Functional studies of ncRNAs in yeast have revealed genetic interactions between certain lncRNAs and the methylation status of H3K4. The PHO84
antisense transcript runs through the body and promoter of PHO84 gene, and can inhibit sense PHO84 transcription in a SET1-dependent manner in cis
(the endogenous configuration) or in trans
(when the antisense is transcribed from a plasmid) [38
]. The Ty1 retrotransposon has an antisense CUT RNA named RTL whose expression is anti-correlated with the Ty1 transcript [40
]. Interesting, silencing of the Ty1 transcript is accomplished in trans
by the RTL RNA through the Set1 methyltransferase complex. More recent work has classified RTL RNA as a member of the Xrn1-sensitive unstable transcripts (XUTs) given their sensitivity to the cytoplasmic 5' to 3' exonuclease Xrn1 [41
]. Many of these XUTs are antisense to coding genes are have properties similar to RTL. The widespread nature of these antisense XUTs in yeast and their connection to the Set1 complex suggest an important link between lncRNAs and H3K4 methylation in yeast, specifically that the antisense XUTs may be positively regulated by Set1 and in turn repress the sense transcript (). One aspect of the XUT mechanism that is not clear is how XUTs, which act at the chromatin level to affect gene expression, are signaled for exported from the nucleus for Xrn1 degradation. Focused studies exploring the specific mechanism for RNA-mediated gene control between cellular compartments will be of interest in the future.
While most of the mammalian lncRNAs characterized to date repress gene activity, recent studies revealed that perhaps many lncRNAs enhance gene expression. The GENCONE database identified numerous new lncRNAs, several of which resulted in examples of activating lncRNAs (ncRNA-a) [42
]. Depletion of these lncRNAs resulted in decreased expression of nearby protein coding genes, and experiments fusing cDNAs encoding ncRNAs to reporter genes suggested several ncRNAs activate gene expression in cis
in an enhancer-like manner, although the mechanism remains unclear. Separately, studies of a novel lncRNA termed HOTTIP has provided a paradigm for how enhancer-like ncRNAs may act. HOTTIP
is located on the very 5' end of the HOXA
homeotic gene cluster [43
]. In cells where 5' HOXA
genes are active, the locus adopts a highly compact form, looping to bring HOTTIP
and the nascent HOTTIP RNA into proximity to multiple 5' HOXA
genes. In turn, HOTTIP RNA directly binds to WDR5, a subunit of the WDR5-Ash2L-RbBP5 (WAR complex) associated with all MLL H3K4 methylases, and recruits it to 5' HOXA to enforce H3K4 methylation and gene expression (). Ectopic expression and RNA tethering experiments showed that HOTTIP can only act in cis,
and thus is likely strictly dependent on the endogenous chromosome looping to dictate its target genes. Thus, lncRNAs such as HOTTIP mediate information transfer of spatial information in chromosomal looping into biochemical information in histone modifications. The recent identification of another lincRNA that activates HoxA6
via direct interaction with MLL1 suggests a common theme [44
Broad domains of active chromatin are hallmarks of developmental genes under epigenetic maintenance [45
]. Once a lncRNA targets the MLL complex, the relatively non-sequence specific DNA binding activity of Ash2L, newly recognized through structural studies, spreads the complex across chromatin. At the HOXA
locus, point mutations that abolish Ash2L DNA-binding activity in vitro allows the complex to target to the HOTTIP
element, but fails to spread properly across HOXA
]. These finding support the notion that lncRNAs have the capacity to enhance gene expression by specifically nucleating H3K4 methylase complex at specific genes, which then spread or stabilize via a separate DNA binding activity in a manner dependent on chromosomal looping. We await future work that delineates in detail the RNA-protein interaction required to specify these events.
Establishing a more complete catalog of lncRNA transcripts will speed the discovery of novel ncRNAs with functional roles (such as HOTTIP), and recently work in mouse and human cells has revealed large numbers of previously unannotated lncRNAs [47
]. Extensive deep sequencing of transcriptomes from diverse human tissues annotated more than 8000 lncRNAs, and importantly revealed that on average, lncRNAs are more tissues specific than protein-coding genes, suggesting that lncRNAs may actually play a significant role in determining cellular fate [48
]. Guttman and colleagues characterized mESC-specific lncRNAs and through functional studies showed many of these lncRNA are regulated by pluripotency factors (Oct4, Sox2, and Nanog) and almost 30% of the ESC-specific lncRNAs appear to interact with at least one repressive chromatin protein complex [49
]. Towards addressing the potential functions of lncRNAs at chromatin in an unbiased manner, a new method termed Chromatin Isolation by RNA Purification (ChIRP) was developed [50
]. Analogous to ChIP-seq, ChIRP-seq provides genome-wide identification of the specific sites of RNA occupancy on chromatin, and has been applied to the telomerase RNA TERC, roX2, and HOTAIR. These lncRNAs bound hundreds to thousands of sites throughout the genome, had focal foot prints (similar to transcription factors) and sequence specificity in their localization. Together, shRNA screens and ChIRP should enable high throughput functional studies of the lncRNA landscape.
Efforts to understand the interplay between ncRNAs and chromatin have established that both the RNA and chromatin can affect one another. While features of the active chromatin state have been well studied, the interactions between RNA and active are only beginning to be explored. Given the pervasive nature of transcription and the near ubiquitous nature of histone methylation, future work is likely to uncover mechanisms that employ both ncRNAs and the state of chromatin methylation to regulate gene expression.