BACKGROUND
transcription of eukaryotic genomes generates a myriad of non-coding rnas that show complex overlapping patterns of expression and regulation  <cit> .
the complexity of the eukaryotic transcriptome, transcribed by rna polymerase  ii, goes far beyond the coding genome and expands to many short rna populations  as well as long non-coding rnas   <cit> .
lncrnas form a heterogeneous group of rnas transcribed from intergenic or intragenic regions, which vary in length from  <dig> nucleotides to over 100 kb  <cit> .
intragenic non-coding transcripts might be further subdivided depending on the way they overlap protein-coding genes and/or the orientation with respect to protein-coding genes   <cit> .
although the biological relevance of many non-coding transcripts has been unambiguously established, this unanticipated level of complexity has led to the notion of pervasive transcription, which refers to the fact that transcription is not restricted to well-defined functional features, such as genes  <cit> .
a large proportion of lncrnas are transcribed in antisense orientation of protein-coding genes, with which they often share sequence complementarities  <cit> .
antisense rnas could potentially exert a regulatory function on their corresponding sense mrna at different levels.
recent findings have shown that some antisense transcripts act as epigenetic regulators of gene expression and chromatin remodeling  <cit> , while others play a role at the level of translation efficiency  <cit> .
besides these transcripts, the existence of non-coding antisense transcripts emanating from the promoters of protein-coding genes  has also emerged as a widespread phenomenon from yeast to mammals  <cit> .
on the one hand, the presence of short bidirectional transcripts appears to be a hallmark of active promoters in mammals  <cit> .
on the other hand, relatively longer non coding antisense transcripts can be detected upstream of most expressed genes in conditions where the rna degradation machinery is inhibited  <cit> .
moreover, lncrnas  are preferentially localized at the vicinity of gene promoters in antisense orientation  <cit> .
for instance, ~60% of lncrnas expressed in es cells were found to originate close to the tss of protein-coding genes  <cit> .
whether long antisense transcripts emanating from bidirectional promoters have general functional implications in gene regulation is currently unknown  <cit> .
in order to systematically identify and characterize bidirectional promoters associated with long non-coding antisense transcription, we took advantage of strand-specific rna-seq experiments, which provide an unprecedented opportunity to analyze and categorize transcripts  <cit> .
thorough analyses of rna-seq data from early developing thymocytes and other mouse tissues indicated that long-range bidirectional transcription is an intrinsic property of a class of promoters whose associated genes mainly encode for transcriptional regulators involved in development and cell differentiation.
accordingly, these promoters are characterized by large cpg islands, high degree of conservation and are generally repressed by polycomb complexes in es cells.
moreover, they display a unique gc skew profile, while the associated coding genes have large first exons, both properties likely reminiscent of their bidirectional activity.
surprisingly, coding genes associated with upstream antisense lncrnas display an increased rate of immature transcription, highlighting an additional level of transcriptional control.
thus, expression of long non-coding antisense transcripts appears as a common feature of a subset of mammalian protein-coding gene promoters with functional implications for gene regulation.
RESULTS
systematic identification of genes associated with long upstream antisense transcripts
we sought to assess whether production of long antisense transcripts is a general feature of mammalian gene promoters.
to this goal, we initially performed strand-specific paired-end total rna-seq with high sequencing depth from Δrag thymocytes , corresponding mainly to cd4-cd8- t-cell precursors .
we selected the set of protein-coding refseq transcripts whose promoter regions  do not overlap with transcripts of any other coding gene .
we then calculated the total rna-seq signal in the sense and antisense orientation for the region -5 kb to +5 kb with respect to each tss and ordered the selection in function of the level of upstream antisense  transcription .
using a stringent threshold  we found  <dig> %  of coding refseq transcripts to be associated with long upstream antisense transcripts , of which  <dig> overlap with previously annotated non-coding transcripts.
several examples are shown in figure 1b.
interestingly, these antisense transcripts are generally polyadenylated as shown by the average profile of strand-specific and polya-enriched rna-seq signal generated from Δrag thymocytes .
to confirm our observation in a different tissue, we analyzed strand-specific total rna-seq data from mouse embryonic kidney  <cit>  and obtained consistent results .
in order to infer the structure of luats, we used the transcript assembly tool cufflinks  <cit> .
we selected antisense cufflinks transcripts starting within the region +/-  <dig>  kb around the tss and longer than  <dig> nt, and inferred antisense transcript models for  <dig> out of  <dig>  refseq genes associated with divergent transcription in Δrag thymocytes, as defined above .
as expected, luats have very low or no coding potential as determined by phylocsf  <cit>  analyses .
assessment of subcellular localization of luats using recently published rna-seq data obtained from fractionated chromatin-associated, nucleoplasmic and cytoplasmic transcripts , showed that they remains mainly associated with the chromatin fraction , consistent with their lack of coding potential.
interestingly, 58% of antisense transcripts start within the region 500 bp upstream the tss of the associated-coding genes , suggesting that both sense and antisense transcripts originate from the same promoter elements.
luats were found to be expressed at relatively low level with a median expression value of  <dig>  fragments per kilobase per million fragments mapped or fpkm .
strikingly however, the luat-associated coding genes are expressed at high levels .
indeed, expression levels of the majority of genes displaying divergent promoters were found to be above the 3rd quartile of expression value distribution in Δrag thymocytes .
overall, these results suggest that antisense transcription is initiated from active coding-gene promoters, leading to concomitant expression of the two divergent transcripts.
luat-associated genes are related to transcription and developmental functions
the above results indicated that a substantial fraction of mammalian promoters sustain divergent transcription of lncrna/mrna gene pairs.
we next assessed whether luat-associated genes were enriched for specific categories of genes.
we found that this set of genes is highly enriched with transcription- and chromatin regulation-related go terms .
this observation was specific to luat-associated genes as compared to a control set composed of genes with similar expression level distribution, but without antisense transcription, which was not significantly enriched for any go terms .
strikingly, the list of luat-associated genes includes most transcription regulators known to be important for early t-cell differentiation  <cit> , including tcf <dig> , lef <dig>  tcf <dig> , satb <dig>  dntt, gfi <dig>  myb, tox, notch <dig>  bcl11a, rorc  and ikzf <dig>  consistent with a tissue-specific function, luats are significantly associated with a higher proportion of thymocyte-specific genes  and with genes involved in t cell differentiation , as compared to the control set.
enrichment for transcription- and development-related functions was also found with luat-associated genes isolated from kidney rna-seq data , comprising important regulators of kidney development, such as irx <dig>  irx <dig>  hnf1b, lhx <dig> and smad <dig>
to obtain a more general view of the functional relevance of luat-associated genes, we analyzed recently published directional and polya-enriched rna-seq data from  <dig> mouse tissues  <cit> .
although the sequencing depth was generally low, we were able to isolate luats for all analyzed tissues and to annotate a non-redundant set of  <dig>  luat-associated genes .
consistent with the initial studies from thymus and kidney , this set of genes was highly enriched for genes involved in transcription regulation and developmental functions .
in contrast, head-to-head coding genes  or randomly selected genes with unidirectional promoters are not enriched for transcription-related functions .
next, we addressed the question of whether luat-associated genes were specifically enriched for developmental genes involved in transcription regulation.
we found that luat-associated genes, but not control genes, were enriched in the set of genes annotated for both “developmental process”  and “transcription factor activity”  go terms .
in conclusion, luat-associated genes are commonly involved in transcription regulation related to developmental functions.
luat and their associated coding-genes are co-regulated throughout cell differentiation and development
the co-expression of the two divergent transcripts suggests that the expression of luats and their neighboring genes might be co-regulated throughout development and cell differentiation.
to address this hypothesis, we first compared two subsequent stages of early t-cell development.
during normal t-cell differentiation, pretcr-induced signaling leads dn thymocytes to cross the so-called β-selection checkpoint, which results in massive cell proliferation and the induction of a developmental process marked by the expression of both cd <dig> and cd <dig> co-receptors, thus generating dp thymocytes  <cit> .
to determine whether luats and associated genes were co-regulated during the β-selection process, we used previously published total rna-seq from dp thymocytes  <cit>  and produced a new set of rna-seq data from Δrag  thymocytes, using the same rna-seq procedure and sequencing platform .
we then selected a non-redundant list of  <dig> luat expressed in either dn or dp cell stages and compared their differential expression ratio along with the expression ratio of the associated coding-genes .
interestingly, we observed a significant association between developmental regulation of luat and their associated genes when considering transcripts with an expression ratio of at least twofold .
examples of co-regulated luat-gene pairs are shown in figure 3c.
to have a more thorough dynamical view of the regulated expression of luats and their associated genes, we analyzed recently published rna-seq data from several stages of early t-cell differentiation  <cit> .
although the absence of strand-specific information did not allow genome-wide isolation of luat in these data sets, visual inspection of the rna-seq revealed clear examples where the luat and the associated gene followed the same kinetics throughout t-cell differentiation .
in the same line, we also observed a tight co-regulation of luat and associated gene pairs between thymocytes and embryonic kidney .
one expectation from this observation is that the expression of luats and their associated genes would be correlated across different tissues.
to address this possibility, we analyzed the expression patterns of the  <dig>  luats and their associated genes found in the multi-tissue analysis.
the vast majority of luats exhibit tissue-specific expression patterns as underlined by unsupervised clustering of expression profiles  and the restricted number of tissues where each of them was found .
moreover, these luats and their coding neighbors are more correlated to each other than random gene pairs, and even slightly more than head-to-head protein-coding gene pairs .
in agreement, we found many examples where luats expression is strictly associated with the expression of their neighboring genes .
taken together, these results suggest that luat expression likely reflects the activity of associated coding-gene promoters throughout cell differentiation and development.
luat-associated promoters share characteristics with those of developmentally regulated genes
the close proximity between luat and tss of associated coding genes, along with the tight correlation of their expression, strongly suggest sharing of common regulatory elements.
hence, we asked whether the bidirectional property of luat-associated promoters might be linked to intrinsic sequence specificities.
we compared several sequence characteristics between the promoters of the three gene sets described above.
we found that gc content differs between these sets.
in the region upstream of the tss, both luat-associated and coding-coding gene promoters have a significantly higher gc content than unidirectional promoters , whereas in the downstream region, luat-associated promoters have a higher gc content than the unidirectional and coding-coding gene sets  .
moreover, 80% of luat-associated and 89% of coding-coding gene promoters are covered by cpg islands within the region , as compared to only 56% of unidirectional gene promoters .
strikingly however, analysis of cpg island size demonstrated that luat-associated gene promoters contain particularly longer cpg islands .
sequence motif analyses revealed that both luat-associated and coding-coding gene promoters are depleted of tata boxes, as compared to unidirectional promoters .
finally, we found that luat-associated promoters contain more conserved elements than the other two sets in regions close to and downstream of the tss .
interestingly, it has been described that developmentally regulated genes are associated with genomic regulatory blocks  which are highly conserved genomic regions characterized by a number of unique features, including very large cpg islands and tata-box depletion  <cit> .
therefore, the bidirectional property of luat-associated promoters might be linked to intrinsic regulatory properties related to genes encoding for transcriptional and developmental regulators.
developmental genes have also been shown to be actively repressed by polycomb complexes in embryonic stem  cells, and therefore are often found associated with trimethylation of h <dig> lysine  <dig>   <cit> .
by analyzing chip-seq data from es cells  <cit> , we found that luat-associated promoters were specifically enriched for h3k27me <dig> within the 2 kb around the tss, as compared to unidirectional and coding-coding gene sets , but not for h3k4me <dig> .
moreover, they were more frequently associated with bivalent domains , composed of concomitant h3k27me <dig> and h3k4me <dig> peaks, a feature related to silencing of developmental genes in es cells, while keeping them poised for activation  <cit> .
one additional expectation for developmentally regulated promoters is that they should be bound by tissue specific transcription factors.
to test this, we analyzed chip-seq data performed in dp thymocytes for a series of lymphoid-specific transcription factors, including gata <dig>  <cit> , ikaros  <cit> , ets <dig>  <cit>  and runx <dig> .
these transcription factors were found in a higher frequency at luat-associated promoters active in dp thymocytes, as compared with a set of control genes .
thus, luat-associated promoters appear to be regulated in a more specific way than other similarly expressed genes.
overall, we found that luat-associated promoters share characteristics with those of developmentally regulated genes.
it seems likely that the divergent transcription observed at luat-associated promoters is linked to intrinsic genomic characteristics of these promoters.
bidirectional transcription at luat-associated promoters is linked to a specific gc skew profile and longer first exon
directionality of transcription is thought to be mediated, at least in part, by an asymmetric distribution of g and c content between the two dna strands around the promoter, a property known as gc skew  <cit> , possibly constraining the orientation of the transcription initiation complex.
gc-rich promoters are characterized by a significant excess of g over c residues  immediately downstream the tsss  <cit> .
to test the link between gc skew and bidirectionality, we computed gc skew profiles for each of the three gene promoter groups described above.
as expected, unidirectional genes show a positive gc skew immediately downstream the tss, while coding-coding genes show two sharp and inverted gc skew peaks, one negative and one positive, respectively upstream and downstream the tss .
strikingly, luat-associated genes also display two inverted gc skew profiles, but the gc bias is less pronounced than at head-to-head coding genes at both sides of the tss .
in addition, the positive gc skew downstream the tss is also less pronounced than at unidirectional genes .
this result suggests that bidirectional transcription at luat-associated promoters might be linked to a unique gc skew profile resulting in lower constraints on the directionality of the transcription initiation complex.
promoter-proximal 5′ splicing sites and first exon length have been recently suggested to play a role in directionality of transcription  <cit> .
we computed the average length of the first exon of genes in each gene set .
strikingly, the set of luat-associated genes has the longest first exon with a median length of 242 bp, compared to 195 bp and 190 bp for the unidirectional and coding-coding gene sets.
consistently, 5′ splicing sites are relatively depleted immediately downstream the tss of luat-associated genes, as compared to the control set of genes .
to assess whether splicing might play a role in controlling the expression of luats, we analyzed recent published data of 3′ ends of polyadenylated rna-seq in mouse es cells in which u <dig> small nuclear ribonucleoprotein  has been functionally inhibited  <cit> .
interestingly, the expression  of a selection of luat normally expressed in es cells  is significantly increased after inhibition of u <dig> snrnp , as compared to control es cells.
taken together, these observations indicate that a unique gc skew profile along with large first exon might both contribute to the bidirectionality of transcription at luat-associated genes.
luat-associated genes harbor specific chromatin features
in order to assess whether luat-associated promoters display specific chromatin features, we analyzed several histone modification marks and general transcription factors in dp thymocytes that were either performed in this study or already published  <cit>  .
we compared the surrounding regions of three sets of promoters displaying similar expression level distribution based on total rna-seq signal at exons  in dp thymocytes: luats-associated promoters, unidirectional promoters, as well as promoters of bidirectional coding gene pairs .
as expected, we found chromatin features common to both sets of bidirectional promoters .
bidirectional promoters display higher and/or wider level of histone modifications linked to open and active chromatin  at the region immediately upstream of the tss , consistent with the bidirectional activity at these promoters.
this property is also associated with additional peaks upstream of the tss for either total or initiating  pol ii, as well as tbp .
furthermore, significant levels of histone modifications linked to early  or late  transcription elongation were observed in the region upstream of bidirectional promoters, confirming the fact that these regions undergo productive transcription .
strikingly, we also found evidences for chromatin features specific to luat-associated promoters.
although the three gene sets are similarly enriched for h3k36me <dig> within the coding gene body, the luat-associated genes display higher levels of h3k79me <dig> .
h3k79me <dig> is generally enriched at the 5′ end of expressed genes comprising the first exon and intron and mark the transition between early  and late  transcription elongation  <cit> .
to have a more precise view of the differential enrichment in h3k79me <dig>  we compared the h3k79me <dig> profiles within rescaled regions comprising the tss to the end of 1st intron .
indeed, we observed that h3k79me <dig> remains significantly higher throughout the first intron of luat-associated genes as compared to the control gene sets.
as the genes from the three sets express similar amounts of productive transcription , our results most likely suggest an actual increased rate of early  transcription elongation from luat-associated promoters.
in agreement with an increased rate of early transcriptional elongation at luat-associated genes, we observed aspecific enrichment of pol ii  and tbp within the 5′ region of luat-associated gene-bodies , indicating extended accumulation of the initiating and/or early elongating transcription complex  <cit> .
to further investigate the possibility of a transcriptional pause immediately downstream the tss, we analyzed the average profiles in dp thymocytes of additional general transcription factors , including taf <dig>  tfiib, tfiie and tfiih .
in all cases, we observed a significant enrichment of the gtfs downstream the tss.
finally, consistently with the transcriptional pause being directly linked to divergent transcription, we also observed a significant and specific enrichment of pol ii and gtfs around a region 1 kb upstream the tss of luat associated genes .
of particular interest is the overall enrichment in tfiih complex around the tss of luat-associated genes, which play a key role in transcription initiation by phosphorylating pol ii at ser <dig>  <cit> .
thus, our results indicate increased pol ii pausing at both sides of luat-associated promoters.
accumulation of immature transcripts at luat-associated genes
early elongation and h3k79me <dig> enrichment are generally associated with the 5′ intronic sequences and splicing events  <cit> .
thus, to further explore the hypothesis of an accumulation of immature transcripts at luat-associated genes, we compared the average profiles around the tss of total and polya rna-seq levels for the three set of equally expressed genes .
we found that total-rna signal downstream of the tss is higher for luat-associated genes, while polya-rna signal is similar among the three gene sets .
while the polya rna-seq signals result only from complete  transcripts, total rna-seq signals result from both immature  and complete transcripts.
thus, a relative enrichment of total rna-seq, as compared to polya rna-seq signal, is indicative of either increased rate of immature transcription or expression of less stable transcripts.
however, the observed results could not be attributed to differences in transcript stability as the three gene sets display equivalent levels of h3k36me <dig>  which is generally coupled to productive elongation .
to directly assess whether luat-associated genes produce increased level of immature transcripts , we computed the splicing index across the three gene sets.
as shown in figure 7b, the splicing index is significantly reduced between the first two exons of luat-associated genes as compared to the other set of genes, while no differences are observed at the middle exons.
this suggests an increased rate of immature transcription at the 5′ region of luat-associated genes.
consistently, we found that first introns of luat-associated genes display higher density of total rna-seq signal as compared to the control gene sets , though there is no significant differences in first intron length between the three set of genes .
significant enrichment of immature transcripts within the first intron of luat-associated genes was also confirmed by rt-qpcr analysis of individual genes .
all in all, luat-associated gene promoters are clearly more prone to induce immature transcription than other bidirectional or unidirectional gene promoters, indicating a less restricted control of pol ii pausing around the tss, likely leading to divergent and pervasive transcription.
discussion
here, we used directional rna-seq from primary mouse tissues to directly and systematically characterize antisense transcripts.
we have found that a significant fraction of gene promoters sustains expression of long non-coding antisense transcripts .
the luat/coding gene pairs are usually co-regulated throughout cell differentiation and development and generally function as transcriptional regulators.
luat-associated promoters share several characteristics of promoters of developmentally regulated genes, including large cpg islands, high degree of conservation and epigenetic regulation during development.
moreover, the bidirectional transcription at these promoters appears to be linked to a specific gc skew profile and particularly longer first exons.
finally, luat-associated promoters display distinctive epigenetic features reflecting an intensified rate of early transcriptional elongation.
overall, our results support the view that promoters of a subclass of transcriptional regulators are characterized by a specialized mechanism of pol ii transcription, in which pol ii recruitment is directly coupled to relaxed bidirectional transcription.
previous studies have shown that lincrnas are preferentially located near protein-coding genes in divergent orientation and that their expression is often correlated  <cit> .
however, the stringent criteria generally used to define lincrnas  likely impaired a comprehensive identification of lncrna transcripts, including those in divergent orientation from coding genes.
a recent study has indeed described the abundance of divergently transcribed lncrna/mrna gene pairs in es cells  <cit> .
although this and the present works likely described the same kind of antisense transcripts, our study largely complement and extend the previous study by using a more direct approach to identify upstream antisense lncrnas solely based on the rna-seq signal and by demonstrating their existence in many primary tissues and throughout t cell differentiation.
moreover, we show here that divergent transcription is clearly associated with a subset of genes coding for transcriptional regulators, and we propose a functional link between divergent transcription and gene expression regulation.
as suggested previously the presence of short bidirectional transcripts appears to be a hallmark of active promoters in mammals  <cit> , generally associated with paused pol ii around the tss.
it has also been previously described that expression of upstream unstable transcripts  are a common characteristic of pol ii transcribed genes  <cit> .
although some overlaps might exist between prompts and luats, luats differ from exosome-sensitive prompts transcripts.
firstly, luats are detected at significant levels without inhibition of the rna exosome degradation machinery.
secondly, they are substantially longer than prompts.
thirdly, they are associated with a specific category of genes.
hence, while many coding gene promoters, essentially those containing cpg islands  <cit> , have the intrinsic property of bidirectional transcription  <cit> , production of long antisense transcript is generally inhibited or are rapidly degraded at most loci.
divergent transcription is linked to intrinsic sequence properties shared with developmental gene promoters
we have found that luat-associated genes frequently encode tfs involved in cell differentiation and development.
this is consistent with previous studies in mouse, human and zebra fish suggesting that large gene deserts flanking tf genes, with roles in embryonic development, preferentially harbor lincrnas  <cit> .
as such, these genes are expected to be subjected to fine tune regulation.
accordingly, we found that their promoters appears to be bound by lineage specific tfs .
another striking characteristic of luat-associated promoters is the presence of very large cpg islands , a feature shared with developmental gene promoters  <cit> , but not observed at bidirectional coding-gene promoters.
as the promoters of these genes are enriched for cpg rich regions and are prone to pervasive transcription, it is expected that dedicated repressive mechanisms might be in place to control their expression.
in line with this, we have found that luat-associated genes are specifically enriched for h3k27me <dig>  and for bivalent domains  in es cells, thus suggesting polycomb-mediated regulation of these genes.
as suggested elsewhere  <cit> , large cpg islands  might represent a favored recruitment platform for polycomb-associated complexes and thus play an important role in transcriptional regulation of key developmental genes.
whether large cpg islands are required for divergent transcription from luat-associated promoters will deserve further investigation.
a link between antisense and immature transcription?
our results indicate an unexpected link between divergent lncrna/mrna transcription and premature termination of coding transcripts.
indeed, luat-associated genes are characterized by a significant accumulation of immature transcripts within the 5′ region of the genes .
our results most likely suggest that intrinsic properties of luat-associated promoters induce a specialized mechanism of pol ii transcription, in which recruitment of the enzyme is directly coupled to pervasive bidirectional transcription.
several arguments point to this direction.
the presence of a tata box is generally linked to strong directionality of transcription.
consistent with their bidirectional feature, luat-associated promoters are essentially depleted of tata box .
although the absence of a tata box might be required for bidirectional transcription, as has been suggested for coding-coding promoters  <cit> , it might not be sufficient as some tata-less promoters still show strong directionality  <cit> .
in addition, we have also observed that luat-associated promoters display two inverted, though moderated, gc skew profiles around the tss .
it has been shown that a positive gc skew immediately upstream the tss favors the formation of so-called r-loop structures  <cit> , which are long, stable and three stranded rna/dna structure formed during transcription  <cit> .
it has also been previously suggested that positive gc skew immediately after the tss, and subsequent r loop formation, may serve to correct the lack of directionality in the initial steps of transcription  <cit> .
this correction might be mediated by the ability of r loops to elicit transcriptional pausing  <cit> .
finally, 5′ splice sites and first exons have been recently shown to play a role in promoter directionality  <cit> .
it is suggested that u <dig> snrnp binding at 5′ splice sites might help to stabilize pol ii recruitment at the promoter and enforce sense transcription.
moreover, the length of first exon appears to influence pol ii stability at promoters and transcription accuracy  <cit> .
indeed, genes with long first exon have pol il accumulation downstream the tss and increased proportion of antisense transcripts  <cit> , reminiscent of what we have observed at luat-associated loci.
strikingly, functional inhibition of u <dig> snrnp in es cells resulted in accumulation of premature cleavage of coding gene transcripts  <cit> , but also increased expression of antisense transcripts .
consequently, spatial separation of promoter and 5′ splice sites might directly impact both directionality and transcription accuracy.
it is plausible that, at luat-associated genes, 5′ splice sites are too distant from the tss to ensure proper directionality of transcription, thus favoring bidirectional transcription.
taking all into account, we favor a hypothesis whereby both immature and bidirectional transcription at a specialized class of gene promoters are directly linked to intrinsic sequence properties, including tata box depletion, unique gc skew profile and long first exon.
functional relevance of bidirectional transcription
the specific association of antisense transcripts with genes related to transcription regulation and development strongly suggests that divergent transcription might be directly or indirectly involved in the tight regulation of these genes.
in line with a direct role of luats, several examples have demonstrated a functional regulation by mammalian antisense transcripts in cis <cit> .
this regulation might act at multiple levels, from modifying local chromatin to enabling regional signal spreading  <cit>  or controlling translation efficiency  <cit> , yet in the latter case an overlapping stretch with coding exons is required.
alternatively, divergent transcription might reflect an intrinsic property of promoters of genes coding for developmental regulators.
the finding that h3k79me <dig>  a mark of early elongation, was found higher at luat-associated genes  suggests the level of early elongating transcription is increased at these genes.
this was further supported by a relative higher ratio of total versus polya levels within the first intron, as well as decreased splicing efficiency .
thus, our findings clearly indicate that luat-associated promoters are more prone to pervasive and/or immature transcription .
what can be the functional relevance of this pervasive transcription?
if divergent transcription and non-productive gene expression are linked, this will imply that the initiation step of transcription is less controlled at these promoters, likely leading to pervasive transcription in both directions.
in this line, an appealing hypothesis will be that expression of luat-associated genes is regulated also at the level of transcript maturation and/or elongation efficiency.
this might reflect a checkpoint for coupling elongation and rna processing, as previously suggested  <cit> .
although some genes are regulated by pol ii pausing in close proximity to the tss  <cit> , others are reported to be efficiently transcribed into precursor transcripts, while the efficiency of transcript processing is being regulated  <cit> .
generally, only a portion of the pol ii that assemble at the promoter enters into early elongation  <cit> .
this entry is characterized by pol ii ser <dig> phosphorylation and chromatin modifications that are specific to transcription initiation and early elongation .
before transcribing further, the polymerase passes a 5′ checkpoint where it pauses, terminates, or commits to productive elongation.
if the polymerase does not proceed through this checkpoint, transcription will be terminated producing an unstable transcript.
if the polymerase proceeds through the checkpoint, it will enter into productive elongation that is associated with characteristic chromatin modifications  and pol ii ser <dig> phosphorylation  <cit> .
in this context, rapid transcriptional induction might be facilitated by the active release of polymerase molecules that have initiated transcription, but are paused downstream the tss.
thus, pausing during early elongation may provide both a kinetic ‘window of opportunity’, as well as an interaction surface, to facilitate additional levels of regulation of the nascent rna before the transition to productive elongation.
another related and not exclusive hypothesis would be that bidirectional promoter activity increase the stochasticity of gene expression, as suggested for antisense expression in yeast  <cit> .
it is well known that expression of developmental regulators, including key transcription factors, is often regulated stochastically during cell differentiation, influencing cell and developmental decisions  <cit> .
it is therefore plausible that luat expression might reflect a dedicated mechanism to induce stochastic expression of developmental regulators by modulating the rate of pervasive  transcription.
in any case, our observations might shed light on a new mechanism representing the outcome of an evolutionary pressure to control the expression of a subclass of genes coding for transcriptional regulators.
CONCLUSIONS
we have found that divergent transcription of lncrna/mrna gene pairs is significantly associated with genes related to transcriptional regulation and development.
their promoters share several characteristics with those of developmental transcription factors, display a unique gc skew profile and are associated with genes harboring long first exons, reminiscent of their bidirectional activity.
unexpectedly, we also found that the 5′ region of the associated coding genes harbor a specific accumulation of h3k79me <dig> epigenetic mark, as well as initiating pol ii complexes, both of which are linked to an intensified rate of early transcriptional elongation.
these results strongly suggest a functional link between divergent transcription and the regulation of genes coding for developmental transcription factors.
altogether, our findings which indicate that, at a subset of transcription factor gene promoters, divergent and pervasive transcription are linked, might illustrate an additional mechanism for regulating the regulators, in a rather sophisticated system for fine-tuning mammalian gene expression.

 transcription of eukaryotic genomes generates a myriad of non-coding rnas that show complex overlapping patterns of expression and regulation. the complexity of the eukaryotic transcriptome, transcribed by rna polymerase ii, goes far beyond the coding genome and expands to many short rna populations as well as long non-coding rnas. lncrnas form a heterogeneous group of rnas transcribed from intergenic or intragenic regions, which vary in length from nucleotides to over 100 kb. intragenic non-coding transcripts might be further subdivided depending on the way they overlap protein-coding genes and/or the orientation with respect to protein-coding genes. although the biological relevance of many non-coding transcripts has been unambiguously established, this unanticipated level of complexity has led to the notion of pervasive transcription, which refers to the fact that transcription is not restricted to well-defined functional features, such as genes. a large proportion of lncrnas are transcribed in antisense orientation of protein-coding genes, with which they often share sequence complementarities. antisense rnas could potentially exert a regulatory function on their corresponding sense mrna at different levels. recent findings have shown that some antisense transcripts act as epigenetic regulators of gene expression and chromatin remodeling, while others play a role at the level of translation efficiency. besides these transcripts, the existence of non-coding antisense transcripts emanating from the promoters of protein-coding genes has also emerged as a widespread phenomenon from yeast to mammals. on the one hand, the presence of short bidirectional transcripts appears to be a hallmark of active promoters in mammals. on the other hand, relatively longer non coding antisense transcripts can be detected upstream of most expressed genes in conditions where the rna degradation machinery is inhibited. moreover, lncrnas are preferentially localized at the vicinity of gene promoters in antisense orientation. for instance, ~60% of lncrnas expressed in es cells were found to originate close to the tss of protein-coding genes. whether long antisense transcripts emanating from bidirectional promoters have general functional implications in gene regulation is currently unknown. in order to systematically identify and characterize bidirectional promoters associated with long non-coding antisense transcription, we took advantage of strand-specific rna-seq experiments, which provide an unprecedented opportunity to analyze and categorize transcripts. thorough analyses of rna-seq data from early developing thymocytes and other mouse tissues indicated that long-range bidirectional transcription is an intrinsic property of a class of promoters whose associated genes mainly encode for transcriptional regulators involved in development and cell differentiation. accordingly, these promoters are characterized by large cpg islands, high degree of conservation and are generally repressed by polycomb complexes in es cells. moreover, they display a unique gc skew profile, while the associated coding genes have large first exons, both properties likely reminiscent of their bidirectional activity. surprisingly, coding genes associated with upstream antisense lncrnas display an increased rate of immature transcription, highlighting an additional level of transcriptional control. thus, expression of long non-coding antisense transcripts appears as a common feature of a subset of mammalian protein-coding gene promoters with functional implications for gene regulation.  systematic identification of genes associated with long upstream antisense transcripts we sought to assess whether production of long antisense transcripts is a general feature of mammalian gene promoters. to this goal, we initially performed strand-specific paired-end total rna-seq with high sequencing depth from Δrag thymocytes, corresponding mainly to cd4-cd8- t-cell precursors. we selected the set of protein-coding refseq transcripts whose promoter regions do not overlap with transcripts of any other coding gene. we then calculated the total rna-seq signal in the sense and antisense orientation for the region -5 kb to +5 kb with respect to each tss and ordered the selection in function of the level of upstream antisense transcription. using a stringent threshold we found % of coding refseq transcripts to be associated with long upstream antisense transcripts, of which overlap with previously annotated non-coding transcripts. several examples are shown in figure 1b. interestingly, these antisense transcripts are generally polyadenylated as shown by the average profile of strand-specific and polya-enriched rna-seq signal generated from Δrag thymocytes. to confirm our observation in a different tissue, we analyzed strand-specific total rna-seq data from mouse embryonic kidney and obtained consistent results. in order to infer the structure of luats, we used the transcript assembly tool cufflinks. we selected antisense cufflinks transcripts starting within the region +/-  kb around the tss and longer than nt, and inferred antisense transcript models for out of refseq genes associated with divergent transcription in Δrag thymocytes, as defined above. as expected, luats have very low or no coding potential as determined by phylocsf analyses. assessment of subcellular localization of luats using recently published rna-seq data obtained from fractionated chromatin-associated, nucleoplasmic and cytoplasmic transcripts, showed that they remains mainly associated with the chromatin fraction, consistent with their lack of coding potential. interestingly, 58% of antisense transcripts start within the region 500 bp upstream the tss of the associated-coding genes, suggesting that both sense and antisense transcripts originate from the same promoter elements. luats were found to be expressed at relatively low level with a median expression value of fragments per kilobase per million fragments mapped or fpkm. strikingly however, the luat-associated coding genes are expressed at high levels. indeed, expression levels of the majority of genes displaying divergent promoters were found to be above the 3rd quartile of expression value distribution in Δrag thymocytes. overall, these results suggest that antisense transcription is initiated from active coding-gene promoters, leading to concomitant expression of the two divergent transcripts. luat-associated genes are related to transcription and developmental functions the above results indicated that a substantial fraction of mammalian promoters sustain divergent transcription of lncrna/mrna gene pairs. we next assessed whether luat-associated genes were enriched for specific categories of genes. we found that this set of genes is highly enriched with transcription- and chromatin regulation-related go terms. this observation was specific to luat-associated genes as compared to a control set composed of genes with similar expression level distribution, but without antisense transcription, which was not significantly enriched for any go terms. strikingly, the list of luat-associated genes includes most transcription regulators known to be important for early t-cell differentiation, including tcf, lef tcf, satb dntt, gfi myb, tox, notch bcl11a, rorc and ikzf consistent with a tissue-specific function, luats are significantly associated with a higher proportion of thymocyte-specific genes and with genes involved in t cell differentiation, as compared to the control set. enrichment for transcription- and development-related functions was also found with luat-associated genes isolated from kidney rna-seq data, comprising important regulators of kidney development, such as irx irx hnf1b, lhx and smad to obtain a more general view of the functional relevance of luat-associated genes, we analyzed recently published directional and polya-enriched rna-seq data from mouse tissues. although the sequencing depth was generally low, we were able to isolate luats for all analyzed tissues and to annotate a non-redundant set of luat-associated genes. consistent with the initial studies from thymus and kidney, this set of genes was highly enriched for genes involved in transcription regulation and developmental functions. in contrast, head-to-head coding genes or randomly selected genes with unidirectional promoters are not enriched for transcription-related functions. next, we addressed the question of whether luat-associated genes were specifically enriched for developmental genes involved in transcription regulation. we found that luat-associated genes, but not control genes, were enriched in the set of genes annotated for both “developmental process” and “transcription factor activity” go terms. in conclusion, luat-associated genes are commonly involved in transcription regulation related to developmental functions. luat and their associated coding-genes are co-regulated throughout cell differentiation and development the co-expression of the two divergent transcripts suggests that the expression of luats and their neighboring genes might be co-regulated throughout development and cell differentiation. to address this hypothesis, we first compared two subsequent stages of early t-cell development. during normal t-cell differentiation, pretcr-induced signaling leads dn thymocytes to cross the so-called β-selection checkpoint, which results in massive cell proliferation and the induction of a developmental process marked by the expression of both cd and cd co-receptors, thus generating dp thymocytes. to determine whether luats and associated genes were co-regulated during the β-selection process, we used previously published total rna-seq from dp thymocytes and produced a new set of rna-seq data from Δrag thymocytes, using the same rna-seq procedure and sequencing platform. we then selected a non-redundant list of luat expressed in either dn or dp cell stages and compared their differential expression ratio along with the expression ratio of the associated coding-genes. interestingly, we observed a significant association between developmental regulation of luat and their associated genes when considering transcripts with an expression ratio of at least twofold. examples of co-regulated luat-gene pairs are shown in figure 3c. to have a more thorough dynamical view of the regulated expression of luats and their associated genes, we analyzed recently published rna-seq data from several stages of early t-cell differentiation. although the absence of strand-specific information did not allow genome-wide isolation of luat in these data sets, visual inspection of the rna-seq revealed clear examples where the luat and the associated gene followed the same kinetics throughout t-cell differentiation. in the same line, we also observed a tight co-regulation of luat and associated gene pairs between thymocytes and embryonic kidney. one expectation from this observation is that the expression of luats and their associated genes would be correlated across different tissues. to address this possibility, we analyzed the expression patterns of the luats and their associated genes found in the multi-tissue analysis. the vast majority of luats exhibit tissue-specific expression patterns as underlined by unsupervised clustering of expression profiles and the restricted number of tissues where each of them was found. moreover, these luats and their coding neighbors are more correlated to each other than random gene pairs, and even slightly more than head-to-head protein-coding gene pairs. in agreement, we found many examples where luats expression is strictly associated with the expression of their neighboring genes. taken together, these results suggest that luat expression likely reflects the activity of associated coding-gene promoters throughout cell differentiation and development. luat-associated promoters share characteristics with those of developmentally regulated genes the close proximity between luat and tss of associated coding genes, along with the tight correlation of their expression, strongly suggest sharing of common regulatory elements. hence, we asked whether the bidirectional property of luat-associated promoters might be linked to intrinsic sequence specificities. we compared several sequence characteristics between the promoters of the three gene sets described above. we found that gc content differs between these sets. in the region upstream of the tss, both luat-associated and coding-coding gene promoters have a significantly higher gc content than unidirectional promoters, whereas in the downstream region, luat-associated promoters have a higher gc content than the unidirectional and coding-coding gene sets. moreover, 80% of luat-associated and 89% of coding-coding gene promoters are covered by cpg islands within the region, as compared to only 56% of unidirectional gene promoters. strikingly however, analysis of cpg island size demonstrated that luat-associated gene promoters contain particularly longer cpg islands. sequence motif analyses revealed that both luat-associated and coding-coding gene promoters are depleted of tata boxes, as compared to unidirectional promoters. finally, we found that luat-associated promoters contain more conserved elements than the other two sets in regions close to and downstream of the tss. interestingly, it has been described that developmentally regulated genes are associated with genomic regulatory blocks which are highly conserved genomic regions characterized by a number of unique features, including very large cpg islands and tata-box depletion. therefore, the bidirectional property of luat-associated promoters might be linked to intrinsic regulatory properties related to genes encoding for transcriptional and developmental regulators. developmental genes have also been shown to be actively repressed by polycomb complexes in embryonic stem cells, and therefore are often found associated with trimethylation of h lysine. by analyzing chip-seq data from es cells, we found that luat-associated promoters were specifically enriched for h3k27me within the 2 kb around the tss, as compared to unidirectional and coding-coding gene sets, but not for h3k4me. moreover, they were more frequently associated with bivalent domains, composed of concomitant h3k27me and h3k4me peaks, a feature related to silencing of developmental genes in es cells, while keeping them poised for activation. one additional expectation for developmentally regulated promoters is that they should be bound by tissue specific transcription factors. to test this, we analyzed chip-seq data performed in dp thymocytes for a series of lymphoid-specific transcription factors, including gata, ikaros, ets and runx. these transcription factors were found in a higher frequency at luat-associated promoters active in dp thymocytes, as compared with a set of control genes. thus, luat-associated promoters appear to be regulated in a more specific way than other similarly expressed genes. overall, we found that luat-associated promoters share characteristics with those of developmentally regulated genes. it seems likely that the divergent transcription observed at luat-associated promoters is linked to intrinsic genomic characteristics of these promoters. bidirectional transcription at luat-associated promoters is linked to a specific gc skew profile and longer first exon directionality of transcription is thought to be mediated, at least in part, by an asymmetric distribution of g and c content between the two dna strands around the promoter, a property known as gc skew, possibly constraining the orientation of the transcription initiation complex. gc-rich promoters are characterized by a significant excess of g over c residues immediately downstream the tsss. to test the link between gc skew and bidirectionality, we computed gc skew profiles for each of the three gene promoter groups described above. as expected, unidirectional genes show a positive gc skew immediately downstream the tss, while coding-coding genes show two sharp and inverted gc skew peaks, one negative and one positive, respectively upstream and downstream the tss. strikingly, luat-associated genes also display two inverted gc skew profiles, but the gc bias is less pronounced than at head-to-head coding genes at both sides of the tss. in addition, the positive gc skew downstream the tss is also less pronounced than at unidirectional genes. this result suggests that bidirectional transcription at luat-associated promoters might be linked to a unique gc skew profile resulting in lower constraints on the directionality of the transcription initiation complex. promoter-proximal 5′ splicing sites and first exon length have been recently suggested to play a role in directionality of transcription. we computed the average length of the first exon of genes in each gene set. strikingly, the set of luat-associated genes has the longest first exon with a median length of 242 bp, compared to 195 bp and 190 bp for the unidirectional and coding-coding gene sets. consistently, 5′ splicing sites are relatively depleted immediately downstream the tss of luat-associated genes, as compared to the control set of genes. to assess whether splicing might play a role in controlling the expression of luats, we analyzed recent published data of 3′ ends of polyadenylated rna-seq in mouse es cells in which u small nuclear ribonucleoprotein has been functionally inhibited. interestingly, the expression of a selection of luat normally expressed in es cells is significantly increased after inhibition of u snrnp, as compared to control es cells. taken together, these observations indicate that a unique gc skew profile along with large first exon might both contribute to the bidirectionality of transcription at luat-associated genes. luat-associated genes harbor specific chromatin features in order to assess whether luat-associated promoters display specific chromatin features, we analyzed several histone modification marks and general transcription factors in dp thymocytes that were either performed in this study or already published. we compared the surrounding regions of three sets of promoters displaying similar expression level distribution based on total rna-seq signal at exons in dp thymocytes: luats-associated promoters, unidirectional promoters, as well as promoters of bidirectional coding gene pairs. as expected, we found chromatin features common to both sets of bidirectional promoters. bidirectional promoters display higher and/or wider level of histone modifications linked to open and active chromatin at the region immediately upstream of the tss, consistent with the bidirectional activity at these promoters. this property is also associated with additional peaks upstream of the tss for either total or initiating pol ii, as well as tbp. furthermore, significant levels of histone modifications linked to early or late transcription elongation were observed in the region upstream of bidirectional promoters, confirming the fact that these regions undergo productive transcription. strikingly, we also found evidences for chromatin features specific to luat-associated promoters. although the three gene sets are similarly enriched for h3k36me within the coding gene body, the luat-associated genes display higher levels of h3k79me. h3k79me is generally enriched at the 5′ end of expressed genes comprising the first exon and intron and mark the transition between early and late transcription elongation. to have a more precise view of the differential enrichment in h3k79me we compared the h3k79me profiles within rescaled regions comprising the tss to the end of 1st intron. indeed, we observed that h3k79me remains significantly higher throughout the first intron of luat-associated genes as compared to the control gene sets. as the genes from the three sets express similar amounts of productive transcription, our results most likely suggest an actual increased rate of early transcription elongation from luat-associated promoters. in agreement with an increased rate of early transcriptional elongation at luat-associated genes, we observed aspecific enrichment of pol ii and tbp within the 5′ region of luat-associated gene-bodies, indicating extended accumulation of the initiating and/or early elongating transcription complex. to further investigate the possibility of a transcriptional pause immediately downstream the tss, we analyzed the average profiles in dp thymocytes of additional general transcription factors, including taf tfiib, tfiie and tfiih. in all cases, we observed a significant enrichment of the gtfs downstream the tss. finally, consistently with the transcriptional pause being directly linked to divergent transcription, we also observed a significant and specific enrichment of pol ii and gtfs around a region 1 kb upstream the tss of luat associated genes. of particular interest is the overall enrichment in tfiih complex around the tss of luat-associated genes, which play a key role in transcription initiation by phosphorylating pol ii at ser. thus, our results indicate increased pol ii pausing at both sides of luat-associated promoters. accumulation of immature transcripts at luat-associated genes early elongation and h3k79me enrichment are generally associated with the 5′ intronic sequences and splicing events. thus, to further explore the hypothesis of an accumulation of immature transcripts at luat-associated genes, we compared the average profiles around the tss of total and polya rna-seq levels for the three set of equally expressed genes. we found that total-rna signal downstream of the tss is higher for luat-associated genes, while polya-rna signal is similar among the three gene sets. while the polya rna-seq signals result only from complete transcripts, total rna-seq signals result from both immature and complete transcripts. thus, a relative enrichment of total rna-seq, as compared to polya rna-seq signal, is indicative of either increased rate of immature transcription or expression of less stable transcripts. however, the observed results could not be attributed to differences in transcript stability as the three gene sets display equivalent levels of h3k36me which is generally coupled to productive elongation. to directly assess whether luat-associated genes produce increased level of immature transcripts, we computed the splicing index across the three gene sets. as shown in figure 7b, the splicing index is significantly reduced between the first two exons of luat-associated genes as compared to the other set of genes, while no differences are observed at the middle exons. this suggests an increased rate of immature transcription at the 5′ region of luat-associated genes. consistently, we found that first introns of luat-associated genes display higher density of total rna-seq signal as compared to the control gene sets, though there is no significant differences in first intron length between the three set of genes. significant enrichment of immature transcripts within the first intron of luat-associated genes was also confirmed by rt-qpcr analysis of individual genes. all in all, luat-associated gene promoters are clearly more prone to induce immature transcription than other bidirectional or unidirectional gene promoters, indicating a less restricted control of pol ii pausing around the tss, likely leading to divergent and pervasive transcription. discussion here, we used directional rna-seq from primary mouse tissues to directly and systematically characterize antisense transcripts. we have found that a significant fraction of gene promoters sustains expression of long non-coding antisense transcripts. the luat/coding gene pairs are usually co-regulated throughout cell differentiation and development and generally function as transcriptional regulators. luat-associated promoters share several characteristics of promoters of developmentally regulated genes, including large cpg islands, high degree of conservation and epigenetic regulation during development. moreover, the bidirectional transcription at these promoters appears to be linked to a specific gc skew profile and particularly longer first exons. finally, luat-associated promoters display distinctive epigenetic features reflecting an intensified rate of early transcriptional elongation. overall, our results support the view that promoters of a subclass of transcriptional regulators are characterized by a specialized mechanism of pol ii transcription, in which pol ii recruitment is directly coupled to relaxed bidirectional transcription. previous studies have shown that lincrnas are preferentially located near protein-coding genes in divergent orientation and that their expression is often correlated. however, the stringent criteria generally used to define lincrnas likely impaired a comprehensive identification of lncrna transcripts, including those in divergent orientation from coding genes. a recent study has indeed described the abundance of divergently transcribed lncrna/mrna gene pairs in es cells. although this and the present works likely described the same kind of antisense transcripts, our study largely complement and extend the previous study by using a more direct approach to identify upstream antisense lncrnas solely based on the rna-seq signal and by demonstrating their existence in many primary tissues and throughout t cell differentiation. moreover, we show here that divergent transcription is clearly associated with a subset of genes coding for transcriptional regulators, and we propose a functional link between divergent transcription and gene expression regulation. as suggested previously the presence of short bidirectional transcripts appears to be a hallmark of active promoters in mammals, generally associated with paused pol ii around the tss. it has also been previously described that expression of upstream unstable transcripts are a common characteristic of pol ii transcribed genes. although some overlaps might exist between prompts and luats, luats differ from exosome-sensitive prompts transcripts. firstly, luats are detected at significant levels without inhibition of the rna exosome degradation machinery. secondly, they are substantially longer than prompts. thirdly, they are associated with a specific category of genes. hence, while many coding gene promoters, essentially those containing cpg islands, have the intrinsic property of bidirectional transcription, production of long antisense transcript is generally inhibited or are rapidly degraded at most loci. divergent transcription is linked to intrinsic sequence properties shared with developmental gene promoters we have found that luat-associated genes frequently encode tfs involved in cell differentiation and development. this is consistent with previous studies in mouse, human and zebra fish suggesting that large gene deserts flanking tf genes, with roles in embryonic development, preferentially harbor lincrnas. as such, these genes are expected to be subjected to fine tune regulation. accordingly, we found that their promoters appears to be bound by lineage specific tfs. another striking characteristic of luat-associated promoters is the presence of very large cpg islands, a feature shared with developmental gene promoters, but not observed at bidirectional coding-gene promoters. as the promoters of these genes are enriched for cpg rich regions and are prone to pervasive transcription, it is expected that dedicated repressive mechanisms might be in place to control their expression. in line with this, we have found that luat-associated genes are specifically enriched for h3k27me and for bivalent domains in es cells, thus suggesting polycomb-mediated regulation of these genes. as suggested elsewhere, large cpg islands might represent a favored recruitment platform for polycomb-associated complexes and thus play an important role in transcriptional regulation of key developmental genes. whether large cpg islands are required for divergent transcription from luat-associated promoters will deserve further investigation. a link between antisense and immature transcription? our results indicate an unexpected link between divergent lncrna/mrna transcription and premature termination of coding transcripts. indeed, luat-associated genes are characterized by a significant accumulation of immature transcripts within the 5′ region of the genes. our results most likely suggest that intrinsic properties of luat-associated promoters induce a specialized mechanism of pol ii transcription, in which recruitment of the enzyme is directly coupled to pervasive bidirectional transcription. several arguments point to this direction. the presence of a tata box is generally linked to strong directionality of transcription. consistent with their bidirectional feature, luat-associated promoters are essentially depleted of tata box. although the absence of a tata box might be required for bidirectional transcription, as has been suggested for coding-coding promoters, it might not be sufficient as some tata-less promoters still show strong directionality. in addition, we have also observed that luat-associated promoters display two inverted, though moderated, gc skew profiles around the tss. it has been shown that a positive gc skew immediately upstream the tss favors the formation of so-called r-loop structures, which are long, stable and three stranded rna/dna structure formed during transcription. it has also been previously suggested that positive gc skew immediately after the tss, and subsequent r loop formation, may serve to correct the lack of directionality in the initial steps of transcription. this correction might be mediated by the ability of r loops to elicit transcriptional pausing. finally, 5′ splice sites and first exons have been recently shown to play a role in promoter directionality. it is suggested that u snrnp binding at 5′ splice sites might help to stabilize pol ii recruitment at the promoter and enforce sense transcription. moreover, the length of first exon appears to influence pol ii stability at promoters and transcription accuracy. indeed, genes with long first exon have pol il accumulation downstream the tss and increased proportion of antisense transcripts, reminiscent of what we have observed at luat-associated loci. strikingly, functional inhibition of u snrnp in es cells resulted in accumulation of premature cleavage of coding gene transcripts, but also increased expression of antisense transcripts. consequently, spatial separation of promoter and 5′ splice sites might directly impact both directionality and transcription accuracy. it is plausible that, at luat-associated genes, 5′ splice sites are too distant from the tss to ensure proper directionality of transcription, thus favoring bidirectional transcription. taking all into account, we favor a hypothesis whereby both immature and bidirectional transcription at a specialized class of gene promoters are directly linked to intrinsic sequence properties, including tata box depletion, unique gc skew profile and long first exon. functional relevance of bidirectional transcription the specific association of antisense transcripts with genes related to transcription regulation and development strongly suggests that divergent transcription might be directly or indirectly involved in the tight regulation of these genes. in line with a direct role of luats, several examples have demonstrated a functional regulation by mammalian antisense transcripts in cis. this regulation might act at multiple levels, from modifying local chromatin to enabling regional signal spreading or controlling translation efficiency, yet in the latter case an overlapping stretch with coding exons is required. alternatively, divergent transcription might reflect an intrinsic property of promoters of genes coding for developmental regulators. the finding that h3k79me a mark of early elongation, was found higher at luat-associated genes suggests the level of early elongating transcription is increased at these genes. this was further supported by a relative higher ratio of total versus polya levels within the first intron, as well as decreased splicing efficiency. thus, our findings clearly indicate that luat-associated promoters are more prone to pervasive and/or immature transcription. what can be the functional relevance of this pervasive transcription? if divergent transcription and non-productive gene expression are linked, this will imply that the initiation step of transcription is less controlled at these promoters, likely leading to pervasive transcription in both directions. in this line, an appealing hypothesis will be that expression of luat-associated genes is regulated also at the level of transcript maturation and/or elongation efficiency. this might reflect a checkpoint for coupling elongation and rna processing, as previously suggested. although some genes are regulated by pol ii pausing in close proximity to the tss, others are reported to be efficiently transcribed into precursor transcripts, while the efficiency of transcript processing is being regulated. generally, only a portion of the pol ii that assemble at the promoter enters into early elongation. this entry is characterized by pol ii ser phosphorylation and chromatin modifications that are specific to transcription initiation and early elongation. before transcribing further, the polymerase passes a 5′ checkpoint where it pauses, terminates, or commits to productive elongation. if the polymerase does not proceed through this checkpoint, transcription will be terminated producing an unstable transcript. if the polymerase proceeds through the checkpoint, it will enter into productive elongation that is associated with characteristic chromatin modifications and pol ii ser phosphorylation. in this context, rapid transcriptional induction might be facilitated by the active release of polymerase molecules that have initiated transcription, but are paused downstream the tss. thus, pausing during early elongation may provide both a kinetic ‘window of opportunity’, as well as an interaction surface, to facilitate additional levels of regulation of the nascent rna before the transition to productive elongation. another related and not exclusive hypothesis would be that bidirectional promoter activity increase the stochasticity of gene expression, as suggested for antisense expression in yeast. it is well known that expression of developmental regulators, including key transcription factors, is often regulated stochastically during cell differentiation, influencing cell and developmental decisions. it is therefore plausible that luat expression might reflect a dedicated mechanism to induce stochastic expression of developmental regulators by modulating the rate of pervasive transcription. in any case, our observations might shed light on a new mechanism representing the outcome of an evolutionary pressure to control the expression of a subclass of genes coding for transcriptional regulators.  we have found that divergent transcription of lncrna/mrna gene pairs is significantly associated with genes related to transcriptional regulation and development. their promoters share several characteristics with those of developmental transcription factors, display a unique gc skew profile and are associated with genes harboring long first exons, reminiscent of their bidirectional activity. unexpectedly, we also found that the 5′ region of the associated coding genes harbor a specific accumulation of h3k79me epigenetic mark, as well as initiating pol ii complexes, both of which are linked to an intensified rate of early transcriptional elongation. these results strongly suggest a functional link between divergent transcription and the regulation of genes coding for developmental transcription factors. altogether, our findings which indicate that, at a subset of transcription factor gene promoters, divergent and pervasive transcription are linked, might illustrate an additional mechanism for regulating the regulators, in a rather sophisticated system for fine-tuning mammalian gene expression. 
we found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding rna /mrna gene pairs. for instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the rna degradation machinery is inhibited. in-depth analysis revealed a unique gc skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. we concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. their promoters share several characteristics with those of transcriptional developmental genes, including very large cpg islands, high degree of conservation and epigenetic regulation in es cells. moreover, many described long non-coding rnas are transcribed antisense from coding gene promoters. here, we used strand-specific rna-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. divergent transcription is a wide-spread phenomenon in mammals. divergent transcriptionlncrnabidirectional promotergc skewdevelopmental transcription factor finally, genes associated with antisense transcription harbor specific h3k79me epigenetic marking and rna polymerase ii enrichment profiles linked to an intensified rate of early transcriptional elongation.
 divergent transcription is a wide-spread phenomenon in mammals. for instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the rna degradation machinery is inhibited. moreover, many described long non-coding rnas are transcribed antisense from coding gene promoters. however, the general significance of divergent lncrna/mrna gene pair transcription is still poorly understood. here, we used strand-specific rna-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues.  we found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding rna /mrna gene pairs. strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. their promoters share several characteristics with those of transcriptional developmental genes, including very large cpg islands, high degree of conservation and epigenetic regulation in es cells. in-depth analysis revealed a unique gc skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. finally, genes associated with antisense transcription harbor specific h3k79me epigenetic marking and rna polymerase ii enrichment profiles linked to an intensified rate of early transcriptional elongation.  we concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. divergent transcriptionlncrnabidirectional promotergc skewdevelopmental transcription factor 