Long non-coding RNA KHPS1 drives a regulatory cascade which activates expression of the protooncogene SPHK1

Abstract

Numerous long noncoding RNAs (lncRNAs) have been discovered, however only a small number of lncRNAs have been explored with respect to their function and little is known how they operate at chromatin. In this study, the function of an E2F1-regulated lncRNA, termed KHPS1, has been investigated. KHPS1 is transcribed in antisense orientation from the SPHK1 (Sphingosine kinase 1) promoter which in sense orientation directs transcription of SPHK1 mRNA. The results demonstrate that KHPS1 activates SPHK1 transcription by recruiting histone acetyltransferase p300 and the transcription factor E2F1 to the SPHK1 enhancer. Binding of p300 and E2F1 is required for transcription of an enhancer-derived RNA, eRNASphk1. Transcription of eRNA-Sphk1 evicts CTCF, a factor that insulates the SPHK1 enhancer from the SPHK1 promoter and facilitates SPHK1 expression. Importantly, the direct association of KHPS1 with a homopurine stretch upstream at the SPHK1 enhancer is essential for SPHK1 expression. Binding of KHPS1 to the SPHK1 enhancer is mediated via Hoogsteen base pairing, forming RNA-DNA-DNA triplex structure. Tethering KHPS1 and associated p300 and E2F1 to the SPHK1 enhancer is a prerequisite for activation of eRNA-Sphk1 transcription and expression of SPHK1. The functional relevance of triplex formation was further studied using reporter plasmids which mimic KHPS1-dependent transcription activation of SPHK1. Deletion or mutation of the triplex forming region (TFR) attenuated recruitment of p300 and E2F1 and compromised transcription of eRNA-Sphk1. Replacement of the TFR by foreign triplex-forming motifs from lncRNAs MEG3 or Fendrr functionally replaced the TFR of SPHK1, i.e. activated eRNA-Sphk1 transcription. Ectopic KHPS1 with the TFR of MEG3 targeted E2F1 and p300 to the MEG3 target gene TGFBR1, underscoring the functional relevance of triplex-forming sequences. Genomic deletion of the TFR or intervention of KHPS1 binding to DNA by ectopic TFR-containing RNA decreased SPHK1 expression and impaired cell viability. Collectively, the results unravel a triplexdependent regulatory feed-forward mechanism, involving lncRNA-mediated activation of eRNA which enhances expression of its target gene. The results underscore the pivotal role of triplex formation in transcription control, supporting a model whereby lncRNAs tethered to specific loci serve as sequence-specific molecular anchors.