Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis.

The human genome encodes thousands of unique long non-coding RNAs (lncRNAs), many of which are emerging as critical regulators of cell fate. However, their functions as well as their transcriptional regulation are only partially understood. The E2F1 transcription factor induces both proliferation and apoptosis, and is a critical downstream target of the tumor suppressor, RB. Here, we provide evidence that a novel lncRNA named GASL1 is transcriptionally regulated by E2F1; GASL1 levels are elevated upon activation of exogenous E2F1 or endogenous E2Fs. Inhibition of GASL1 expression induced cell cycle progression, and in particular, G1 exit. Moreover, GASL1 silencing enhanced cell proliferation, while, conversely, its ectopic expression inhibited proliferation. Knockdown of GASL1 also enhanced E2F1-induced apoptosis, suggesting the existence of an E2F/GASL1 negative feedback loop. In agreement with this notion, silencing of GASL1 led to increased levels of phosphorylated pRB and loss of Rb impaired the effect of GASL1 silencing on G1 exit. Importantly, xenograft experiments demonstrated that GASL1 deletion enhances tumor growth. Moreover, low levels of GASL1 are associated with decreased survival of liver cancer patients. Taken together, our data identify GASL1 as a novel lncRNA regulator of cell cycle progression and cell proliferation with a potential role in cancer.

Graphical Abstract Highlights d Crystal structure of the intact human Robo2 ectodomain at 3.6 Å d Dimerization through domain 4 (D4) is required for Robo axon guidance d Robo receptors have an auto-inhibited conformation in which D4 is blocked d Slit may relieve Robo auto-inhibition, followed by dimerization and signaling SUMMARYProper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from ''off'' to ''on'' states remains unclear.Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.

26Background: Diverse biological processes and transcriptional programs are 27 regulated by RNA polymerase II (Pol II), which is recruited by the general transcription 28 machinery to the core promoter to initiate transcription. TRF2 (TATA-box-binding 29 protein-related factor 2) is an evolutionarily conserved general transcription factor that 30 is essential for embryonic development of Drosophila melanogaster, C. elegans, 31 zebrafish and Xenopus. Nevertheless, the cellular processes that are regulated by 32 TRF2 are largely underexplored. 33Results: Here, using Drosophila Schneider cells as a model, we discovered that TRF2 34 regulates apoptosis and cell cycle progression. We show that TRF2 knockdown 35 results in increased expression of distinct pro-apoptotic genes and induces apoptosis. 36Using flow cytometry, high-throughput microscopy and advanced imaging-flow 37 cytometry, we demonstrate that TRF2 regulates cell cycle progression and exerts 38 distinct effects on G1 and specific mitotic phases. RNA-seq analysis revealed that 39 TRF2 controls the expression of Cyclin E and the mitotic cyclins, Cyclin A, Cyclin B 40 and Cyclin B3, but not Cyclin D or Cyclin C. To identify proteins that could account for 41 the observed regulation of these cyclin genes, we searched for TRF2-interacting 42 proteins. Interestingly, mass spectrometry analysis of TRF2-containing complexes 43 identified GFZF, a nuclear glutathione S-transferase implicated in cell cycle regulation, 44and Motif 1 binding protein (M1BP). TRF2 has previously been shown to interact with 45 M1BP and M1BP has been shown to interact with GFZF. Furthermore, available ChIP-46 exo data revealed that TRF2, GFZF and M1BP co-occupy the promoters of TRF2-47 with TRF2, it is TRF2, rather than GFZF or M1BP, that is the main factor regulating 50 the expression of Cyclin E and the mitotic cyclins. 51 Conclusions: Our findings uncover a critical and unanticipated role of a general 52 transcription factor as a key regulator of cell cycle and apoptosis. 53 54 Keywords 55 Basal transcription machinery, RNA polymerase II, gene expression, TATA box-56 binding protein (TBP), TBP-related factor 2 (TRF2), cyclin genes. 57 58 BACKGROUND 59 Multiple biological processes and transcriptional programs are regulated by RNA 60 polymerase II (Pol II). The initiation of transcription of protein-coding genes and 61 distinct non-coding RNAs occurs following the recruitment of Pol II to the core 62 promoter region by the general/basal transcription machinery (1-4). The core 63promoter, which directs accurate initiation of transcription and encompasses the 64 transcription start site (TSS), may contain short DNA sequence elements/motifs, 65 which confer specific properties to the core promoter (1, 4-10). The first step in the 66 recruitment of Pol II to initiate transcription is the binding of TFIID, which is 67 composed of TATA-box-binding protein (TBP) and TBP-associated factors. 68Remarkably, although TBP is considered a universal general transcription factor, 69 robust Pol II transc...