To examine whether MAF1 has the potential to repress Pol II-transcribed genes, we first examined the knockdown effect of MAF1 by quantitative RT-PCR (qRT-PCR) and immunoblot using multiple siRNAs (Figure 1A,B). The siRNA with the strongest knockdown effect was used to perform expression analysis using microarray. 124 Pol II-transcribed genes were upregulated more than twofold after MAF1 knockdown. Among them, CDKN1A was significantly upregulated, resulting in the downregulation of positive cell cycle regulators. Consistent with expression data, flow cytometry analysis showed that MAF1 knockdown arrested cells at the G₁ phase (Figure 1C). We carried out qRT-PCR to confirm whether CDKN1A expression was upregulated by MAF1 knockdown. Efficiency of MAF1 knockdown was verified by the strong upregulation of two products of Pol III, pretRNA^Tyr and pretRNA^Leu (Reina et al., 2006) (Figure 1D). Consistent with microarray analysis, qRT-PCR and immunoblot analysis showed that CDKN1A expression was upregulated about 10-fold after MAF1 knockdown (Figure 1D,E). GAPDH, ACTB, and TAF5, genes that were not affected by MAF1 knockdown in the microarray, were chosen as the control for qRT-PCR. Expression of these genes was not affected by MAF1 knockdown (Figure 1D).

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Because CDKN1A is a downstream target of p53 (Allen et al., 2014), we further performed MAF1 knockdown in HCT116^p53+/+ (wild-type) and HCT116^p53−/− (p53-null) cell lines to analyze whether the induced CDKN1A expression is dependent on p53. The absence of p53 in HCT116^53−/− was confirmed by immunoblot (Figure 1—figure supplement 1A). CDKN1A expression was induced after MAF1 knockdown in both wild-type and p53-null HCT116, which indicates that the activation is independent of p53 (Figure 1—figure supplement 1B,C). Immunoblot analysis also showed that CDKN1A protein level was upregulated after MAF1 knockdown in p53-null HCT116 (Figure 1—figure supplement 1D). CDKN1A activation by MAF1 knockdown was also found in a non-tumorigenic cell line (MCF-10A) and a p53 mutant breast cancer cell line (MDA-MB-231) (Figure 1—figure supplement 1E,F). Together, these results demonstrate that MAF1 can regulate CDKN1A expression in a variety of cell types independent of p53.

CDKN1A activation after MAF1 knockdown could be due either to interference of binding of transcription factors to the CDKN1A promoter by MAF1 or to the active recruitment or activation of Pol III after MAF1 knockdown. To determine which of these two mechanisms are involved in this process, we carried out simultaneous knockdown of both MAF1 and Pol III. The former mechanism would not be affected by the double knockdown, whereas the latter would be. The effect of Pol III knockdown was analyzed by qRT-PCR and immunoblot using multiple siRNA sequences (Figure 1—figure supplement 1G,H). Simultaneous knockdown of Pol III and MAF1 indeed abolished the induction of CDKN1A expression by knockdown of only MAF1 (Figure 1D and Figure 1—figure supplement 1B,C,E,F) in five different cell lines. A control experiment using two Pol III genes, pretRNA^Tyr and pretRNA^Leu, confirmed the efficiency of Pol III knockdown (Figure 1D and Figure 1—figure supplement 1B,C,E,F). Knockdown of Pol III alone did not significantly affect CDKN1A expression (Figure 1D). This result indicates that Pol III plays a critical role in activation of CDKN1A expression by MAF1 knockdown.

Because mRNA levels can be affected by transcription, post-transcriptional processing as well as RNA turnover rate, upregulation of gene expression after MAF1 knockdown could be due to post-transcriptional mechanisms other than transcription activation. To demonstrate that the induced CDKN1A expression indeed occurs at the transcriptional level, we analyzed the rate of nascent transcription by conducting a nuclear run-on experiment after MAF1 knockdown or simultaneous knockdown of MAF1 and Pol III. The run-on nascent RNA was labeled with biotin, affinity purified, and analyzed by RT-PCR. A negative control without biotin labeling was used. Consistent with qRT-PCR analysis, nascent transcription of CDKN1A was indeed induced after MAF1 knockdown, whereas transcription of ACTB and TAF5 was not affected (Figure 2A,B). Simultaneous knockdown of MAF1 and Pol III diminished the induced nascent RNA transcription of CDKN1A by knockdown of MAF1 alone (Figure 2A,B). These results indicate that Pol III is required for the induction of CDKN1A upon MAF1 knockdown.

Figure 2

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Recent evidence indicated that R-loop formation positively correlates with active transcription in human cells by maintaining the unmethylated state at promoters with skewed guanine-cytosine (GC) content (Ginno et al., 2012). The high GC skew of the CDKN1A promoter prompted us to test whether the R-loop was present in this region during the activation of transcription by MAF1 knockdown (Figure 2C). We performed R-loop foot-printing by native sodium bisulfite treatment, which converts cytosine to uracil only on the single-stranded DNA (Yu et al., 2003). MAF1 knockdown resulted in the formation of the extended R-loop in the gene body of CDKN1A, which indicates active transcription, whereas simultaneous knockdown of Pol III and MAF1 inhibited R-loop formation (Figure 2C–E). The R-looping of the control gene ACTB, an active housekeeping gene with high GC skew and expression, was not affected by MAF1 knockdown (Figure 2F–H). The results of R-loop formation and nuclear run-on described above further confirmed that the upregulation of CDKN1A expression by MAF1 knockdown and recruitment of Pol III occurred at the transcriptional level.

Expression analysis indicated that CDKN1A expression is strongly upregulated after MAF1 knockdown, and simultaneous knockdown of MAF1 and Pol III diminished the induced expression. These results indicate that removal of MAF1 may induce recruitment of Pol II and Pol III to activate transcription. To examine this possibility, chromatin-immunoprecipitation (ChIP) analysis followed by quantitative PCR (qPCR) was performed under various knockdown conditions. Efficiency of MAF1 or Pol III knockdown as well as simultaneous knockdown of MAF1 and Pol III was verified by the binding of Pol III to pretRNA^Arg and pretRNA^Leu genes. The results showed enhanced binding of Pol III at pretRNA^Arg and pretRNA^Leu after MAF1 knockdown, and the binding was diminished after double knockdown (Figure 3A,B). Examination of CDKN1A gene in the UCSC Genome Database shows that there are two transcription start sites, NM_001220777 (long form) and NM_001220778 (short form), which are 2.25 kb apart (Figure 3—figure supplement 1A). Although the expression of both forms was induced after MAF1 knockdown, the short form had higher expression level and stronger promoter activity in the MCF-7 cell line (Figure 3—figure supplement 1B–D). ChIP analysis showed that MAF1 was associated with both transcription start site regions (Figure 3C,D). Furthermore, MAF1 knockdown resulted in the depletion of this regulatory factor with concomitant increase in the binding of both Pol II and Pol III polymerases to both transcription start site regions (Figure 3E,F).

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Consistent with the expression data, there was significant increase in the binding of active, Serine-5-phosphorylated Pol II at the CDKN1A promoter after MAF1 knockdown, which indicates that the gene was in the active transcription state. The binding of active Pol II was abolished after simultaneous knockdown of Pol III and MAF1 (Figure 3F). Simultaneous knockdown of MAF1 and BRF1, a subunit of TFIIIB that associates with Pol III and is required for binding of Pol III to the DNA template, also abolished the enhanced binding of Pol III and Pol II after MAF1 knockdown (Figure 3E,F). ChIP analysis also indicated induced binding of BRF1 after MAF1 knockdown, whereas the binding was diminished under simultaneous knockdown of either Pol III or BRF1 with MAF1 (Figure 3G). These results therefore support the mechanism that recruitment of Pol III to the promoter after MAF1 knockdown enhances CDKN1A expression. Expression of ACTB and TAF5 was not affected because MAF1 and Pol III did not bind to their promoters (Figure 3H,I) and their expression was not affected by MAF1 knockdown; therefore, they were used as negative controls. The transcription of these two control genes was not affected by single or double knockdown of Pol III and/or MAF1 (Figure 1D).

Because ChIP data revealed that MAF1 may directly bind to the promoter, we searched for potential binding sites in the CDKN1A promoter region. We noticed a MIR3 element (a SINE with Pol III promoter) that could be transcribed by Pol III and therefore might represent the target-binding site of MAF1. To test this possibility, we used an in vitro DNA binding reaction (Britten, 1996; Toth and Biggin, 2000) to examine whether purified MAF1 protein could bind to the cloned CDKN1A promoter. Consistent with ChIP, in vitro DNA binding assay showed that purified MAF1 protein could indeed bind to the CDKN1A promoter that contained the MIR3 element (Figure 4A,B), but the binding was abolished when the MIR3 repeat element was deleted, which indicates specificity of MAF1 binding to the MIR3 element (Figure 4A,B). To further show that the Pol III promoter of the MIR3 element is responsible for the binding, an in vitro binding assay was carried out with DNA in which the MIR3 DNA sequence from the CDKN1A promoter had a deleted or mutated A-box sequence (Pol III promoter element). In vitro DNA-protein binding assays performed by the above method or colorimetric assay (Abcam, ab117139) both showed that the DNA with deleted or mutated A-box sequences exhibited significantly lower binding of MAF1, which indicates the specificity of MAF1 for the Pol III promoter element (Figure 4A–C). Moreover, consistent with ChIP data, MAF1 did not bind in vitro to the ACTB promoter that did not contain a SINE or sequences that would resemble the Pol III promoter element (Figure 4A,B). To the best of our knowledge, this is the first demonstration of direct binding of MAF1 to a specific DNA sequence.

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Although the several types of evidence discussed above strongly support the regulation of CDKN1A by recruitment of Pol III to the promoter, the effect observed in vivo nevertheless could be due to some other indirect effect. To directly demonstrate the enhancement of Pol II transcription by removing MAF1 and recruiting Pol III, we carried out in vitro transcription using commercial HeLa cell nuclear extract. Constructed DNA templates of the genes analyzed are described in the ‘Materials and methods’. The in vitro, newly transcribed RNA was labeled with biotin, and the products were affinity purified. The nature of the affinity-purified nascent RNA was then analyzed by qRT-PCR. Negative control without biotin labeling was used. First, we showed that the in vitro transcription was indeed mediated by Pol II by inhibition of transcription either by α-amanitin treatment (Figure 4D) or by depletion of Pol II in the extract using anti-Pol II antibody (Figure 4E). When purified MAF1 protein was pre-incubated with DNA template prior to the addition of nuclear extract, CDKN1A transcription was repressed with respect to the control (Figure 4D). This result is consistent with in vivo expression analysis and the in vitro binding assay. Together these results suggest that MAF1 protein serves as repressor of CDKN1A transcription. As a control, pre-incubation with MAF1 protein did not affect TAF5 transcription (Figure 4D) because MAF1 did not bind to this DNA template in vivo (Figure 3I).

When nuclear extract was pre-incubated with an anti-MAF1 antibody to deplete MAF1 during in vitro transcription, CDKN1A transcription was significantly upregulated compared with that of the control, which was pre-incubated with IgG or no antibody (Figure 4E). Simultaneous depletion of Pol III and MAF1 by pre-incubation nuclear extract with Pol III and MAF1 antibodies abolished the enhancement of transcription after depletion of MAF1 alone (Figure 4E). Specificity of the Pol III antibody was demonstrated by the inhibitory effect of anti-Pol III antibody on in vitro transcription of the Pol III-transcribed RPPH1 promoter in a pSUPER plasmid. Addition of the anti-MAF1 antibody did not induce RPPH1 transcription (Figure 4E) because binding of MAF1 was not detected in this gene by the in vitro MAF1 binding assay (Figure 4B).

These experiments recapitulated the in vivo transcription regulation of CDKN1A by MAF1 and Pol III. Taken together with the in vivo and in vitro nuclear run-on expression analyses, these results unambiguously demonstrate that MAF1 can serve as a repressor of the CDKN1A promoter, and that recruiting Pol III after MAF1 depletion is crucial for the activation of CDKN1A transcription.

The data above indicate that binding of Pol III to the CDKN1A promoter after MAF1 knockdown is crucial for enhanced transcription. This indicates that Pol III may help recruit the regulatory factors necessary for efficient Pol II transcription. To test this hypothesis, we carried out ChIP analysis to examine the Pol III-dependent recruitment of transcription activators after MAF1 removal. ChIP analysis showed that MAF1 knockdown resulted in significantly enhanced levels of active histone modifications, including H3K4me3, H3K9Ace, and H3K27Ace, in the 5′ regions of CDKN1A (Figure 5A,B). The enhanced active histone marks after MAF1 knockdown were abolished under simultaneous knockdown of Pol III and MAF1 (Figure 5B). The histone repression marker, H3K27me3, was detected at the 5′ regions and decreased after MAF1 knockdown, but the level was restored under simultaneous knockdown of Pol III and MAF1 (Figure 5B).

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Because H3K4 methylation is catalyzed by the SET1/MLL family of histone methyltransferases in humans (Shilatifard, 2012), we performed knockdown assays to investigate which methyltransferase is responsible for H3K4me3 modification after MAF1 knockdown. Previously, CFP1 (SET1C-specific subunit) and p300 were shown to act cooperatively to regulate H3K4me3 modification and CDKN1A transcription (Tang et al., 2013). Indeed, the MAF1 knockdown-induced transcription of CDKN1A was downregulated after CFP1 (CXXC1) knockdown (Figure 5—figure supplement 1A).

p300 and PCAF have been shown to regulate K27 and K9 acetylation, respectively, of the CDKN1A promoter (Love et al., 2012). Thus, we next analyzed whether CFP1, p300, and PCAF could bind to the CDKN1A promoter after MAF1 knockdown. As expected, the removal of MAF1 by knockdown induced binding of CFP1, p300, and PCAF to the CDKN1A promoter (Figure 5C–E). Furthermore, simultaneous knockdown of MAF1 and Pol III abolished the induced binding of these factors along with active histone marks, which indicates that Pol III is required to recruit these factors to the CDKN1A promoter for histone modifications (Figure 5C–E).

Because TBP is an important factor required in both Pol II (part of TFIIB) and Pol III (part of TFIIIB) transcription (Zhao et al., 2003), we next determined whether binding of TBP was enhanced after MAF1 knockdown. Indeed, enhanced binding of TBP was observed at the CDKN1A promoter after MAF1 knockdown, and the binding was abolished when there was simultaneous knockdown of Pol III and MAF1 (Figure 5F). Simultaneous knockdown of MAF1 and TBP also abolished the enhanced expression of CDKN1A by knockdown of only MAF1 (Figure 5—figure supplement 1B), which indicates that TBP is important for CDKN1A transcription activation. We also observed enhanced binding of a common subunit of all three RNA polymerases, that is, POLR2E (RPB5), after MAF1 knockdown, and this enhanced binding was also abolished when there was simultaneous knockdown of Pol III and MAF1 (Figure 5G), indicating interplay between Pol II and Pol III polymerases.

Because binding of Pol III and Pol II was detected at the 5′ flanking regions of both long and short CDKN1A forms in the UCSC Genome Database after MAF1 knockdown, we performed 3C analysis to investigate whether chromatin looping occurs between these two regions. Chromatin looping was detected between the regions after MAF1 knockdown, but not in adjacent regions (Figure 6A,B). Moreover, simultaneous knockdown of MAF1 with either Pol III or BRF1 (a subunit of TFIIIB) disrupted the looping formation (Figure 6C). These results demonstrate that Pol III is required for induced chromatin looping after MAF1 knockdown.

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The above results demonstrate that MAF1 knockdown can activate CDKN1A expression by recruiting Pol III and Pol II along with histone-modifying factors. To demonstrate that this type of mechanism also regulates expression of other Pol II genes, we performed expression analysis of GDF15, which is another cell proliferation-related gene that is upregulated after MAF1 knockdown as found by microarray analysis. As expected, qRT-PCR analysis showed that GDF15 expression was strongly upregulated after MAF1 knockdown, and simultaneous knockdown of MAF1 with Pol III diminished the induced expression (Figure 1D). ChIP analysis also indicated binding of MAF1 at the 5′ flanking region of GDF15 (Figure 7A,B).

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We also employed an in vitro transcription assay using HeLa cell nuclear extract to demonstrate the importance of the Pol III promoter element in regulation of GDF15 transcription after MAF1 knockdown. When nuclear extract was pre-incubated with anti-MAF1 antibody to deplete MAF1 during in vitro transcription, GDF15 transcription was significantly upregulated compared with the control with pre-incubation with IgG or no antibody (Figure 4E). We also noticed an MIR repeat element in the 5′ flanking region of GDF15. As in the case of CDKN1A, deletion of the Pol III A-box element associated with this repeat also abolished the enhancement of GDF15 in vitro transcription when anti-MAF1 antibody was added to the extract (Figure 4E), indicating that this element mediated MAF1 binding. Indeed, the in vitro binding assay using purified MAF1 protein indicated that MAF1 did not bind to this deletion mutant but did bind to the wild-type sequence (Figure 4B).

A 3C assay was performed to further investigate whether MAF1 knockdown induces chromatin looping. The analysis indicated that there was chromatin looping between a promoter region and a region that is 12-kb upstream of the GDF15 promoter after MAF1 knockdown (Figure 7C). Furthermore, the looping was abolished under simultaneous knockdown of MAF1 with either Pol III or BRF1 (Figure 7D). Similar to the CDKN1A results, induced binding of Pol III and Pol II to the GDF15 promoter was observed after MAF1 knockdown, whereas the binding was diminished after simultaneous knockdown of Pol III and MAF1 (Figure 7E,F). These results show that transcription of GDF15, like CDKN1A, is upregulated after MAF1 knockdown by recruiting Pol III, and Pol III is required for chromatin looping at the GDF15 promoter.

To demonstrate the role of the Pol III promoter element in Pol III-mediated activation of the Pol II gene, we analyzed the effect of deletion of the Pol III promoter element using a reporter assay. The promoter regions of CDKN1A, GDF15, and TAF5, as indicated in the ‘Materials and methods’, were cloned into the reporter plasmid pGL3. Promoter-driven luciferase activities of CDKN1A and GDF15 were upregulated, whereas TAF5 promoter-driven expression was not affected after MAF1 knockdown (Figure 8A). Simultaneous knockdown of both Pol III and MAF1 abolished the upregulation caused by knockdown of MAF1 alone (Figure 8A). These results thus recapitulated the results of in vivo endogenous gene analysis.

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Because the majority of SINEs are transcribed by the type II internal Pol III promoter that contains an A-box and B-box (Okada and Ohshima, 1995), our model indicates that mutation of the Pol III promoter element in the promoter-associated SINE should abolish the enhancement of reporter expression after MAF1 knockdown. To test this possibility, we chose the GDF15 promoter for analysis because it was shown (Ichikawa et al., 2008) that deletion of the −465 to −429 sequence, which contains the Pol III promoter element, did not affect promoter activity. We mutated the A-box (−447 to −437) in the GDF15 promoter (−889 to +110) in the reporter and examined the effect of mutation on reporter expression. Under regular cell culture conditions, no significant change in GDF15 promoter activity was observed in our results when the A-box was mutated or deleted, in consistent with the results of Ichikawa et al. (2008). However, deletion of A-box in the GDF15 promoter diminished the upregulation of the reporter after MAF1 knockdown (Figure 8B). These results further demonstrate that MAF1 represses CDKN1A and GDF15 promoter activity by binding to the Pol III promoter element. Moreover, recruitment of Pol III after MAF1 depletion is crucial for transcription activation of these genes.

MCF-7, MCF-10A, and MDA-MB-231 cell lines were originally obtained from ATCC (Manassas, VA), and cultured in RPMI, HuMEC and DMEM medium (Invitrogen; Waltham, MA), respectively. HCT-116^p53+/+ (wild-type) and HCT116^p53−/− (p53-null) cell lines were originated from Bert Vogelstein (John Hopkins University) and cultured in McCoy's 5A medium (Bunz et al., 1998). Each medium was supplemented with 10% of fetal bovine serum and incubated in a humidified 37°C incubator with 5% CO₂.

Knockdown assay was performed using siRNA obtained from MISSION RNA (Sigma-Aldrich; St. Louis, MO). Inhibition of expression of MAF1 ([#1] SASI_Hs01_00135954, [#2] SASI_Hs01_00135956 and [#3] SASI_Hs01_00135958), Pol III (POLR3A) ([#1] SASI_Hs01_00046568, [#2] SASI_Hs01_00046571 and [#3] SASI_Hs01_00046572), BRF1 (SASI_Hs01_00131187), CFP1 (SASI_Hs02_00322879), and TBP (SASI_Hs01_00122768) was achieved by transfection with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol for 72 hr. MISSION siRNA Universal Negative Control (Sigma) was used as knockdown control. Cells were transfected in serum-free medium. After 8 hr, the siRNA containing medium was replaced with complete medium.

Cells were lysed at 4°C in RIPA lysis buffer (50 mm Tris-HCl, pH 7.2, 150 mm NaCl, 5 mm EDTA, 1% [wt/vol] NP-40, 1% [wt/vol] SDS and protease and phosphatase inhibitor mixtures [Roche Applied Science; Penzberg, Germany]). The lysates were cleared by centrifugation (15,000×g for 15 min), resolved on a 10% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The antibody dilutions used were rabbit anti-POLR3A (1:1000; ab96328, Abcam; Cambridge, England), rabbit anti-MAF1 (1:1000; GTX106776, Acris; Herford, Germany), rabbit anti-CDKN1A (1:1000; ab18209, Abcam), and mouse anti-tubulin (1:10,000; ab7291, Abcam).

Cells were grown to 85% confluence in 6 cm tissue culture dish. Each 6 cm dish was washed with 1× phosphate buffered saline (PBS) for three times. Total RNA was extracted using TRIreagent (Invitrogen) protocol. The integrity of the RNA extract was checked by 1.2% (wt/vol) agarose gel electrophoresis and the concentration of RNA was estimated by ultraviolet spectrophotometry.

Affymetrix microarray was performed using Human U133 plus 2.0 (Affymetrix; Santa Clara, CA). Details of the methods for RNA quality, sample labeling, hybridization, and expression analysis were according to the manual of Affymetrix Microarray Kit. All Affymetrix data are MIAME compliant and that the raw data have been deposited in a MIAME compliant database, GEO. The microarray data were deposited at the NCBI GEO website (GEO accession number GSE42239).

Reverse transcription was performed by using superScript III RNase H- Reverse Transcriptase (Invitrogen) and random hexamer according to the manufacturer's protocol. Quantitative PCR was performed using KAPA SYBR FAST (KK4603, KAPA Biosystems; Wilmington, MA) on ABI StepOnePlus Real-Time PCR System (Invitrogen). All reactions were performed in triplicate with KAPA SYBR FAST plus 10 μM of both the forward and reverse primer according to the manufacturer's recommended thermo cycling conditions, and then subjected to melting curve analysis. The calculated quantity of the target gene for each sample was divided by the average sample quantity of the housekeeping genes, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) or 18S to obtain the relative gene expression.

MCF-7 knockdown cells were collected by trypsinization and washed twice with ice-cold PBS. The cells were resuspended in 0.3 ml of PBS and fixed by slowly adding 3 ml of 70% cold ethanol. Cells were fixed at −20°C for 1 hr. The fixed cells were washed with ice-cold PBS and rehydrated for 15 min. After centrifuging at 200×g for 5 min, cells were resuspended in 0.1 mg/ml of propidium iodide and 0.6% of Triton X-100 in 500 μl of PBS. Then add 500 μl of 2 mg/ml of RNase A and incubate in the dark for 45 min. Data were collected using a FACScan flow cytometry system (BD; Franklin lakes, NJ).

Nuclear run-on reactions were performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads as described by Patrone et al. (2000) with minor modifications. Nuclei were prepared from MCF-7 cells by resuspension in Nonidet P-40 lysis buffer (10 mM HEPES, pH 7.3, 10 mM NaCl, 3 mM MgCl₂, 150 mM sucrose, and 0.5% Nonidet P-40). Nuclei were isolated, and the pellets were resuspended in 1 ml of glycerol buffer (50 mM Tris-Cl, pH 8.3, 40% glycerol, 5 mM MgCl₂, and 0.1 mM EDTA). 1 ml of transcription buffer (20 mM Tris-Cl, pH 8.0, 200 mM KCl, 5 mM MgCl₂, 4 mM dithiothreitol, 4 mM each of ATP, GTP, and CTP, 200 mM sucrose, and 20% glycerol) was added in the nuclei along with 10 μl of biotin-16-UTP or UTP for run-on reaction or negative control, respectively (Roche). After incubation at 29°C for 30 min, the reaction was terminated by the addition of 12 μl of 250 mM CaCl₂, and 12 μl of RNase-free DNase I and incubated at 29°C for 10 min. To purify RNA, a TRIreagent extraction, phenol-chloroform extraction, and isopropanol (Sigma) precipitation were then performed. A small aliquot (5 μl from a total of 50 μl) was saved as input control. Dynabeads M-280 streptavidin (Invitrogen) were mixed with an equal volume of the isolated RNA samples for 20 min at 42°C for 20 min and 2 hr at room temperature. After washing with 15% formamide and 2× SSC, the beads were resuspended in 45 μl of nuclease-free water. Reverse transcription was performed by using superScript III RNase H—Reverse Transcriptase (Invitrogen). Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. The gel pictures were quantified with ImageJ (provided by NIH: http://imagej.nih.gov/ij/). The purified run-on products where normalized with internal control (GAPDH) to obtain the relative transcription levels for each gene.

Knockdown assay was performed using siRNA obtained from MISSION RNA (Sigma). Inhibition of expression of Pol III (SASI_Hs01_00046568) and MAF1 (SASI_Hs01_00135954) was achieved by transfection with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol for 72 hr. DNA purification and single-stranded R loop foot-printing were carried out as previously described with slight modifications (Yu et al., 2003). 500 ng of purified genomic DNA was bisulfite converted by adding CT Conversion Reagent from the EZ DNA Methylation-Gold Kit (Zymo Research; Irvine, CA) at 37°C for 16 hr in the dark. PCR amplified region for cloning is shown in Figure 2A,F as foot-printing region. The PCR product was gel eluted and ligated to sequencing vector yT&A (Sigma). Approximately, 20 individual clones were sequenced for all PCR products, and the sequencing data were analyzed and aligned to CDKN1A or ACTB genomic sequence. The sequence of the beginning and end of each clone is trimmed due to low quality of sequencing. A background conversion (approximately 5% of cytosine) may be seen possibly due to DNA breathing during the prolonged incubation at 37°C in our data and others (Yu et al., 2003). Approximately, 1–2 clones showed both cytosine to thymine and guanine to adenine conversions, which is known as ‘mosaic molecules’ (Yu et al., 2003).

ChIP assay was performed according to the manufacturer's protocol (Upstate Biotechnology, Inc.; Lake Placid, NY) with slight modifications. Human MCF-7 cells were fixed with 1% of formaldehyde at room temperature for 10 min. The cells were lysed and the chromatin was sonicated to 200–500 bp fragments by Bioruptor sonicator (cycle condition of 25 s on and 25 s off in a total of 25 min at highest output). Chromatin was immunoprecipitated by using Pol III (ab96328, Abcam), Pol II (ab5131, Abcam), MAF1 (GTX106776, Acris), H3K4me3 (04–745, Millipore; Billerica, MA), H3K27me3 (ABE44, Millipore), TBP (ab28175, Abcam), H3K9Ace (06–942, Millipore), H3K27Ace (07–360, Millipore), CFP1 (ABE211, Millipore), p300 (05–257, Millipore), POLR2E (ab180151, Abcam) BRF1 (ab74221, Abcam)or IgG (ab46540, Abcam) antibody, with 10 μg/ml of BSA and 50 μl of Dynabeads Protein A and G (Invitrogen) for overnight at 4°C. The beads were washed once with each washing buffer, including low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer, and twice with 1× TE buffer. Precipitates were eluted with 1% of SDS and 100 mM of NaHCO₃. Proteinase K was added to the samples, and rotated at 65°C for 2 hr followed by 95°C for 10 min and cooled down to room temperature. RNase A was added and samples were incubated at 37°C for 1 hr. After genomic DNA extraction, qPCR was performed. The degree of enrichment is calculated relative to the ratio of signals obtained in the input DNA fraction subtracting IgG-immunoprecipitated DNA.

3C assay was performed according to (Dekker et al., 2002) with some modifications. MCF-7 cells were fixed in 2% formaldehyde for 10 min at room temperature and quench with 0.125 M glycine. After centrifugation for 15 min at 3500 rpm, the cells were suspended in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Nonidet P-40 and 1:500 Complete protease inhibitor cocktail; Roche) for 90 min on ice. Next, the nuclei were pelleted by centrifugation for 15 min at 2500 rpm, resuspended in 500 μl of 1× NEB buffer 4 plus 0.3% SDS and incubated at 37°C for 1 hr. After the addition of Triton-X to a final concentration of 1.8% to sequester the SDS, the mixture was incubated at 37°C for 1 hr, which was followed by the addition of 800 U of PstI and incubation at 37°C overnight to digest the chromatin. The reaction was terminated by adding SDS to a final volume of 1.6% and then the solution heated to 65°C for 20 min. Ligation of DNA in situ was carried out using 0.5–2.0 ng/μl of chromatin in 800 μl of ligation buffer (NEB; Ipswich, MA) plus 1% Triton-X and 30 Weiss Units of T4 ligase (NEB) for 4 hr at 16°C. After reversing of the crosslinks with proteinase K digestion at 65°C overnight, the DNA was purified by phenol-chloroform extraction and ethanol precipitation. The ligation products were detected by PCR using primers located near Pst1 cutting sites. The PCR products were purified from an agarose gel, cloned and sequenced.

CDKN1A (with or without MIR3), ACTB, GDF15 (including deleted or mutated A-box) and RPPH1 template DNA was obtained by PCR followed by gel elution (Qiagen; Venlo, Netherlands) according to the manufacturer's protocol. The deletion of MIR3 was performed as described by PCR-mediated deletion and checked by sequencing (Lee et al., 2004). The purified DNA was further used for in vitro DNA binding reactions as described previously with slight modifications (Britten, 1996; Toth and Biggin, 2000). The in vitro protein-DNA binding assay coupled with immunoprecipitation was performed as following: 20 ng of DNA template, 400 ng of MAF1 protein (His tag) (80R-1955, Cantor Fitzgerald; New York, NY), 400 ng of Anti-6X His tag antibody (ab18184, Abcam), protease inhibitor (539134, Calbiochem; La Jolla, CA), and 200 ng of BSA was added into 50 μl of binding buffer (20 mM HEPES [pH7.6], 150 mM NaCl, 0.25 mM EDTA, 10% glycerol, 0.2% NP40, and 1 mM DTT). A negative control was performed by substituting IgG antibody for Anti-6× His tag antibody (Protein + IgG) or with only the Anti-6× His tag antibody for the MAF1 protein (Ab only). The mixture was rotated at 4°C for 10 min and on ice for 30 min. 10 μl of Dynabeads Protein G (Invitrogen) was added to the mixture and rotated at 4°C for 10 min and on ice for 30 min. The immunoprecipitated DNA-protein complexes were then washed twice with washing buffer (20 mM Tris [pH 7.5], 0.25 mM EDTA, 10% glycerol, and 0.2% NP40) and once with TE buffer by each rotating at 4°C for 5 min. Elution was performed with 1% of SDS and 0.1 M of NAHCO₃. Input DNA was prepared as 1 ng of template DNA (5% of 20 ng). Proteinase K was added to the samples, and rotated at 65°C for 2 hr followed by 95°C for 10 min and cooled down to room temperature. DNA isolated from immunoprecipitated protein-DNA complex was subjected to qPCR. The degree of enrichment is calculated relative to the ratio of signals obtained in the input DNA fraction.

Biotin-labeled (labeled at 5′) and non-labeled CDKN1A (5′-AATCAACAACTTTGTATACTTAAGTTCAGTGGACCTCAATTTCCTCATCTGTGAAATAAA-3′) as well as mutated A-box template DNA (5′-AATCAACAACTTTGTATA CTTCCCATCCCAAAACCTCAATTTCCTCATCTGTGAAATAAA-3′) was obtained by oligo synthesis from Genomics (Houston, TX). The oligos were annealed and used for in vitro DNA-protein binding assay by the DNA-Protein Binding Assay Kit (Colorimetric) provided by Abcam (ab117139). The assay was performed according to manufacturer's protocol. In brief, 40 ng of biotin-labeled DNA template and 500 ng of MAF1 protein (His tag) (80R-1955, Fitzgerald) was used for the binding assay. For competition assay, 200 ng of competitor DNA was added to the mixture. Anti-6× His tag antibody (ab18184, Abcam) and Goat anti-Mouse IgG2b heavy chain (HRP) antibody (ab97250, Abcam) were prepared and added according to manufacturer's protocol. Blank control was performed without the addition of protein as specified by the kit and the degree of enrichment is calculated by subtracting with blank control.

The upstream promoter regions of CDKN1A (−864 to +41 of NM_001220778 for short form and −1249 to +92 of NM_001220777 for long form), GDF15 (−889 to +110), and TAF5 (−998 to +157) genes were cloned into the pGL3-basic Reporter Vector (Promega; Fitchburg, WI). Knockdown assay was performed as mentioned above for 24 hr before MCF-7 cells were transfected with the plasmids using Lipofectamine LTX (Invitrogen), along with a plasmid expressing β-galactosidase for normalization. The plasmids were transfected for 48 hr, and the cells were lysed and luciferase assay was conducted using the Luciferase Assay System (Promega) using a fluorimetric plate reader.

In vitro transcription was performed by using HeLaScribe^R Nuclear Extract in vitro Transcription System (Promega Cat. #E3110) according to the manufacturer's protocol with slight modifications. Template DNA was prepared by linearizing the constructed promoter region of CDKN1A, GDF15, and TAF5 as used in Luciferase assay, as well as RPPH1 promoter as used in in vitro MAF1 binding assay. In vitro transcription was performed by incubation with linear form of constructed promoter region with nuclear extract, transcription buffer, magnesium ion, GTP, CTP, ATP, biotin-16-UTP, RNase inhibitor, and 30 μg of yeast tRNA. Negative control was performed by incubation with non-biotin labeled NTPs. 0.2 μg of α-amanitin was added during in vitro transcription for inhibition of Pol II transcription. 3 μg of MAF1 protein (His tag) (80R-1955, Fitzgerald) used in in vitro MAF1 binding assay was pre-incubated with template DNA before adding nuclear extract to enable binding of MAF1 to the template DNA. Anti-Pol III (ab96328, Abcam), anti-Pol II (ab5131, Abcam), anti-MAF1 (GTX106776, Acris), or anti-IgG (ab46540, Abcam) antibody was pre-incubated with nuclear extract for 15 min to deplete the target protein of interest. After incubation at 30°C for 1 hr, the reaction was terminated by the addition of 175 μl of HeLa Extract Stop Solution (Promega Cat. #E3110). A TRIreagent extraction, phenol-chloroform extraction, and isopropanol (Sigma) precipitation were then performed to purify RNA. A small aliquot (2 μl from a total of 22 μl) was saved as ‘total nuclear RNA’ for each condition. The biotinylated RNA was isolated using streptavidin-coated magnetic beads as described in Run-on assay. Reverse transcription was performed by using superScript III RNase H- Reverse Transcriptase (Invitrogen). Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This work is supported by a grant from the Ministry of Education, Aim for the Top University Plan, and by a grant from High-throughput Genome Analysis Core Facility (NSC 102-2319-B-010-001). The authors acknowledge the High-throughput Genome Analysis Core Facility of National Core Facility Program for Biotechnology, Taiwan (NSC 102-2319-B-010-001), for sequencing. The authors declare no competing financial interests.

© 2015, Lee et al.

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