microarray chip (microarray format: 11K) contained 3896 probes that represented genes in 1948 exon-overlapping SAT (sense-antisense transcript) pairs.The same total RNA samples were reciprocally labeled with Cy3 or Cy5, hybridized to the oligo DNA on the chip, and dye-normalized. The data on all genes on the chip were used to enable the Feature Extraction software to produce the processed signals. The total mean signal on the mouse chip in each hybridization experiment was adjusted with to a value of 4872.3. The RNA samples used for human microarray experiments came from brain, HF19 cells (fibroblast cell line), heart, liver and testis. The total brain, heart, and testis RNA used in the array experiments was purchased from Ambion. Total RNA was isolated from the fibroblast cells by using Trizol reagent (Invitrogen). The same total RNA samples were labeled with single color Cy3, hybridized to the oligo DNA on the chip, and dye-normalized; the processed signals were obtained by using Feature Extraction software (Agilent Technologies). The custom oligo DNA microarray chip (microarray format: 11K) contained 2793 probes for 1486 pairs of exon-overlapping SATs. The data on all genes on the chip were used to enable the Feature Extraction software to produce the processed signals. For further analysis, the Cy3-labeled processed signal was used as the processed signal from the expression of a particular gene. The total mean signal on the human chip in each hybridization experiment was adjusted to 1491.6, so that the relative differences in gene expression could be compared among cell lines and tissues.

Project description:Sense-antisense transcript (SAT) pairs are pairs of transcripts that fully or partially overlap but are transcribed in opposite directions. Although SAT expression occurs in various species, most SAT pairs have not been examined in detail. Because our previous studies revealed some tissue specificity in SAT expression, SAT pairs might be involved in cell differentiation and the maintenance of tissue-specific gene expression programs. To analyze such tissue-specific SAT pairs, we assessed the expression profiles of SATs from 12 tissues of normal mice at a genome-wide scale and found that a considerable number of SAT pairs showed expression patterns unique to testis. This finding prompted us to study the relationship between SAT expression pattern and another epigenetic gene regulatory mechanism, DNA methylation. We conducted a comparative global analysis of the DNA methylation status of CpG island (CGI)-associated SAT loci from various tissues and found that in 99 of the 4911 SAT pairs studied, DNA methylation could cooperatively suppress its downstream “sense” transcription together with the “antisense” transcriptional activity in a tissue-specific manner. The tissue-specific differentially methylated regions (T-DMRs) of these SAT pairs mainly occurred outside of the defined CGIs. In addition, some of these T-DMRs are situated at the 5′ region of the antisense transcripts. This positioning implies a novel mechanism for DNA methylation to regulate gene transcription, in which DNA methylation inhibits an opposing transcriptional activity and therefore maintains stable expression of a tissue-specific gene. Twelve tissues (brain, thymus, heart, lung, liver, spleen, stomach, kidney, small intestine, testis, and placentae [10.5 and 13.5 days postcoitum (dpc)]) of C57BL/6J Jc1 and a mouse fibroblast cell line (SL10) were used for the mouse expression oligo-microarray. The expression array data were obtained by both oligo-dT and random priming methods, except for SL10 (olio-dT only). Twelve tissues (brain, thymus, heart, lung, liver, spleen, stomach, kidney, small intestine, testis, and placentae [10.5 and 13.5 days postcoitum (dpc)]) were used for DIP chip experiments. Nine of twelve tissues were separately analyzed classified on the basis of sex. A technical duplication of DIP chip analysis was performed by using the same genomic DNA of male kidney.

Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions. Transcriptomes of two yeast species under mid-log phase, early stationary phase, and after heat shock treatment were generated by Illumina HiSeq 2000 paired-end sequencing

Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions.

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Expression of Mouse and Human Sense-Antisense Transcript

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