Cell transfection with siRNA and plasmids

HEK 293T cells were seeded on poly-l-lysine (Sigma-Aldrich, Oakville, ON, Canada) and transfected with plasmid using the calcium phosphate technique. The vector pGL033-p65 was a kind gift from Dr Warner Greene lab (Gladstone Institute of Virology and Immunology, San Francisco, USA). For JMJD3 rescue experiments plasmids encoding for JMJD3 wild-type and mutant JMJD3 H1390A were kindly shared with us by Dr Charlie Degui Chen lab (Institute of Biochemistry and Cell Biology, Shangaï, China) (27). For DDX21, wild-type plasmid was ordered from GeneCopoeia (cat# EX-E1618-M08, MD, USA). Mutant DDX21 S375L/A376E (28,29) was created by site-directed mutagenesis (Civic Bioscience Ltee, QC, Canada). The vector RnaseH (#108699) and the plasmid encoding for HA-JMJD3 (#24167) used for analysis of the interactome of JMJD3 were obtained from Addgene (MA, USA). For siRNA treatment, cells were transfected by use of Lipofectamine RNAiMax (Invitrogen Thermofisher, Canada). siRNAs targeting JMJD3 (siJMJD3#1 cat no. #SI03181136 and siJMJ3#2 cat no.#SI04133836), DDX21 (siDDX21#1 cat no. #SI04311412 and siDDX21#2 cat no.#SI04155781), UTX (siUTX#1 cat no#SI04306743 and siUTX#2 cat no. #SI04314303), and EZH2 (cat no. #SI02665166) were purchased from QIAGEN (ON, Canada). For rescue experiments, HEK293T cells were transfected with siRNA targeting the 3′untranslated region (UTR) of intended mRNA target, followed by expression of siRNA-resistant wild-type or mutants constructs for DDX21 and JMJD3.

Luciferase reporter assay

HEK 293T cells were transfected with vectors encoding for 2.5 kb of ENPP2 promoter. In some constructions κB1 (CTGGCATTTCCAGTAT) or/and κB2 (CTGGTGGAAAGCCCTT) sites were deleted by directed mutagenesis. For analysis of enhancer activity, HEK 293T cells were transfected with the pGL4.10 luciferase vector (Promega, WI, USA) containing a minimal promoter sequence with or without the enhancer region located at chr8:120733232-120735226 on build 37 from ENCODE (Gene synthesis and subcloning, Bio Basic, ON, Canada). In these experiments, a vector coding for renilla luciferase was used as a reporter for transfection efficiency. Luciferase activities were measured at 48 h after transfection with the Promega Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol.

Chromatin immunoprecipitation (ChIP) and detection of R-loops

HEK 293T cells were crosslinked by adding formaldehyde solution to a final concentration 0.2%, dropwise directly to the media for 10 min at room temperature (Sigma-Aldrich). Then cells were harvest in 1 ml of phosphate-buffered saline (PBS) 1× (Sigma, ON, Canada) and were centrifuged 5 min at 800 g at 4°C. The supernatants were removed, pellets were then resuspended in 500 μl lysis buffer (50 mM Hepes-KOH, pH7.5, 140 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) pH8.0, 1% triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), PIC) and sonicated to shear the DNA to an average length of 100–400 base pairs (bps). One microgram of H3K27me3 (#9733, Cell signaling), 2 μg of H3K4me3 (#9751, Cell signaling), 2 μg of H3K4me1 (#5326, Cell signaling), 8 μg of RNA pol II (#Ab26721, Abcam), 8 μg of UTX (#Ab36938, Abcam), 8 μg of JMJD3 (#Ab38113, Abcam), 2 μg p65 (#3012-100, Biovision), 2 μg of DDX21 (#NBP1-83310, Novus Biologicals) antibodies or isotype IgG (Cell Signaling, MA, USA) were incubated with proteins G dynabeads (Life technologies, Thermofisher, ON, Canada) for 6 h before DNA samples were added to the antibodies/dynabeads mixtures and incubated at 4°C overnight on a rotator. R-loops detection was performed using 8 μg of S9.6 (#ENH001, Kerafast) antibody as previously described (30–34). The next day, the samples were washed with low salt buffer (0.1% SDS, 1% triton X100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0 and 150 mM NaCl), then with high salt buffer (0.1% SDS, 1% triton X100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0 and 500 mM NaCl), followed with LiCl buffer (10 mM Tris–HCl, pH8.0, 250 mM LiCl, 1 mM EDTA, pH 8.0, 1% NP-40) and finally with TE buffer (10 mM Tris–HCl, pH8.1, 1 mM EDTA, pH 8.0). The DNA–proteins complex were then eluted from dynabeads by adding 200 μl of elution buffer (1% SDS, 100 mM NaHCO₃) at 65°C for 1 h. Eluted samples were reverse cross-linked at 65°C overnight by adding 4.8 μl of 5M NaCl. The final mixture containing DNA was digested with 0.33 mg/ml of RNase A (QIAGEN, ON, Canada) for 1 h at 37°C and proteins digested for 1 h at 55°C with 0.5 mg/ml of proteinase K (QIAGEN, ON, Canada). Samples were extracted by phenol–chloroform and analyzed by quantitative PCR assay using primers listed in Supplementary Table S1.

ChIP-ReChIP

Re-chromatin immunoprecipitation (Re-ChIP) assays was performed according the same protocol except one step. Briefly, HEK 293T cells were crosslinked, lysed and sonicated as described in the ChIP protocol. Chromatin was incubated with first antibody (anti-HA) in the presence of magnetic beads. The beads were washed, and the material was eluted with Re-CHIP elution buffer (2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% NP-40) at 37°C for 30 min. Eluted samples were further incubated with the second antibody (anti-DDX21 or IgG).

Co-immunoprecipitation

HEK 293T were transfected with HA-JMD3 plasmids and harvested with TNGT buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4 1M, 1% Glycerol, 0.1% Triton X100, PIC). Whole cellular extracts were treated with 40U/ml of RNaseH, 25U/ml of DNaseI for 1 h at 37°C or not and immunoprecipitated with 8 μg of antibodies against HA-epitope (Sigma-Aldrich, ON, Canada), or isotype IgG (Cell Signaling, MA, USA) in the presence of 40μl magnetic beads (BioRad, CA, USA) overnight at 4°C. The next day, samples were washed three times with TNGT. Purified proteins were analyzed by western blot.

Western blot

Samples were boiled for 5 min, and proteins were loaded onto polyacrylamide gels followed by electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either HA primary antibodies (#H9658, Sigma-Aldrich), RNA polymerase II (#Ab26721, Abcam), DDX21 (#NBP1-83310, Novus Biologicals) using a 1:1000 dilution, overnight at 4°C. Membranes were then washed and incubated with HRP-labeled secondary antibodies (Cell Signaling Technology, MA, USA) using a 1:2000 dilution. Detection was done using clarity western ECL substrate (BioRad, ON, Canada).

Purification and identification of JMJD3-associated proteins

Fifty million cells were used per condition and were transfected with a plasmid coding for HA-JMJD3 for 24 h. LPS treatment was performed for 6 h at 100 ng/ml. The cells were then pelleted at 1200 g for 5 min (4°C) and the supernatant removed. The resulting pellet was then resuspended in 10 ml of TNGT buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4 1M, 1% Glycerol, 0.1% Triton X100, PIC) and immunoprecipitated with 360 μg of antibodies against HA-epitope (Sigma-Aldrich, ON, Canada) conjugated to 1800 μl of magnetic beads (BioRad, CA, USA). This IP mixture was then incubated overnight at 4°C (rotating). The next day, samples were washed three times with TNGT, and three times with ammonium bicarbonate 50 mM (freshly prepared). After the last wash, the beads were briefly spun down and any residual liquid was removed. Beads were frozen (−20°C) and later processed for mass spectrometry analysis (Proteomics platform, CHU de Québec Research Center, Qc, Canada). The MS/MS spectra, were filtered by the ‘Exclusive Unique Peptide Count’ from Scaffold3 software, corresponding to the number of different amino acid sequences (unique peptides), regardless of any modification that are attributed to a single protein. Raw data are provided in Supplementary Table S2. Peptides-associated with JMJD3 were considered valid if they contained more than two unique peptide fragments and matches of 95% probability. At last, data were processed with a Gene Ontology (GO) analysis from David interface (https://david.ncifcrf.gov/summary.jsp).

Capture nascent RNAs

To capture nascent RNAs, HEK 293T cells were labeled by adding 0.2 mM ethynyl uridine (EU) into the culture medium for 6 h simultaneously with LPS. The EU-labeled RNA were extracted and 8 μg biotinylated with 0.8 mM biotin azide by using the Click-it Nascent RNA Capture Kit (Life technologies, Thermofisher, ON, Canada). The biotinylated RNAs were then precipitated with ethanol and 1 μg bound to 50 μl of Dynabeads MyOne Streptavidin T1 magnetic beads in Click-iT RNA binding buffer containing 80U of RNAseOUT Recombinant Ribonuclease Inhibitors (Life technologies, Thermofisher, ON, Canada). The beads were washed with Click-iT wash buffer1 and 2, resuspended in 50 μl of Click-iT wash buffer 2 and used for cDNA synthesis using SuperScript VILO cDNA synthesis kit (Life technologies, Thermofisher, ON, Canada) in accordance with the manufacturer's instructions.

Promoter capture Hi-C (PCHi-C)

Data on ENPP2 chromosome interactions were retrieved from the Promoter Capture HiC (www.chicp.org) (35). PCHi-C studies provided high-resolution analysis of interactions between 31 253 annotated promoters (Fantom5, ENCODE) and distal DNA regulatory elements in 17 human primary blood cell types. Data from CD34+ cells were retrieved and analyzed. The data visualization results in a circularized overview of interactions and annotations in chromosome. Each interaction is indicated with colored arcs.

CRISPR/Cas9 editing

HEK 293T cells were transfected with Cas9-PuroR constructs #62988 (Addgene, MA, USA) along with a plasmid encoding for two RNA guide specific of enhancer region located at chr8:120733232-120735226 on build 37. After 24 h, the cells were selected with 2 μg/ml puromycin for 48 h (Wisent, QC, Canada), and q-PCR or ChIP experiments were performed. To monitor the enhancer deletion, we performed a PCR-based strategy as described previously (36,37). Briefly, we used q-PCR with five sets of primers covering the enhancer region and two additional primer sets located upstream and downstream of the predicted cut sites (control) as depicted in Supplementary Figure S9A.

Chromosome conformation capture (3C)

Chromosome conformation capture (3C) experiments was performed as described previously (38). Briefly, 1 × 10⁷ HEK 293T cells were harvested in 10 ml of Dulbecco's-modified Eagle's medium, centrifuged 5 min at 800 g at 4°C and supernatants were removed. Cell pellets were resuspended in 10 ml of PBS 1×/SVF 10% and cross-linked by adding paraformaldehyde solution to a final concentration 1%, directly to the media for 15 min at room temperature (Sigma-Aldrich). Quenching step was performed by adding 1.425 ml of 1M glycine. Next, cells were centrifuged 5 min at 400 g at 4°C, resuspended in 5 ml of lysis buffer (10 mM Tris–HCl pH7.5; 10mM NaCl; 0.2% NP40; PIC 1×) and incubated for 10 min on ice. Nuclei were isolated by centrifugation for 5 min at 400 g, resuspended with 500 μl of CutSmart Buffer 1.2× (New England BioLabs) supplemented with 0.3% SDS. Nuclei were incubated 1 h at 37°C while being shaken at 900 r.p.m. before the addition of 50 μl of 20%Triton X-100 (2% final) and an additional incubation was performed. DNA was then digested overnight at 37°C with 400U of NspI (New England BioLabs, ON, Canada). The next day, restriction enzyme was inactivated by the addition of 1.6% SDS at 65°C for 25 min and digested nuclei were diluted 20-fold with T4 DNA Ligase buffer 1.15× (New England BioLabs, ON, Canada) containing 1% of Triton X-100. Ligation step was performed with 400U of T4 DNA Ligase (New England BioLabs, ON, Canada) at 16°C overnight. Samples were reverse cross-linked at 65°C overnight in presence of proteinase K. The final mixture containing DNA was digested with 300 μg of RNaseA (QIagen, ON, canada), extracted by phenol-chloroform. Quantification of ligated products was performed by qPCR using the NspI cutting site, located in the TSS of ENPP2 as an anchor primer combined with primers positioned to each NspI cutting site. The sequences of primers used are listed in Supplementary Table S1. Results were expressed as frequency of random interactions between sites separated by increasing genomic distances ranging from 0.3 to 85 kb with the anchor primer. For all fragments, we first normalized using a GAPDH loading control, as described previously (38). Second, we used the level of ligation products between the anchor and the nearest downstream NspI site (#1) as a strong normalizer to quantify interaction frequency. qPCRs were performed in quadruplicates to improve sensitivity. Enhancer-promoter interactions are demonstrated as a peak across a region of multiple primers

ENPP2 promoter has bivalent histone marks

To investigate the chromatin changes of ENPP2 locus associated with transcription process, we analyzed the histone modifications at ENPP2 promoter. In ChIP-qPCR experiments, we found that the promoter region was enriched for H3K27me3 and H3K4me3 marks and bound by promoter-proximal paused RNA Pol II at the TSS (Figure ​(Figure2A2A–C). Hence, these data suggest that ENPP2 has characteristics of a bivalent gene: paused RNA Pol II around TSS and the coexistence of H3K27me3 with the activating histone modification H3K4me3. Interestingly, after LPS activation the level of H3K27me3 was substantially reduced at κB consensus sites and TSS (Figure ​(Figure2A).2A). We next investigated the involvement of H3K27me3-specific enzymes in the regulation of ENPP2: EZH2 (methyltransferase), JMJD3 (demethylase) and UTX (demethylase). The knockdown of EZH2 with small interfering RNA (siRNA) increased by 3-fold the expression of ENPP2 after LPS stimulation (Figure ​(Figure2D).2D). Inversely, the knockdown of specific H3K27 demethylase JMJD3 and UTX using two non-overlapping siRNA prevented LPS-mediated expression of ENPP2 (Figure ​(Figure2E2E and F). As confirmed by q-RT-PCR and western blot, the transfection of HEK 293T cells with RNAis against EZH2, JMJD3 and UTX efficiently depleted their respective target RNA transcripts and protein levels (Supplementary Figures S2A–C, 3A–C and 4A–C). To assess whether JMJD3 and/or UTX promoted the loss of H3K27me3 at ENPP2 locus during LPS stimulation we performed ChIP-qPCR assay with anti-H3K27me3 antibody, after a depletion of JMJD3 or UTX. The level of H3K27me3 was robustly increased with UTX depletion (Figure ​(Figure2G),2G), but not significantly different in absence of JMJD3 (Figure ​(Figure2H2H).

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Figure 2.

ENPP2 promoter has bivalent histone marks. (A) ChIP-qPCR measurement of H3K27me3 enrichment at the ENPP2 locus and a negative locus (NEG; downstream of SNRPN) in HEK 293T cells following 6 h of LPS exposure (B and C) ChIP-qPCR assays showing (B) enrichment of the H3K4me3 histone mark and (C) RNA pol II recruitment at the ENPP2 TSS. (D–F) Effect of siRNA against (D) EZH2, (E) UTX and (F) JMJD3 on LPS mediated expression of ENPP2, qPCR analyses. (G and H) H3K27me3 enrichment at the ENPP2 promoter, determined by ChIP-qPCR, in response to siRNA targeting (G) UTX and (H) JMJD3. (I) qPCR measurements of ENPP2 expression in response to JMJD3 siRNA transfection followed by rescue with constructs encoding for JMJD3 wild-type or JMJD3 H1390A (catalytic dead mutant). LPS 100 ng/ml.

To reinforce this finding, we carried out rescue experiments with JMJD3 catalytic dead mutant. First we performed a siRNA-mediated JMJD3 knockdown, and after 24 h we transfected cells again, either with a vector encoding for wild-type JMJD3, or for a catalytically inactive JMJD3 mutant (JMJD3 H1390A) (Figure ​(Figure2I).2I). Indeed, enzymatically inactive JMJD3 (JMJD3 H1390A) completely re-established the response to LPS on the expression of ENPP2 (Figure ​(Figure2I).2I). In these experiments, we confirmed that JMJD3 mutant was similarly expressed compared to wild-type plasmid construct (Supplementary Figure S5A–C). These data are thus consistent and shows that UTX promotes the removal of H3K27me3 at the ENPP2 locus, whereas JMJD3 controls the expression of ENPP2 through a nonenzymatic process. At last, we investigated whether a crosstalk scenario between UTX and JMJD3 could explain these findings. Hence, we explored whether the binding of UTX to ENPP2 promoter upon LPS treatment (Supplementary Figure S6A) was JMJD3-dependant and vice versa in ChIP-qPCR assays. Interestingly, gene knockdown of JMJD3 and UTX, lowered respectively the recruitment of UTX and JMJD3 at the promoter and TSS of ENPP2 (Supplementary Figure S6B and C). These data thus indicate a co-dependency in the recruitment of JMJD3 and UTX at this locus.

JMJD3 and DDX21 are recruited to the promoter and gene body of ENPP2

Previous reports showed that JMJD3 binds to promoters, but also to gene bodies, in neural stem cells and macrophages to instruct gene expression changes by promoting RNA pol II progression (11,17). In accordance with previous data, in ChIP-qPCR, we found that in response to LPS JMJD3 was recruited to the TSS and at intron–exon junctions throughout the gene body (Figure ​(Figure3A).3A). We thus hypothesized that JMJD3 controlled the expression of ENPP2 through its protein interactome, which could help promote transcription elongation. We transfected HEK 293T cells with a vector encoding for HA-epitope tagged JMJD3 in basal condition and after LPS stimulation and purified interaction partners by HA pull-down. Mass spectrometry results showed that 563 proteins interacted with JMJD3, and 260 were enriched after LPS treatment (with fold change ≥1.5) (Supplementary Figure S7A). Peptide coverage of specific associated proteins is shown in (Supplementary Figure S7B). A full list of JMJD3-interacting proteins is provided in Supplementary Table S2. GO analysis of JMJD3-associated proteins revealed strong association with RNA processing and unwinding of RNA secondary structure (Figure ​(Figure3B).3B). DDX21, a RNA helicase, was among the first 10 JMJD3-associated proteins that were enriched after LPS stimulation (Figure ​(Figure3C).3C). To confirm the mass spectrometry results, we performed co-immunoprecipitation experiments. Pull-down of HA-JMJD3 recovered DDX21, and an association already described with RNA Pol II (Figure ​(Figure3D)3D) (17). Interestingly, DNaseI and RNAseH treatment did not abolish the interaction between JMJD3 and DDX21 (Supplementary Figure S7C).

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Figure 3.

JMJD3 and DDX21 are recruited to the promoter and gene body of ENPP2. (A) JMJD3 enrichment on promoter and gene body of ENPP2 in HEK 293T cells treated with LPS during 6 h compared to a negative locus (NEG; downstream of SNRPN), ChIP-qPCR analysis. (B) GO analysis of JMJD3 interactome identified by mass spectrometry analysis (LCMS/MS). (C) Table of the first ten JMJD3-associated proteins that were enriched after LPS stimulation, as determined by mass spectrometry analysis. (D) DDX21 and RNA pol II co-immunoprecipitated with HA-JMJD3 in HEK 293T cell extracts. (E) ChIP-qPCR experiment demonstrating DDX21 enrichment at ENPP2 promoter and gene body following 6 h of LPS treatment. Two set of primers targeting an intergenic region downstream of SNRPN (NEG; downstream of SNRPN) and promoter region of DDX23 (DDX23) were used as negative and positive control, respectively. (F) qPCR quantification of ENPP2 expression in response to DDX21 siRNA transfection and LPS exposure in HEK 293T cells. LPS 100 ng/ml.

Previous studies have highlighted that DDX21 controls the expression of ribosomal RNA (rRNA) genes and genes transcribed by RNA pol II (26). We examined whether DDX21 could also exert a control over the expression of ENPP2. In ChIP-qPCR, we documented that DDX21 was recruited to the TSS and the gene body of ENPP2 following a treatment of cells with LPS compared to negative control region (Figure ​(Figure3E).3E). In these experiments, the promoter of DDX23, which is bound to DDX21 was used as a positive control region (Figure ​(Figure3E).3E). In cells with siRNA-mediated knockdown of DDX21, we found that LPS-mediated expression of ENPP2 was abrogated (Figure ​(Figure3F).3F). The efficiency of these two non-overlapping DDX21 siRNAs was confirmed by q-RT-PCR and western blot analysis (Supplementary Figure S8A–C). Hence, these data showed that JMJD3 interacts with the RNA helicase DDX21, which controls the expression of ENPP2.

The complex formed by JMJD3 and DDX21 is required for R-loops resolution at ENPP2 locus

Recent work has highlighted that DDX21 unwinds R-loop, thus safeguarding genome integrity and prevent stalling of RNA Pol II on estrogen-responsive genes in breast cancer cells (26). To document the presence of R-loops at the ENPP2 locus we immunoprecipitated RNA:DNA hybrids with the S9.6 antibody (39). Interestingly, R-loops were increased after LPS treatment at TSS and within the gene body (ranged from ~0.6 to 10% of input depending on the gene section while negative locus in intergenic region ranged from 0.1 to 1%) (Figure ​(Figure4A).4A). As expected, treatment of lysates with RnaseH before immunoprecipitation abolished the signal with S9.6 antibody and confirmed the presence of R-loops during the transcriptional process at ENPP2 locus (Figure ​(Figure4A).4A). We next hypothesized that JMJD3 may participate to the recruitment of DDX21 at ENPP2 locus and thus could prevent R-loop formation. Consistently, siRNA mediated knockdown of DDX21 increased R-loops formation-sensitives to RNAseH after a treatment of cells with LPS (Figure ​(Figure4B).4B). In addition, knockdown of JMJD3 led to a striking reduction in DDX21 occupancy at the TSS and gene body of ENPP2 (Figure ​(Figure4C).4C). In these experiments, the knockdown of JMJD3 also led to a rise in R-loop formation at the TSS and gene body (Figure ​(Figure4D4D).

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Figure 4.

The complex formed by JMJD3 and DDX21 is required for R-loops resolution at ENPP2 locus. (A) ChIP-qPCR indicating increased R-loops formation at ENPP2 TSS, gene body and a negative locus (NEG; downstream of SNRPN) following LPS treatment (6 h) in HEK 293T cells; treatment of lysates with RNase H before immunoprecipitation abolished R-loops. (B) Effect of DDX21 siRNAs on R-loop formation, as measured by ChIP-qPCR, in presence of LPS. (C) JMJD3 siRNAs treatment hinders on DDX21 recruitment at ENPP2 TSS and gene body in presence of LPS, ChIP-qPCR analysis. (D) ChIP-qPCR showing the effect of JMJD3 siRNA on R-loop formation at ENPP2 TSS and gene body in the presence of LPS. (E) Scheme of the ChIP-ReChIP main steps. (F) ChIP-ReChIP-qPCR analysis indicating simultaneous binding of DDX21 and HA-JMJD3 at ENPP2 TSS, enhanced upon addition of LPS. LPS 100 ng/ml, RNaseH 60U/ml.

To further assess whether JMJD3 and DDX21 are co-recruited at ENPP2, we performed ChIP/re-ChIP experiments in HEK 293T cells transfected with HA-JMJD3 plasmid and treated with LPS for 6 h. Chromatin was sequentially immunoprecipitated with anti-HA (JMJD3) and then re-chromatin immunoprecipitated (re-ChIP) with antibodies specific to DDX21 as depicted in Figure ​Figure4E.4E. Re-ChIP experiments confirmed simultaneous binding of DDX21 and HA-JMJD3 at the TSS of ENPP2, suggesting that DDX21 and HA-JMJD3 might form a protein complex recruited at similar genomic region in HEK293T cells (Figure ​(Figure4F).4F). Importantly, the co-recruitment of HA-JMJD3 and DDX21 is increased by 2.5-fold after 6 h of LPS, compared to basal conditions (Figure ​(Figure4F).4F). Hence, these results show that DDX21 and JMJD3 form a complex necessary for R-loops resolution and transcription elongation upon LPS treatment.

R-loops resolution at ENPP2 locus promotes the synthesis of nascent transcript

During the transcription process, R-loops are naturally formed and constitute a regulatory system. Although, previous studies revealed that R-loops have beneficial functions in gene expression (23), growing evidence suggest that R-loops detected at exon 2 slowed Pol II progression by creating a chromatin environment favorable for Pol II pausing (40). To investigate the functional role of R-loop in ENPP2 expression we depleted DDX21 with two non-overlapping siRNAs (Supplementary Figures S8A–C). In some conditions, a plasmid coding for RNaseH1 was also transfected. In these experiments, RNaseH1 completely rescued the expression of ENPP2 upon LPS treatment, indicating that LPS-induced ENPP2 expression relies on the resolution of R-loops (Figure ​(Figure5A).5A). Moreover, to investigate whether the helicase activity of DDX21 is involved in the regulation of ENPP2 via R-loops resolution, we performed a DDX21 rescue experiment with a helicase-dead mutant DDX21 (S375L/A376E) or with a vector encoding for the wild-type DDX21. Following the knockdown of DDX21 with two non-overlapping siRNAs, we found in rescue experiments that the helicase activity of DDX21 was required to partly restore the expression of ENPP2 (Figure ​(Figure5B).5B). In these experiments, we confirmed that DDX21 mutant was similarly expressed compared to wild-type plasmids constructs in cells treated with siRNAs (Supplementary Figure S8D–F).

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Figure 5.

R-loops resolution at ENPP2 locus promotes the synthesis of nascent transcript. (A and B) Endogenous ENPP2 expression after transfection of siDDX21 and rescue with a vector encoding (A) RNaseH1 or in (B) DDX21 WT or a DDX21 S375L/A376E (catalytic dead mutant), qPCR analyses. (C) Schematic representation of the experimental protocol for transcriptional run-on assay using the Click-it technology. HEK 293T cells were pulse labeled with 5-ethynyl uridine (EU) for 6 h, and the EU-incorporated RNAs were biotinylated and captured with streptavidin conjugated beads, followed by RT-PCR. (D–F) Transcriptional run-on assay indicates that depletion of DDX21 alters ENPP2 nascent transcript formation. LPS 100 ng/ml, EU 0.2 mM.

Next, we performed a transcription run-on experiment to assess the impact of siRNA-mediated depletion of DDX21 on the level of ENPP2 nascent transcript. To capture nascent RNAs, HEK 293T cells were pulse labeled with 5-ethynyl uridine (EU) for 6 h and the EU-incorporated RNAs were biotinylated and purified with streptavidin conjugated beads (Figure ​(Figure5C).5C). Interestingly, we found that the knockdown of DDX21 impaired the production of nascent ENPP2 transcripts suggesting that DDX21 promotes the elongation of transcription at ENPP2 locus (Figure ​(Figure5D5D–F).

A distant-acting enhancer contacts the promoter region of ENPP2 and controls its expression

To evaluate whether ENPP2 locus has chromatin interactions we used available data obtained from promoter capture Hi-C (PCHi-C) experiments (www.chicp.org) (35). PCHi-C is a web-based tool that allows the detection of physical chromatin interactions between distal DNA regulatory elements and 31 253 annotated promoters in 17 human primary blood cell types. Interrogation of PCHi-C in CD34+ cells revealed a physical interaction of the promoter region of ENPP2 with a putative regulatory element, which is located at 82 kb from the TSS (Figure ​(Figure6A).6A). To determine if this physical interaction was conserved in HEK 293T cells we conducted a 3C assay (41). 3C technology employs formaldehyde crosslink to preserve endogenous three-dimensional structures and digestion of genomic DNA using a restriction enzyme (in this case NspI). Samples were ligated under conditions that favor the union of DNA fragments that are physically connected and qPCR was used to determine the frequency of interaction of each restriction fragments with an anchor primer located at the TSS of ENPP2. In HEK 293T cells, we found that the TSS of ENPP2 and fragment #3–4, corresponding to the 5′ portion of the putative regulatory element, were positioned in close proximity on chromatin (Figure ​(Figure6B).6B). The treatment with LPS did not modify the chromatin looping pattern (Figure ​(Figure6B).6B). Therefore, these results suggested that a distant regulatory element could function as an enhancer for ENPP2.

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Figure 6.

A distant-acting enhancer contacts the promoter region of ENPP2 and controls its expression. (A) Circos plot showing interactions between ENPP2 TSS and a putative enhancer located at 82 kb, dataset from CD34+ cells. (B) 3C experiments replicating this interaction in HEK 293T cells. LPS treatment did not modulate this interaction. (C) H3K4me1 enrichment at the enhancer region, as measured by ChIP-qPCR, using six sets of primers covering the putative enhancer region. (D) Luciferase reporter assay demonstrating that the region has enhancer activity, independently of its orientation. (E) Scheme depicting the deletion of the enhancer region by using the CRISPR-Cas9 technology. Cas9 enzyme was transfected simultaneously with two gRNA targeting 5′ end and 3′ end of the enhancer region. (F) Endogenous expression of ENPP2 after CRISPR-Cas9-mediated deletion of the enhancer, as measured by q-RT-PCR. LPS 100 ng/ml.

To verify this hypothesis we performed ChIP-qPCR with an H3K4me1 antibody with six different primer sets to cover this potential regulatory locus. We found that this region was strongly enriched for H3K4me1, which is consistent with a potential enhancer (Figure ​(Figure6C).6C). In addition, we cloned this sequence in a reporter plasmid containing a minimal promoter and the luciferase gene (Figure ​(Figure6D).6D). Importantly, this region, both in forward and reverse orientation, increased by 2-fold the luciferase activity before LPS activation (Figure ​(Figure6D).6D). Upon LPS stimulation, the enhancer effect on luciferase activity was more pronounced (Figure ​(Figure6D).6D). Together, these results suggest that the putative enhancer region has a transcriptional effect. At last, to investigate whether this enhancer controls the expression of ENPP2, genome editing was performed with the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system to specifically delete this enhancer (Figure ​(Figure6E).6E). CRISPR-Cas9-mediated deletion of the enhancer region decreased the rise of ENPP2 after LPS stimulation (Figure ​(Figure6F).6F). In these experiments, the efficiency of enhancer deletion was confirmed by q-RT-PCR using 5 sets of primers spanning the enhancer region and control regions located upstream and downstream of the predicted cut sites (Supplementary Figure S9A). The residual signal in selected cells is likely related to monoallelic deletion (Supplementary Figure S9B). Taken together, these data revealed that a distant-acting enhancer contacts the promoter region of ENPP2 and exerts a control on gene expression.

We thank Dr Warner Greene lab (Gladstone Institute of Virology and Immunology, San Francisco, United States) who kindly give us p65 cDNA. We are grateful to Dr Charlie Degui Chen (Institute of Biochemistry and Cell Biology, Shangaï, China) for plasmids encoding for JMJD3 wild-type and JMJD3H1390A (27).