1. Volume 60, Issue 4, Pages 626-636 (November 2015)
LncRNA Khps1 Regulates Expression of the Proto-oncogene SPHK1 via Triplex- Mediated Changes in Chromatin Structure 
Anna Postepska-Igielska, Alena Giwojna, Lital Gasri-Plotnitsky, Nina Schmitt, Annabelle Dold, Doron Ginsberg, Ingrid Grummt 
Molecular Cell 
Volume 60, Issue 4, Pages (November 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 60, 626-636DOI: (10.1016/j.molcel.2015.10.001)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Khps1 Is an E2F1-Regulated lncRNA
(A) Schematic of the human SPHK1 locus. Arrows mark TSSs, boxes represent exons, Khps1 is depicted in red. The potential triplex-forming regions TFR1 (−45/–86) and TFR2 (−327/−349) and E2F1 binding sites are indicated.
(B) Khps1 is a >10-kb lncRNA. Northern blot (NB) using a Khps1-specific RNA probe (nucleotides −1787/–1717 upstream of the TSS of SPHK1-B). The left lane shows an ethidium bromide (EtBr) stain of cellular RNA.
(C) The Khps1 promoter is located in the first intron of SPHK1-B. Promoter activity of different fragments of SPHK1 (+456/+1242, +1219/+1795, and +2492/+3235) was measured in HepG2 cells using dual-luciferase reporter assays. The empty pGL4.10 vector was set to 1 (n = 2). FL, firefly luciferase; RL, Renilla luciferase.
(D) Depletion of E2F1 and E2F3 decreases transcription of Khps1 and Sphk1 mRNA. U2OS cells were depleted of E2F1 and E2F3, and Khps1 and Sphk1 mRNA levels were determined by qRT-PCR. Error bars denote means ± SD (n = 3).
(E) E2F1 activates Khps1 transcription. qRT-PCR data showing the levels of Sphk1 mRNA and Khps1 after overexpression of wild-type E2F1 (wt) or a DNA-binding-deficient mutant E2F1 (mut) in U2OS cells (n = 3).
(F) Ectopic E2F1 stimulates expression of a luciferase reporter. HepG2 cells were co-transfected with Khps1 or SPHK1-B promoter-luciferase fusions and expression vectors encoding wild-type or mutant E2F1. Bars show changes in dual-luciferase activity relative to cells without E2F1 overexpression (−) (n = 2).
(G) E7 oncoprotein stimulates Khps1 transcription. Levels of Khps1 and Sphk1 mRNA after overexpression of wild-type or mutant E7 protein in WI38 fibroblasts (n = 2).
Data are presented as mean ± SEM unless specified differently. See also Figure S1.
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4. Figure 2 Khps1 Activates SPHK1 Transcription
(A) Khps1 correlates with SPHK1 expression. Data represent normalized levels of Khps1 and Sphk1 mRNA in the indicated cell lines (n = 3).
(B) Knockdown of Khps1 downregulates SPHK1 transcription. Relative levels of Khps1 and Sphk1 mRNA in cells transfected with siRNA against Khps1 compared with control (ctrl) siRNA (n = 4).
(C) Exosome knockdown enhances SPHK1 transcription. Shown are Khps1 and Sphk1 mRNAs in HeLa cells transfected with siRNA against hExoSc3 (n = 3).
(D) Depletion of Khps1 leads to loss of E2F1 from the SPHK1-B promoter. ChIP shows binding of E2F1 to the SPHK1-B and CDC2 promoter in U2OS cells transfected with siRNA against Khps1 (n = 3).
(E) ChIP showing binding of E2F1 to SPHK1-B in exosome-depleted HeLa cells (n = 3).
All data are presented as mean ± SEM. See also Figure S2.
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5. Figure 3
An E2F1-Driven Regulatory Loop Links Khps1 Transcription to SPHK1 Expression
(A) Activation of E2F1 upregulates transcription of Khps1 and SPHK1. Inset: RNA-seq data from untreated and 4-OHT-treated (100 nM, 16 hr) U2OS/ER-E2F1 cells. Reads mapped to both strands on chr17:74,378,109–74,383,973 are depicted, denoting Sphk1 mRNA (blue) and Khps1 (red). Bar diagram: Khps1 and Sphk1 mRNA levels in U2OS cells expressing ER-tagged wild-type E2F1 (ER-E2F1) or mutant E2F1-E132 incubated with 4-OHT for the indicated times. Error bars denote means ± SD.
(B) Induction of E2F1 augments binding of E2F1 and Pol II to the SPHK1-B promoter. ChIP shows E2F1 and Pol II occupancy in untreated and in 4-OHT-treated U2OS/ER-E2F1 cells (n = 3).
(C) E2F1-dependent activation of SPHK1 transcription requires Khps1. U2OS/ER-E2F1 cells depleted of Khps1 by siRNA were treated with 4-OHT for 2 hr. Relative levels of Sphk1 mRNA and Khps1 are shown (n = 3).
(D) Khps1 is required for E2F1 binding to the SPHK1-B promoter. ChIP shows E2F1 occupancy at the SPHK1-B promoter in Khps1-depleted (siKhps1) and flavopiridol-treated (1 μM, 1 hr) U2OS/ER-E2F1 cells (n = 3).
(E) Binding of E2F1 and Pol II to the SPHK1-B promoter requires Khps1. Cells were treated with flavopiridol or depleted of Khps1 before 4-OHT treatment for 2 hr (n = 3).
(F) Reporter assay showing Khps1-dependent transcription of sense RNA. NIH 3T3 and HeLa cells were transfected with a reporter construct (pTet-Khps1 +1448/−592) driving transcription of Khps1 (see schematic above). Levels of reporter-derived Khps1 and sense transcripts as well as endogenous Sphk1 mRNA were monitored (n = 3). The positions of primers used for cDNA synthesis and PCR amplification are marked by blue and black arrows. Dox, doxycycline.
(G) ChIP showing increased binding of E2F1 to the reporter upon induction of Khps1 (tTA, n = 3).
Data are presented as mean ± SEM unless specified differently. See also Figure S3.
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6. Figure 4 Khps1 Mediates Epigenetic Changes at the SPHK1-B Promoter
(A) Induction of E2F1 triggers changes of histone marks. ChIP shows SPHK1-B promoter occupancy of the indicated histone marks at the SPHK1-B promoter before and after treatment of U2OS/ER-E2F1 with 4-OHT for 10 hr. Data were normalized to input values and are presented in reference to untreated cells (n = 3).
(B) Khps1 is required for active histone modifications at the SPHK1-B promoter. ChIP shows histone marks at the SPHK1-B promoter after treatment of U2OS/ER-E2F1 cells with flavopiridol or depletion of Khps1 by siRNA (n = 3).
(C) E2F1-dependent changes of chromatin structure require Khps1. ChIP shows histone marks in Khps1-depleted U2OS/ER-E2F1 cells 2 hr after induction of E2F1 by 4-OHT (n = 3).
(D) Khps1 is associated with p300/CBP. Shown is RIP comparing the association of Khps1 and a control lncRNA (HOTAIR) with p300 precipitated from 4-OHT-treated U2OS/ER-E2F1 cells (n = 2).
(E) Khps1 recruits p300/CBP to the SPHK1-B promoter. Shown is ChIP comparing binding of p300 to the SPHK1-B promoter in untreated U2OS/ER-E2F1 cells with mock-transfected or Khps1-depleted cells (siKhps1) that were treated for 2 hr with 4-OHT (n = 3).
(F) p300/CBP activity is required for Khps1-dependent transcription of Sphk1 mRNA. qRT-PCR shows normalized levels of Khps1 and Sphk1 mRNA in U2OS/ER-E2F1 cells treated for 2 hr with curcumin (30 μM), followed by tamoxifen treatment for 4 hr (n = 3).
(G) Khps1-dependent changes in histone acetylation and E2F1 binding require p300 activity. ChIP shows histone modifications and E2F1 binding in curcumin-treated NIH 3T3 cells comprising an integrated reporter plasmid driving Khps1 transcription (n = 3).
(H) Transcription of Khps1 induces chromatin decompaction. FAIRE assay comparing the extractability of SPHK1-B in U2OS/ER-E2F1 cells before and after E2F1 induction in the absence or presence of flavopiridol. The data present levels of recovered DNA measured by qPCR using the indicated amplicons. A, −592/−425; B, −406/−241; C, −175/−65; ctrl, 18S rRNA (n = 3).
Data are presented as mean ± SEM. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Khps1 Binds to the SPHK1 Promoter by Forming RNA-DNA Triplexes
(A) Schematic depicting the position of TFRs and synthetic RNAs used. The nucleotides involved in Hoogsteen base-pairing between the TFR2 sequence and Khps1 are indicated. The arrowed lines illustrate in vitro-transcribed Khps1 derivatives used to capture the SPHK1-B promoter (A, −373/+7; B, −137/+7; C, −406/–241; D, +1050/+1239).
(B) Khps1 binds to TFR2 via Hoogsteen base-pairing. Biotinylated Khps1 versions (A, B, C, or D, see above) were incubated with SPHK1-B fragments (−592/+7 and +596/+1242) generated by standard PCR (left and center) or in the presence of deaza-7-dATP and deaza-7-dGTP (right). Upon binding to streptavidin beads, associated DNA was analyzed by qPCR using promoter-specific (−592/–425) or intronic-specific (+1132/+1242) primers. Data represent the fold increase in DNA bound to the respective RNAs compared with assays without RNA (n = 3).
(C) Khps1 associates with SPHK1-B in nuclei. Biotinylated Khps1 (A or D) was incubated with nuclei, RNA was bound to streptavidin beads, and RNA-associated DNA was quantified by qPCR using promoter-specific (−406/−304) or intronic-specific (+1132/+1242) primers (n = 3).
(D) Khps1 forms DNA-RNA triplexes with TFR2. Left: increasing amounts (40-, 80-, and 160-fold molar excess) of synthetic Khps1 (−373/−241) were incubated with a double-stranded 32P-labeled oligonucleotide comprising TFR2 (−357/−319), and formation of RNA-DNA triplexes was monitored by EMSA. Right: reactions containing labeled TFR2 oligonucleotide and a 40-molar excess of Khps1 (−373/−241) were treated with 0.5 U RNase H (H) or with 0.5 ng RNase A (A) for 30 min at room temperature.
(E) Sequence-specific interaction of Khps1 with TFR2. EMSA shows the mobility of 32P-labeled WT or mutant (Table S3) SPHK1-B promoter fragment (−357/−319) after incubation with wild-type or mutant Khps1 (−373/−241).
(F) Khps1 interacts with the SPHK1-B promoter in vivo. HeLa cells were transfected with biotinylated Khps1 (A or D) and RNA-associated DNA was quantified as in (C) (n = 3).
(G) Khps1 forms DNA-RNA triplexes in vivo. HeLa cells transfected with a 5′-6C-psoralen-3′-biotin-labeled control RNA oligo (ctrl) or an oligo comprising Khps1 sequences (−322/−352, TFR2) were UV-crosslinked, and RNA-associated DNA was quantified by qPCR. Data represent enrichment of the SPHK1-B promoter. As negative controls, non-crosslinked cells or cells transfected with an unrelated modified RNA oligo were used (n = 2).
All data are presented as mean ± SEM. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Khps1-Dependent SPHK1 Expression Is Required for Cell-Cycle Progression and Cell Viability
(A) Khps1 and Sphk1 mRNA transcription fluctuates during the cell cycle. WI38 cells were arrested in G0 and released by serum addition, and then Khps1 and Sphk1 mRNA were monitored by qRT-PCR.
(B) Knockdown of Khps1 or Sphk1 impairs cell proliferation. U2OS cells were transfected with nonspecific siRNA (ctrl), Khps1-specific siRNA (siKhps1), or SPHK1-specific (siSphk1) siRNA, and cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye assay (n = 3).
(C) FACS analysis of U2OS cells treated with siRNA against Khps1 or Sphk1 mRNA. The percentage of cells in different phases of the cell cycle and cells with sub-G1 DNA content 72 hr post-transfection is displayed (n = 3).
(D) Ablation of Khps1 or Sphk1 mRNA enhances apoptosis. Shown is a western blot monitoring cleaved Caspase-3 in U2OS cells treated with the indicated siRNAs.
(E) Knockdown of Khps1 augments E2F1-dependent apoptosis. Khps1-depleted U2OS/ER-E2F1 cells were treated for the indicated times with 4-OHT. Annexin V-positive apoptotic cells were determined by FACS (n = 3).
(F) Model illustrating the E2F1- and antisense RNA-driven feedforward loop that induces SPHK1 expression. Elevated levels of E2F1 induce transcription of Khps1, initiating an lncRNA-mediated regulatory loop that activates SPHK1 expression. Khps1 tethered to the purine-rich sequence (TFR2) upstream of the transcription start site of SPHK1-B via triplex formation guides Khps1-associated p300/CBP to the SPHK1-B promoter. As a consequence, hyperacetylated decompacted chromatin facilitates E2F1 binding and transcription activation of SPHK1.
Data are presented as mean ± SEM unless specified differently. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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