Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway

    Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway Pallavi Rajput, Vijaya Pandey, Vijay Kumar PII: DOI: Reference:

S1874-9399(16)30082-7 doi: 10.1016/j.bbagrm.2016.05.002 BBAGRM 1031

To appear in:

BBA - Gene Regulatory Mechanisms

Received date: Revised date: Accepted date:

16 January 2016 23 April 2016 4 May 2016

Please cite this article as: Pallavi Rajput, Vijaya Pandey, Vijay Kumar, Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway, BBA - Gene Regulatory Mechanisms (2016), doi: 10.1016/j.bbagrm.2016.05.002

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ACCEPTED MANUSCRIPT Stimulation of ribosomal RNA gene promoter by transcription factor Sp1

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involves active DNA demethylation by Gadd45-NER pathway

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Running Title: rRNA gene transcription by Sp1

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Pallavi Rajput, Vijaya Pandey and Vijay Kumar* Virology Group, International Centre for Genetic Engineering and Biotechnology,

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Aruna Asaf Ali Marg, New Delhi- 110067, India

*Address correspondence to: Vijay Kumar, Ph. D, J.C. Bose Fellow, Virology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi – 110067, India. E-mail: [email protected]; Tel: +91-11-26742360; Fax: +9111-26742316.

Abbreviations used are: ChIP, chromatin immunoprecipitation; NER, nucleotide excision repair; NoRC, nucleolar remodeling complex; PBS, phosphate-buffered saline; Pol I, RNA polymerase I; qRT-PCR, quantitative real time-polymerase chain reaction; rDNA, ribosomal DNA; Sp1, Specificity protein 1; TIP5, TTF-I-interacting protein 5; TTF-I, transcription termination factor-I; UBF, upstream binding factor; XPG, Xeroderma pigmentosum complementation group G.

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ACCEPTED MANUSCRIPT Abstract The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role

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in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated

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the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatic active state. More

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importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA.

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Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel

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role of Sp1 in rDNA transcription involving promoter demethylation.

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Keywords: ribosomal RNA; DNA demethylation; Sp1; Gadd45; nucleolar remodeling complex;

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epigenetic regulation; ribosome biogenesis.

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ACCEPTED MANUSCRIPT 1. Introduction Proliferating cells require continuous supply of new ribosomes for protein biosynthesis through coordinated activity of the three forms of RNA polymerases [1, 2]. RNA polymerase I (Pol I)

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apparently plays a central role in ribosome biogenesis by synthesizing the precursor 45S ribosomal RNA (rRNA) which is processed into mature 28S, 18S and 5.8S rRNAs. Whereas Pol

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III synthesizes 5S rRNA, all ribosomal proteins are synthesized by Pol II [3, 4]. We still know

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little about how cells allow co-transcriptional assembly of pre-ribosomes and adjust the production of each ribosomal constituent in time and space. Although the transcription of rRNA

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genes is a highly regulated process affected by general cell metabolism, it is equally responsive to specific environmental challenges [5]. rDNA transcription starts in the G1 phase of cell cycle,

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continues during S and G2 phases, shuts down in mitosis and again starts recovering as cells reenter G1 phase [6]. Alterations in cell proliferation are accompanied by profound changes in the transcription rate of rRNA genes. Not surprisingly, dysregulation of Pol I transcription and

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ribosome biogenesis is associated with etiology of many of the human diseases including cancer

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[3, 7].

Initiation of rDNA transcription requires the assembly of the preinitiation complex at the rDNA

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promoter via the synergistic action of the upstream binding factor (UBF) and the promoter selectivity factor SL1 – a complex containing the TATA binding protein and associated factors [4, 8]. Activation of Pol I transcription involves phosphorylation of UBF at specific serine residues by protein kinases associated with G1 cyclins [3]. Other protein kinases including PI3K and mTOR, also contribute to enhanced rDNA transcription [9]. In addition, Pol I transcription can be upregulated by an interplay between oncogenes and tumor suppressor proteins. For example, c-Myc can bind to the nucleolar ‘E box elements’ of rRNA genes, associate with SL1/TIF-IB and facilitate rDNA transcription [10, 11] whereas tumor suppressors like pRb and p53 repress Pol I transcription [12]. However, studies on the involvement of other transcription factors or their ‘cis’ elements in Pol I transcription has not received adequate attention. In mammalian cells, the tandemly repeated multiple copies of rRNA genes are transcribed with high efficiency to keep up with the cell’s metabolic activity and demand for ribosomes [13]. The intracellular level of rRNA is usually regulated by changing the rate of transcription initiation at active rDNA genes rather than by activating silent transcription units [14]. The promoter of

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ACCEPTED MANUSCRIPT active rRNA genes is free of CpG methylation and associated with histones that are acetylated whereas an opposite pattern is seen among silent genes [15]. The silencing of rRNA gene is epigenetically regulated by a nucleolar remodeling complex (NoRC) that consists of two

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subunits, the ATPase SNF2h and TIP5 (TTF-I-interacting protein 5). NoRC is recruited to rDNA promoter by the transcription termination factor-I (TTF-I) to function as a chromatin-specific

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repressor of rDNA transcription through recruitment of histone deacetylase and DNA methyl-

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transferase activities [16, 17]. However, the molecular mechanism governing the silencing and activation of individual rDNA transcription units during cell proliferation and differentiation

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remains elusive.

More recently, we have reported synchronized synthesis of both rRNAs and ribosomal proteins

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during the G1 phase of cell cycle and identified Sp1 as a major transcriptional activator of ribosomal protein genes [18]. However, despite a clear understanding of the regulation RNA

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polymerase II-dependent ribosomal protein genes by Sp1, the regulation of RNA polymerase Idependent rRNA promoter remains largely unpursued. In the present study, we investigated the

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role of Sp1 as a regulator of Pol I- dependent rDNA transcription. We show that Sp1 is recruited

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to rDNA during G1 phase in association with RNA polymerase I and promotes epigenetic modifications on the rDNA promoter through crosstalk of various chromatin modifiers which further lead to transcriptional activation. The report elucidates the novel role of Sp1 in triggering DNA demethylation through Gadd45a-NER pathway. The current study provides mechanistic insight into the molecular events orchestrated by Sp1 for epigenetic activation of rDNA, thus revealing an interesting link between Sp1 and ribosome biogenesis.

2. Materials and Methods 2.1. Plasmids and siRNA The EGFP expression pEGFP-N1 and pSilencerU6 1.0 plasmid were procured from Clonetech and Ambion respectively. The development of reporter construct human rDNA-pGL3 was described previously. The pCS2-myc-Gadd45a and pCMV-Flag-Sp1-HA expression vectors were a kind gift from Dr. Christof Niehrs and Dr. M. Spengler and respectively [19, 20]. The shRNA directed against human Sp1 mRNA was obtained by cloning the two correspondingly annealed oligos into pSilencerU6 1.0 between ApaI and EcoRI sites. The sequences of the two 4

ACCEPTED MANUSCRIPT oligos

for

Sp1

shRNA

were

as

follows:

5’-

GATCACTCCATGGATGAAATTCAAGAGATTTCATCCATGGAGTG 5'-

ATCTTTTTTTT-3’and

TTCATCCAGGAGTGATCGGCC-3'.

UBF

siRNA

obtained

from

Santa

cruz

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biotechnologies.

was

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AATTAAAAAAAAGATCACTCCATGGATGAAATCTCTTGAAT

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2.2. Cell culture and DNA transfection

The human embryo kidney HEK293 and HEK293T, human hepatoma Huh7, human

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osteosarcoma U2OS, lung carcinoma A549, colorectal HCT116 p53-/- cells were cultured in DMEM media supplemented with 10% fetal bovine serum at 37o C in a humidified incubator

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with 5% CO2. Cells were seeded at density of 0.6 X 106 or 1.5 X 106 in a 60- or 100- mm culture dish. 2 or 5 g of plasmid DNA was transiently transfected in a 60- or 100- mm culture dish

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respectively using Lipofectamine 2000 (Invitrogen) as per manufacturer’s instructions. pEGFPN1 and pSilencerU6 1.0 were used as transfection vector control for overexpression and

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knockdown studies respectively. For luciferase assay, 0.25 g of reporter plasmid was

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transfected with 1.0 g of indicated expression plasmid. 2.3. Antibodies and reagents

Antibodies were obtained from the following sources. Santa Cruz biotechnologies for UBF, RPL5, TAFIp110 and GAPDH; EMD Millipore for Sp1, H3K9ac, H4K12ac, H3K4me3, H4k20me3 and HDAC1; Abcam for RNA polymerase I (RPA135), TBP and DNMT1; Cell signaling for Gadd45a; and Proteintech for XPG protein. The chemical inhibitors used and their working concentration were: Actinomycin D (5 nM, 24 h), RG108 (200 M, 24 h) and Trichostatin A (200 nM, 24 h) from Sigma-Aldrich, Cisplatin (35 M, 24 h) from Calbiochem. 2.4. Cell synchronization and flow cytometry For cell cycle studies, HEK293T cells were serum starved for 24 hours followed by 10% serum stimulation. Flow cytometry was performed as described elsewhere [21]. Briefly, cells were fixed in 70% ethanol and processed for propidium iodide staining. The cell profile was analyzed using cell quest software.

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ACCEPTED MANUSCRIPT To analyze cell size in asynchronous cells, U2OS cells were transiently transfected with vector control or Sp1 plasmid. After 48 hours post-transfection, cells were processed for flow cytometry. The G1 phase cells were gated and the mean FSC-H was determined as a measure of

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relative cell size.

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2.5. Cell Viability assay

Cell viability was determined using MTT assay. Cells were incubated with MTT (1 mg/ ml) at

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37°C for 15-30 min. Crystals were solubilized using molecular grade DMSO and the absorbance was recorded at 560 nm using spectrophotometer. The mean absorbance values of three

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experiments were expressed as percentage of viability in relative to control. 2.6. Luciferase assay

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Luciferase assay was performed according to supplier’s instructions (Promega). The relative luciferase activities were normalized with the total protein amount.

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2.7. Transcription analysis by RT-qPCR

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Total RNA was isolated from cells using TRIzol reagent as per manufacturer’s instructions. Total RNA was reverse transcribed using random hexamer with M-MuLV Reverse transcriptase

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(Fermantas) according to manufacturer’s guidelines. RT-qPCR was performed using Universal SYBR green mix (Biorad). Acidic ribosomal phosphoprotein P0 (ARPP) was used as an internal control and the results were analysed using comparative ΔΔCt method [22]. Primer sequences used are listed in Supplementary Table S1. 2.8. Immuno-precipitation and Western Blotting Cells were harvested and lysed in cell lysis buffer and quantified for protein amount by Bradford assay. Equal amount of protein was then taken and immunoprecipitated with 1 g of desired antibody overnight at 4◦C. Further Protein A or G sepharose beads were added and incubated at 4◦C. Subsequently, the beads were washed with the lysis buffer and then suspended in 2X SDS dye. Western Blotting of protein samples were done as described elsewhere [21]. 2.9. Immunoflourescence Huh7 cells were seeded on a coverslip in a 12-well plate at a density of 0.1 X 106 cells /well. Before fixing, cells were serum starved for 48 h followed by 8 hours of 10% serum release. For immunofluorescence, cells were fixed in 2% paraformaldehyde for 15 minutes at room 6

ACCEPTED MANUSCRIPT temperature, followed by permeabilization with Triton-X100 in PBS for 5 minutes on ice. Primary antibodies were then added at desired dilution. Alexa488 (anti-mouse) or Alexa594 (anti-rabbit) were used as secondary antibodies. DAPI staining was used to visualize nuclei.

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Pictures were acquired with a Nikon ECLIPSE TE 2000-U fluorescence microscope equipped

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with a 60X objective lens. 2.10. Chromatin immunoprecipitation- qPCR

(Upstate

Biotechnology).

The

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Chromatin immuno-precipitation assay was performed as per manufacturer’s instructions immunoprecipiated

chromatin

was

purified

through

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phenol/chloroform extraction and ethanol precipitation. The purified DNA were amplified by real time PCR using Univerasl SYBER green mix (Biorad) with indicated primers listed in

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Supplementary Table S1. The data obtained was normalized with input DNA and expressed as fold DNA enrichment over mock (pre immune sera).

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2.11. Methylation sensitive restriction analysis (MSRA)

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To examine CpG methylation at rDNA, genomic DNA was isolated and digested with HpaII or Msp1. Real time-qPCR was performed to amplify rDNA from undigested and digested samples

control.

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using primer B. The result was expressed as mean HpaII uncut DNA with reference to vector

2.12. Bisulphite sequencing

The genomic DNA isolated from HEK293T cells transiently transfected with vector or Sp1 expression plasmid was subjected to bisulphite conversion using The EZ DNA Methylation TM Gold Kit (Zymo Research) as per supplier’s instructions. The modified genome was PCR amplified using primers for rDNA loci (Supplementary Table S1). The 246 bp PCR product was cloned into pGEM-T Easy vector (Promega) according to the manufacturer’s protocol. Plasmid DNA from individual clones was sequenced using 3730xl DNA analyzer (Macrogen Inc.). 2.13. In vitro methylation of plasmid DNA The reporter rDNA-pGL3 plasmid was in vitro methylated using SssI CpG methyltransferase (New England Biolabs) as per manufacturer’s instructions. The efficacy of the enzymatic reaction was confirmed by digestion with HpaII enzyme. The methylated DNA was purified using Qiagen DNA purification kit. The concentration and purity of DNA was calculated by 7

ACCEPTED MANUSCRIPT measuring Absorbance at 260 nm (A260) and ratio of absorbance A260:A280 nm and

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A260:A230.

2.14. Polysome/Ribosome Profiling

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The protocol for ribosome profiling has been previously described [23]. Briefly, HEK293T cells

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were transiently transfected with vector or Sp1 expression plasmid. After 48h post-transfection, cells were rinsed and harvested in 1X PBS containing 100g/ml cycloheximide (CHX). Cells

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were lysed in 1ml PEB buffer (20mM Tris-HCl pH7.5, 50mM KCl, 10mM MgCl2, 1mM DTT, 100 g/ml CHX, 200 g/ml Heparin) containing 1% Triton- X100 and kept at ice for 30 mins.

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Centrifuged at 13000 rpm for 30 min at 4 ºC and collected supernatant. Simultaneously, prepared 50%, 40%, 30%, 20%, 10% sucrose solutions in PEB (without Triton- X100). The sucrose gradient was prepared by adding 2 ml of each solution (50% on bottom, 10% on top) into

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ultracentrifuge tube. Added 1 ml of cell extract to the top of each gradient and gently placed it in

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SW41 rotor. Centrifuged samples at 38,000 rpm for 2 h at 4 ºC. After centrifugation collected fractions from bottom to top manually and quantified spectrophotometerically (O.D254). The

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values obtained were plotted against the density gradient from low to high. 2.15. Ribosome isolation

U2OS cells were transfected with vector or Sp1 expression plasmid. Total ribosome was isolated from equal number of U2OS cells as described elsewhere [24]. Estimation of ribosome content was done using spectrophotometer and total ribosomal amount was calculated according to the formula. O.D260 = 14 corresponds to 1mg of ribosomes and about 500 g of ribosomal proteins. The mean quantified value of two experiments were expressed relative to vector control. 2.16. Bioinformatic analysis TFSEARCH software relating to the TRANSFAC database (http://www.cbrc.jp/research/ db/TFSEARCH.html) was used to predict transcription factor binding sites on the human complete rDNA unit (Gene accession no. U13369.1). 2.17. Statistical analysis 8

ACCEPTED MANUSCRIPT Data are expressed as mean ± S.D. The statistical significance of our data was calculated using Student’s t-test. ** and * - indicate statistically significant difference at p < 0.01 and p < 0.05

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respectively.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Sp1 binds to rDNA inside the nucleolus To understand the role of Sp1 in Pol I- dependent rDNA transcription, we did in silico analysis

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of the complete human rDNA unit. Our bioinformatics analysis (threshold score of 85.0 or

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above) predicted a total of 106 Sp1 binding sites in both transcribed as well as in the nontranscribed promoter and intergenic regions of rDNA (Fig. 1A). The non-random distribution of

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Sp1 binding sites in rDNA unit, suggested its functional importance in rDNA transcription. To confirm whether Sp1 binds to rDNA in vivo, we next examined the recruitment of Sp1 in

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different rDNA regions by ChIP-qPCR (Fig. 1B). The specific association of Sp1 with rDNA (and not with rRNA transcripts) was ensured by RNase treatment of ChIP samples. Sp1

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prominently bound to the regulatory region (Primer a, b and c) where the binding sites are clustered. As expected there was negligible enrichment in region (primer d) that had no Sp1

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binding sites. A significant enrichment was also observed in the intergenic region that carries

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Sp1 binding sites (primer e). Furthermore, in cell line transiently transfected with HA-Sp1, we observed a similar enrichment of ectopic Sp1 on rDNA (Fig. 1C). The specificity of Sp1 binding

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to its cognate element on rDNA was further confirmed in the presence of actinomycin D- a potent DNA intercalating drug well-known to inhibit Sp1 interaction with its cognate elements [25]. As expected, treatment of HEK293T cells with actinomycin D (25 nM for 24h) led to a decreased rDNA occupancy of endogenous Sp1 (Fig. 1D). Note at that low concentrations, actinomycin D treatment is known to cause nucleolar stress and inhibit Pol I transcription [26]. Therefore, to abolish the possibility of less Pol I transcription at the rRNA gene leading to reduced binding of Sp1 to rDNA in the presence of ActD, we checked the recruitment of Sp1 on Pol I transcriptional inhibition by siRNA mediated knockdown of UBF. As shown in Supplementary Fig. S1, knockdown of UBF had not effect on Sp1-rDNA association. Thus, we convincingly indicate that Sp1 specifically binds to rDNA. As nucleolus is the prime site of rRNA transcription, we wondered about the subcellular localization of Sp1 in the nucleolar sub-compartment. Earlier studies have suggested a physical interaction between Sp1 and the nucleolar proteins such as nucleophosmin (NPM) and ADPribosylation factor (ARF) [27, 28]. However, endogenous Sp1 generally exhibits a pan nuclear distribution without much presence inside nucleolus. Therefore, to examine the sub-nuclear 10

ACCEPTED MANUSCRIPT localization of Sp1, we transfected cells with HA-Sp1 expression plasmid and analyzed its presence inside the nucleolus. As shown in Supplementary Fig. S2, HA-Sp1 specifically colocalized with UBF suggesting its nucleolar distribution. The nucleolar distribution of Sp1 was

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further enhanced by treating cells with inhibitor of proteasome machinery (MG132). Thus, it clearly indicates that under normal conditions, Sp1 seems to be present inside the nucleolus

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albeit at a relatively lower level. However, elevating its levels either by overexpression or

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inhibiting its degradation could lead to visibly enhanced nucleolar localization of Sp1. 3.2. Sp1 activates RNA pol I- dependent rDNA transcription

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As our ChIP experiments suggested specific binding of Sp1 to rDNA, we next investigated its physiological relevance on Pol I- dependent rDNA transcription using an rDNA promoter-

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reporter construct. In this luciferase-reporter construct, the human rDNA promoter carries four putative Sp1 binding sites and an IRES sequence upstream of the rDNA region (-406 to +378 bp)

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ensuring the expression of only Pol I transcripts and not any cryptic Pol II transcript (Supplementary Fig. S3). In HEK293T cells, the luciferase activity was found to be linearly up-

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regulated with increasing concentration of the Sp1 expression plasmid (Fig. 2A). As the reporter

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gene stimulation by Sp1 could be observed in different cell lines used, it suggested the redundancy of the Sp1-dependent Pol I regulation (Fig. 2B). As expected, there was a sharp decline in rDNA promoter activity following down-regulation of Sp1 levels by specific shRNA (Fig. 2C and Supplementary Fig. S4). To further validate the effect of Sp1 on rDNA transcription, we directly monitored the rRNA levels following Sp1 overexpression by qRTPCR. As shown in Fig. 2D, the 45S pre-rRNA levels were significantly elevated in presence of Sp1. Importantly, the observed increase in rRNA expression showed resistance to -amanitin (RNA Polymerase II inhibitor) treatment in comparison to the expression of Pol II- dependent RPS27a transcripts. The ARPP P0 mRNA levels remain unchanged on - amanitin treatment. Therefore, it evidently negates the indirect effects of Sp1 mediated Pol II dependent response in rDNA activation. The specificity of endogenous Sp1 on rDNA stimulation was further confirmed by a significant decrease in the rRNA levels following Sp1 depletion. Apparently, the Sp1mediated Pol I transcriptional stimulation also appeared to be a p53-independent process as evident from stimulation of rDNA promoter in p53-/- HCT116 cell line (Supplementary Fig. S5).

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ACCEPTED MANUSCRIPT Altogether, these results strongly suggest that Sp1 has a direct and essential role in stimulating

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rDNA transcription.

3.3. Sp1 activates rDNA transcription in G1 phase

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The synchronized synthesis of ribosomal components is essential during G1 phase for cells

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committed to cell division. Therefore, to examine the temporal expression pattern of pre-rRNA, cells were synchronized by serum starvation and the distribution of cells in different phases was

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determined by FACS (Supplementary Fig. S6). Accordingly, pre-rRNA expression peaks up in G1 phase with maximum levels corresponding to late G1 phase (G1/S transition) (Fig. 3A).

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Interestingly, the expression pattern of pre-rRNA coincided with that of transcription factor Sp1 [29]. Next, we examined the recruitment of Sp1 on rDNA in a cell cycle dependent manner. We observed the maximal binding of Sp1 on rDNA during G1 phase (Fig. 3B). This was found to be

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in complete coherence to the observed expression profiles. Collectively, it suggests that Sp1

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binds to rDNA during G1 phase to drive Pol I transcription.

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3.4. Sp1 interacts with Pol I and modulates the recruitment of transcription machinery Since RNA polymerase I, SL1 (TBP-TAF) complex and UBF constitute the basal transcription machinery for rDNA, we next probed the molecular mechanism underlying the Sp1-dependent rRNA gene expression. Sp1 interaction with TBP (an integral part of SL1 complex) is well known [30]. As shown in Fig. 4A, anti-Sp1 antibody specifically co-immunoprecipitated RPA135 (a Pol I subunit). Conversely, RPA-135 antibody was able to pull down recombinant Sp1 from the HA-Sp1 expressing cells as opposed to vector transfected cells (Fig. 4B). These results suggested a strong and specific interaction between Sp1 and RNA Pol I. Further as shown in Fig. 4C, Sp1 also co-precipitated with a TAF specifically present in SL1 (TAFIp110). However, no interaction between Sp1 and UBF could be observed in these co-immunoprecipitation experiments (data not shown). Nevertheless these studies indicate that Sp1 could interact with the RNA Pol I transcription machinery in order to stimulate rRNA synthesis. Therefore, we examined the rDNA occupancy of pre-initiation complex in presence and absence of Sp1. As shown in Fig. 4D, there was a marked increase in the occupancy of UBF, TBP and RPA-135 in the presence of Sp1. Further, the recruitment of these factors was abrogated when Sp1 was

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ACCEPTED MANUSCRIPT knocked down by shRNA (Fig. 4E). Together, these results suggested that Sp1 facilitates the recruitment of the basal transcription complex in order to stimulate rDNA transcription.

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3.5. Sp1 regulates ribosome biogenesis It has been recently reported that ribosomal protein genes carry multiple Sp1 binding sites that

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play a key role in the expression of these Pol II-dependent genes [18]. As Sp1 also bound to rDNA and stimulated rRNA gene expression, we anticipated a profound role of Sp1 in

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synchronized production of ribosomal components (both rRNAs and ribosomal proteins) during the cell cycle. Therefore, we did ribosome profiling of vector or Sp1 transfected cells in the G1

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phase of cell cycle. As shown in Fig. 5A, the ribosome profile of Sp1-transfected cells was conspicuously enhanced in comparison to vector control cells as indicated by an upward shift in

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the 80S ribosome peak. The increase in total ribosomal content was also evident from the commassie blue staining and spectrophotometric measurement of the ribosomal fractions isolated

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from vector and Sp1-transfected cells (Fig. 5B).

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Ribosomes constitutes the major protein machinery of the cell. Therefore, the ribosomal content is the most crucial indicator of the rate of protein synthesis in a cell, thereby, a determinant of

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cell growth and proliferation [1]. Therefore, we next examined the effect of Sp1 overexpression on cell size by calculating the mean FSC-H of the G1 gated cells. As shown in Fig. 5C, ectopic expression of Sp1 led to remarkable rightward shift in the FSC-H histogram of the G1-gated Sp1-transfected cells in comparison to control cells. Consequently, quantification of the mean FSC-H from three independent experiments depicts a significant increase in Sp1 transfected cells in comparison to the control vector, indicating an overall increase in cell size in the presence of Sp1. Furthermore, in agreement to the pro-proliferative role of Sp1, we also observed increased cell proliferation and survival in presence of Sp1. As shown in Supplementary Fig. S7, the Sp1 transfected cells exhibited accelerated entry into the S-phase and relatively better survival in comparison to control cells. Collectively, we suggest that Sp1 plays a major role in ribosome production which may further strengthen its role in cell growth and proliferation. 3.6. Sp1 regulates the epigenetic status of rDNA Epigenetic modifications are promiscuously associated with transcriptional activation to enable the binding of transcription machinery. Epigenetic silencing of rDNA unit is primarily mediated by nucleolar remodelling complex (NoRC)-dependent recruitment of repressor molecules such as 13

ACCEPTED MANUSCRIPT HDACs and DNMTs which lead to heterochromatin formation [31]. Given that Sp1 activates rDNA transcription, we next investigated the association of NoRC complex to rDNA in the presence of Sp1. We observed that the binding of TIP5 protein, which is a major component of

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NoRC complex, was significantly reduced following Sp1 overexpression. This was accompanied by a decreased association of other co-repressor molecules such as HDAC1 and DNMT1 (Fig.

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6A). In addition, treatment of Sp1 depleted cells with known epigenetic inhibitors of rDNA

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transcription- TSA (HDAC inhibitor) and RG108 (DNMT inhibitor) was able to restore the decreased rRNA levels resulting from Sp1 depletion (Fig. 6B). These results suggest that Sp1

recruiting Pol I transcriptional machinery.

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plays an important role in antagonizing the role of NoRC and stimulating rDNA transcription by

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As NoRC- dependent rDNA silencing plays a pivotal role in regulating the epigenetic landscape of rDNA, we next examined the status of various histone modifications which embark the active and repressive state of rDNA [3]. We found that the ectopic expression of Sp1 was associated

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with increase in histone acetylation (H3K9ac, H4K12ac) and active histone methylation

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(H3K4me3) marks concomitantly with disappearance of repressive marks such as H4K20me3 (Fig. 6C). Further, as expected down regulation of Sp1 led to an increase in the H4K20me3 mark

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and depreciation of activation marks, viz. H3K9ac, H4K12ac, and H3K4me3 (Fig. 6D). DNA methylation has long been considered as an epigenetic switch for inhibiting transcriptional activity. It is generally regarded that DNA demethylation generally correlates with transcriptional activation. Accordingly, the present premise correlates transcription factor binding to maintenance of unmethylated CpG sites in gene promoters [32-34]. Quite interestingly, the Sp1 binding sites have been found in the CpG islands of a number of gene promoters [35, 36]. In an attempt, to elucidate the correlation between Sp1 binding and methylation of CpG sites on rDNA, we examined the methylation status of the HpaII/Msp1 sites by methylation sensitive restriction analysis (MSRA). There were a total of four HpaII/Msp1 sites located in vicinity of Sp1 sites in the desired amplicon. We observed that Sp1 expression inversely correlated with the HpaII resistance (Fig.7A and 7B). Moreover, bisulfite sequencing of all CpGs in the rDNA promoter region showed a dramatic increase in the number of unmethylated CpG residues in the Sp1-transfected cells as compared to the control cells (Fig. 7C). In light of the above observation, we further examined the effect of Sp1 on stimulation of 14

ACCEPTED MANUSCRIPT methylated gene promoter. We performed in vitro methylation of Pol I promoter- reporter construct by SssI methyltransferase and confirmed its status by MSRA (Supplementary Fig. S8). Interestingly, Sp1 was able to stimulate gene transcription from an in vitro methylated Pol I

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promoter- reporter construct (Fig. 7D). Thus, our data strongly suggest that Sp1 can stimulate rDNA demethylation leading to rRNA gene expression. Collectively, we provide compelling

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evidences confirming the role of Sp1 in euchromatin formation and epigenetic activation of

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rRNA genes.

3.7. Sp1 triggers Gadd45a-Nucleotide excision repair pathway for demethylation of rDNA

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DNA demethylation may result due to decreased activity of DNMTs which is a relatively slower process. Further, it may not fully account for the rapid demethylation following ectopic

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expression of Sp1 [33]. On the other hand, active demethylation is a rapid process tightly coupled with the DNA repair processes. Having shown that Sp1 can hypomethylate rDNA and

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stimulate rDNA transcription, we next probed into the involved molecular mechanism of DNA

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demethylation.

A direct involvement of nucleotide excision repair (NER) machinery in bringing active rDNA

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demethylation via Gadd45a had been previously reported [37]. Gadd45a is a DNA damage response protein which senses the damaged site and recruits the NER complex to enzymatically remove the methylated base. The Xeroderma pigmentosum complementation group G (XPG) protein of NER complex is a 3’ endonuclease which nicks the damaged DNA. Therefore, we next investigated the kinetics of Gadd45a and XPG binding on rDNA. As shown in Fig. 8A, Gadd45a bound to rDNA during early G1 phase which was succeeded by XPG recruitment in late G1 phase. The rDNA methylation profile also indicates that the demethylation events takes place in late G1 phase in concord to XPG binding. The temporal recruitment of these proteins along with Sp1 in G1 phase therefore suggest the presence of a synchronized and orchestrated mechanism leading to DNA demethylation and transcriptional activation. We next examined the interaction between Sp1, Gadd45a and XPG in a cellular environment. Our immunofluorescence studies showed that Sp1 was essentially present inside the nuclear sub-compartment along with Gadd45a and XPG as a complex (Supplementary Fig. S9). Further, our co-immunoprecipitation studies confirmed the interaction among Sp1, Gadd45a and XPG (Fig. 8B). These results also

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ACCEPTED MANUSCRIPT imply that the interaction of Sp1 with Gadd45a and NER- associated proteins in vivo could

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regulate the downstream demethylation events.

To ascertain the direct role of Gadd45a-NER pathway in Sp1 dependent rDNA demethylation,

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we first analyzed the effect of Sp1 depletion on Gadd45a mediated epigenetic activation. As

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shown in Fig. 8C, ectopic expression of Gadd45a led to a significant activation of methylated rDNA promoter in control cells, whereas, no significant change (p>0.05) was observed in the

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reporter activity of Sp1 depleted cells. This was further supported by the observation that treatment with cisplatin- a well-known inducer of NER pathway led to a significant increase in

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both Sp1 and Gadd45a rDNA occupancy. However, downregulation of Sp1 in presence of cisplatin, not only led to decreased binding of Sp1 but also Gadd45a (Fig. 8D and Supplementary Fig. S10). Therefore, Sp1 seemed to engage Gadd45a-NER pathway to induce DNA

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demethylation. Not surprisingly, ectopic expression of Sp1 led to significant increase in loading

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of both Gadd45a and XPG proteins on rDNA (Fig. 8E). Thus, our results confirmed that Sp1 play a major role in recruitment of Gadd45a and NER machinery to rDNA, which in turn

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stimulates rDNA demethylation and transcription. The indication that Sp1 interacts with NER-associated proteins under cellular environment contended upon the redundancy of NER pathway in the epigenetic regulation of other RNA pol II dependent Sp1 target gene promoters. Therefore, we extended our study to Pol II dependent oct4 gene promoter to detect whether a similar demethylation mechanism was operative. We chose oct4 gene promoter which is a known target of Sp1 [38]. Intriguingly, epigenetic activation of oct4 promoter is also elicited through Gadd45a-NER dependent DNA demethylation [19]. To confirm whether Sp1 affects the methylation status of oct4 gene promoter, we examined the oct4 methylation pattern in presence of Sp1 by MSRA and quantifying 5-methyl cytosine levels. Indeed, over expression of Sp1 lead to significant decline in percent HpaII resistance on oct4 promoter (Supplementary Fig. S11A). This was found to be in perfect agreement to the observed increase in the binding of Gadd45a and XPG to oct4 promoter on Sp1 overexpression (Supplementary Fig. S11B). Altogether, the present study unveils a novel role of Sp1 to engage Gadd45a-NER pathway to control the dynamic DNA methylation-demethylation events, which

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ACCEPTED MANUSCRIPT may regulate the transcriptional activity of a number of other target promoters through an

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epigenetically programmed landscape.

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4. Discussion

RNA Pol I-dependent transcription of rRNA genes is a tightly regulated process which accounts

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for nearly 70 per cent of the total transcriptional activity in a cell. However, in certain pathological conditions such as cancer, increased rate of rDNA transcription is seen in cells in

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order to meet their enhanced proliferation rate and metabolic requirements [1, 39]. Therefore, it is of immense importance to understand the molecular mechanisms underlying the rDNA

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transcriptional machinery. Despite so much information available on the regulatory mechanisms of rDNA expression, the involvement of classical Pol II-associated transcription factors has

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factor Sp1 in rDNA transcription.

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remained largely unappreciated. In this study, we elucidate new engagements of transcription

We found that Sp1 is an important transcriptional activator of RNA Pol I-dependent rDNA

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transcription. Sp1 directly bound to the rDNA promoter region, facilitated the loading of Pol I transcription initiation complex and induced rRNA expression during the G1 phase of cell cycle. Since synchronized synthesis of rRNAs and ribosomal proteins is essential for ribosome biogenesis, our earlier observation that Sp1 can stimulate the expression of several ribosomal protein genes during the same time window should have important physiological consequences [18]. Ribosome production is orchestrated in a cell cycle dependent manner. The G1 phase being the preparatory phase and the most decisive period of the cell cycle, the ribosome production during this phase determines the fate of cell growth and proliferation. Indeed, it is not surprising that various physiological and pathological conditions such as development and cancer are characterized by deregulated ribosome biogenesis and elevated expression of Sp1 [1, 39-41]. Thus, our study further substantiates the pro-proliferative potential of Sp1 and exemplifies its implications in cancerous growth. The growth dependent regulation of rRNA synthesis is mainly governed by dynamic association of TIF-1A to the pre-initiation complex, which in turn is regulated by various post-translational modifications. Previous reports suggest that TIF-1A is a transcriptional target of c-Myc 17

ACCEPTED MANUSCRIPT oncoprotein [42]. In addition, c-Myc coordinates with S6 kinase to regulate TIF-1A activity [43]. Thus, it will be quite interesting to study the effect of Sp1 on TIF-1A activity and its role in

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driving Sp1-mediated rDNA transcription. Transcriptional regulation of genes require chromatin remodeling through covalent modification

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of histones and DNA by allowing the binding of activator and repressor molecules on the target gene promoters [44]. Moreover, in case of rDNA genes, NoRC is well known to epigenetically

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regulate the silencing of rDNA via histone modifications and/or CpG methylation of genes [17]. The present study provides new evidences supporting the role of Sp1 in the maintenance of

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active euchromatin state of rDNA. We found that Sp1 antagonized and abated the association of NoRC silencing complex leading to decreased association of co-repressor molecules. More

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importantly, Sp1 also induced gene-specific DNA demethylation by engaging Gadd45a-NER pathway [19, 37]. There was enhanced recruitment of Gadd45a on to the rDNA promoter in the

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presence of Sp1 that in turn stimulated the NER pathway to cascade active DNA demethylation. The inhibition of NoRC-dependent rDNA silencing combined with the recruitment of Gadd45a-

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NER complex ultimately affected the post-transcriptional histone modifications and CpG

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methylation on rDNA and set the stage for Pol I transcription. Thus, the binding of Sp1 appears to specify the epigenetic landscape of rDNA unit commensurate with a transcriptionally active gene which allows the binding of pre-initiation complex on gene promoter for rRNA expression. Further, this substantiated the involvement of Sp1 in Pol I-driven rDNA transcription. Gadd45a abates gene silencing by promoting DNA repair and removing methylated cytosines [37, 45]. Interestingly, Gadd45a overexpression does not induce global DNA demethylation rather it affects certain genes such as rDNA, Fgf-1B, oct-4, Runx-2 and BGP [19, 37, 46, 47]. However, what determines the site specificity for Gadd45a binding remains enigmatic? This question can be accurately explained based on our observations that Sp1 can facilitate the recruitment of Gadd45a on gene promoters (including rDNA and oct4). Thus, Sp1 can also play the role of a chromatin adaptor protein that facilitates the binding of Gadd45a-NER complex to demethylate the methylated DNA sites. Note that most gene promoters known to be demethylated and epigenetically activated by Gadd45a contain Sp1 binding sites, thus reinforcing the possible role of Sp1 in specifying the loading of Gadd45a for DNA demethylation [38, 48-52]. Besides, the enrichment of H3K4me3 and H3K9ac marks on the 18

ACCEPTED MANUSCRIPT rDNA promoter in the presence of Sp1 can be easily reconciled with targeted binding of Gadd45a and NER to these sites [53, 54].

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Previous reports have suggested an interesting role of transcription factor Sp1 in DNA methylation. Sp1 binding is known to confer resistance against CpG methylation. However,

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despite the clue that Sp1 links DNA methylation to transcription, very little is known about the mechanistic details of the same. In line with this recent studies has implicated transcription factor

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Sp1 in DNA damage and repair response. The first clue came from the study demonstrating the ATM- dependent phosphorylation of Sp1 in response to DNA damage inducing agents [55].

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ATM (Ataxia telangiectasia mutated) is a Ser/Thr kinase activated in response to DNA damage. It phosphorylates several proteins initiating check point activation and DNA repair. ATM

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phosphorylates Sp1 at Ser101 residue on induction of DNA damage. In addition, another study demonstrate the recruitment of Sp1 on DNA damage sites and assist the repair process [56].

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Depletion of Sp1 is able to inhibit the repair of DNA breaks. Together, these data indicate a specific role of Sp1 in activation of intrinsically linked network constituting DNA methylation,

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repair and transcription pathways [57].

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Pol I transcription accounts for nearly 70 per cent of cell’s transcriptional activity which makes it most susceptible to DNA damage [57]. Analysis of some recent studies seem to suggest that DNA damage induced through the transcriptional events might be bridged through specific binding of Sp1, albeit its molecular underpinnings still remain elusive [58]. Based on our study, we propose a new model that closely depicts the molecular mechanisms by which Sp1 binding favors the recruitment of repair machinery to stimulate transcription. Therefore, the current role of transcription factor Sp1 in coupling rDNA gene transcription with DNA repair might shed some light on our understanding of transcription-coupled DNA repair. However, the possible role of Sp1 in linking rDNA transcription to replication or genotoxic stress induced DNA damage, needs further investigation.

COMPETING INTERESTS The authors declare no competing interests.

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ACCEPTED MANUSCRIPT AUTHORS’ CONTRIBUTION KS performed the experiments and drafted the manuscript. VK conceived the study, designed the

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experiments and finalized the manuscript. Acknowledgements

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This work was supported in part by a J.C. Bose National Fellowship (Grant No. SR/S2/JCB-

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80)/2012) from the Department of Science and Technology, Government of India, New Delhi (to V.K.). Pallavi Rajput and Vijaya Pandey have respectively been senior research fellow and

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research associate of the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India. We thank Dr. S. K. Shukla for valuable suggestions. We are grateful to the following scientists for kindly providing us the expression plasmids: Dr M.

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Spengler (Roswell Park Cancer Institute, Buffalo, NY, USA) for Sp1 and Dr. Christof Niehrs (Institute of Molecular Biology, Mainz, Germany) for Gadd45a. The HCT116 p53-/- cell line was

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kindly provided by Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore). Technical

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assistance by R. Kumar and T. Choedon is gratefully acknowledged.

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ACCEPTED MANUSCRIPT 56. Beishline, K., Kelly, C.M., Olofsson, B.A., Koduri, S., Emrich, J., Greenberg, R.A., and Azizkhan-Clifford, J. (2012) Sp1 facilitates DNA double-strand break repair through a nontranscriptional mechanism. Mol. Cell. Biol. 32, 3790-3799.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Fig. 1. Identification and validation of Sp1 binding sites on the rDNA unit. (A) Schematic

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representation of in silico predicted Sp1 binding sites (vertical bars) in relation to the transcribed and non-transcribed regions of rDNA. (B) Cross linked chromatin from asynchronously growing

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HEK293T cells was pulled down with either anti-IgG (Mock) or anti-Sp1 antibody and assayed by ChIP-qPCR using different sets of primers (a to e) as shown in panel A and Supplementary

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Table 1. GAPDH and EPO gene promoters were used as positive and negative controls respectively. (C) Cells were transfected with vector or HA-Sp1 expression plasmids and Sp1

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recruitment was measured as above following the immunoprecipitation of chromatin with antiHA antibody. (D) ChIP-qPCR analysis of cells treated with DMSO or Actinomycin D (5nM, 24

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h). The fold Sp1 enrichment was measured by ChIP- qPCR using primer b. All results are represented as the mean ± SD of three independent experiments for each group. Statistical

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significance was calculated using Student’s t-test. ** and * represent p < 0.01 and p < 0.05

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respectively.

Fig. 2. Transcriptional regulation of rDNA promoter by Sp1. (A) HEK293T cells were

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transiently transfected with increasing concentrations of Sp1 expression plasmid (0.25, 0.5, 1 g) along with rDNA- pGL3 reporter construct (0.25 g) and the relative luciferase activity was measured. (B) Regulation of rDNA promoter by Sp1 under different cellular environment. Huh7, HEK293, A549 cells were transfected with vector control or Sp1 expression plasmid (1 g) along with rDNA-pGL3 reporter construct (0.25 g) and relative luciferase activity was measured. (C) HEK293T cells were transfected with Sp1 expression plasmid and Sp1- shRNA (0.5 g each) and luciferase activity was measured. (D) Cells transfected either with control or Sp1 expression vectors (1 g) and treated with - amanitin (2 g/ml) for 6 h before harvesting. Total RNA was used to measure 45S pre-rRNA and RPS27a transcripts by RT-qPCR after normalization with ARPP P0 mRNA level. (E) Cells were transfected with vector or Sp1-shRNA (1 g) and the levels of 45S pre-rRNA were determined by RT-qPCR. All results are represented as mean of three independent experiments ± S.D. ** represents p < 0.01 by Student’s t-test. Fig. 3. Expression of rDNA during the cell cycle. HEK293T cells were starved for 24 h followed by stimulation with 10% serum for indicated time periods and then harvested. (A) Total 27

ACCEPTED MANUSCRIPT RNA was isolated from these cells and the levels of transcripts for Sp1 and pre-rRNA were measured by RT-qPCR. (B) The binding of endogenous Sp1 and Gadd45a to rDNA unit was

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determined by ChIP-qPCR assay using primer b (Supplementary Table 1). Fig. 4. Interaction of Sp1 with RNA Pol I transcription machinery. (A-C) Whole cell lysate

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of HEK293T cells were immunoprecipitated either with control (IgG) or anti-Sp1, RPA135 or TAFIp110 antibodies followed by immunoblotting with indicated antibodies. (D-E)HEK293T

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cells were transiently transfected with vector or Sp1 plasmid (2 g) (D) and vector or Sp1shRNA plasmid (2 g) (E), followed by chromatin immunoprecipitation with anti- Sp1, UBF,

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TBP and RPA-135 antibodies. The samples were analyzed by ChIP-qPCR assay using primer b (Supplementary Table 1). Results are represented as mean of three independent experiments ±

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S.D. ** and * represent p < 0.01 and p < 0.05 respectively by Student’s t-test. Fig. 5. Regulation of ribosome biogenesis in the presence of Sp1. (A) Representative

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polysomal profiles from HEK293T cells transfected with vector control or Sp1 expression

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plasmid (2 g). The absorbance of the collected fractions was measured at 254 nm and plotted from low to high density. (B-C) Cells were transiently transfected with vector control or Sp1

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expression plasmids. The ribosomes were isolated from equal number of cells and the protein content of purified ribosome was measured spectrophotometrically. Commassie staining was used to visualize ribosomal fraction after SDS-PAGE. Ribosomal fraction was probed using antiRPL5 antibody (B). Data shown is represented as mean ± SD (n=2). The cell size represented as mean FSC-H of G1 gated cells was determined by flow cytometry (C). Data shown is represented as mean ± SD (n=3). Statistical significance was calculated using Student’s t-test. ** and * represent p < 0.01 and p < 0.05 respectively. Fig. 6. Epigenetic regulation of rDNA by Sp1. (A) HEK293T cells were transiently transfected with vector or Sp1 expression plasmid (2 g). After 48 h, cells were processed for ChIP assay using anti-TIP5, HDAC1 and DNMT1 antibodies. The fold enrichment over mock was quantified by ChIP-qPCR using primer b. (B) Cells were transiently transfected with control vector or Sp1-shRNA plasmids (2 g) and after 24h treated with TSA (200nM, 24h) and RG108 (200mM, 24h). Total RNA was isolated and quantified by RT-qPCR after normalization with Actin mRNA level. (C-D) Cells were transiently transfected with vector or Sp1 expression 28

ACCEPTED MANUSCRIPT plasmid (2 g) (C) and vector or Sp1-shRNA plasmid (2 g) (D). Cross linked chromatin was immunoprecipitated with anti- H3K9ac, H4K12ac, H3K4me3 and H4K20me3 antibodies and analyzed by ChIP-qPCR assay using primer b (Supplementary Table 1). Results are represented

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as mean of three independent experiments ± S.D. ** and * represent p < 0.01 and p < 0.05

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respectively by Student’s t-test.

Fig.7. Effect of Sp1 on rDNA methylation. (A-B) HEK293T cells were transiently transfected

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with vector or Sp1 expression plasmid (2 g) (A) and vector or Sp1-shRNA (2 g) (B). After 48h post-transfection, genomic DNA was isolated and digested either with mock or HpaII/ Msp1

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enzymes. Purified DNA was analysed by RT-qPCR assay using primer b. (C) Bisulphite mapping of rDNA unit (42805 to 52 bp) in vector or Sp1 transfected cells. Each horizontal row

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represents an individual clone. The open squares represent unmethylated CpGs while the closed squares represent methylated CpGs. (D) Cells were transiently transfected with vector or Sp1

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Fig. 8. Involvement of Gadd45a-NER pathway in the Sp1-mediated rDNA demethylation. (A) The serum synchronized HEK293T cells were stimulated with 10% serum for indicated time points. The samples were processed either for the presence of Gadd45 and XPG by ChIP-qPCR using primer b following immunoprecipitation with anti-Gadd45a and anti-XPG, or for the methylation sensitive restriction analysis for HpaII enzyme. (B) Whole cell lysates of HEK293T cells were immunoprecipitated with anti-IgG or anti-Sp1 antibodies, followed by immunoblotting with Sp1, Gadd45a and XPG antibodies.(C) Cells were transiently transfected with vector or Sp1-shRNA (2g) in presence or absence of Gadd45a expression plasmid along with in vitro methylated rDNA reporter plasmid (0.25 g) and the luciferase activity was measured. (D) Cells were transfected with vector or Sp1-shRNA followed by cisplatin treatment (35 M, 24h) and the binding of endogenous Sp1 and Gadd45a was determined by ChIP-qPCR assay using primer b (Supplementary Table 1). (E) Cells transiently transfected with vector or Sp1 expression plasmid were processed for ChIP-qPCR (primer b) assay using anti-Gadd45a and

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Graphical abstract

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