1. Volume 33, Issue 3, Pages 344-353 (February 2009)
TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation 
Kerstin-Maike Schmitz, Nina Schmitt, Urs Hoffmann-Rohrer, Andrea Schäfer, Ingrid Grummt, Christine Mayer 
Molecular Cell 
Volume 33, Issue 3, Pages (February 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Ectopic Gadd45a Triggers Demethylation of rDNA
(A) Gadd45a triggers demethylation of rDNA. Left panel: DNA from HEK293T cells overexpressing human HA-Gadd45a was digested with HpaII, and distinct regions of rDNA were amplified by qPCR using the primer pairs A–D that are depicted in the scheme above. Right panel: Gadd45a is associated with the rDNA promoter. Crosslinked chromatin from HEK293T cells overexpressing HA-Gadd45a was incubated with IgGs (light bar) or anti-HA antibodies (gray bars), and HA-Gadd45a occupancy was assayed by qPCR. Data are represented as mean ± SD.
(B) Overexpression of Gadd45a enhances pre-rRNA synthesis. RNA was isolated from HEK293T cells overexpressing hGadd45a. Pre-rRNA and β-actin mRNA were analyzed on Northern blots (inset), quantified, and compared to mock transfected cells. Gadd45a expression is shown on the western blot above. Data are represented as mean ± SD.
(C) Ectopic Gadd45a induces transcription activation and hypomethylation of premethylated Pol I reporter plasmids. Left panel: luciferase activity was monitored in lysates from HEK293T cells that were cotransfected with increasing amounts of pA-HA-hGadd45a and the premethylated reporter pHr-IRES-Luc-T4 (1 μg). Expression of HA-Gadd45a is presented in the western blot above. Middle and right panels: NIH 3T3 cells were transfected with a premethylated murine Pol I reporter (pMr131-BH) in the absence (−) or presence (+) of 2 μg of pA-HA-hGadd45a. Transcripts were monitored on northern blots and quantified (light bars). The methylation level of pMr131-BH was determined by HpaII digestion and qPCR (dark bars). Data are expressed as the mean ± SD of at least two independent experiments.
(D) Gadd45a is required to maintain active rRNA genes hypomethylated. Gadd45a was depleted from HEK293T cells by siRNA, and CpG methylation of rDNA was analyzed by HpaII digestion prior to qPCR (left panel). C, control siRNA (light bar); G45, Gadd45a-specific siRNA (dark bar). In parallel, rDNA transcription was monitored on northern blots (top panel). The bar diagram below shows pre-rRNA levels normalized to β-actin mRNA. Data are expressed as the mean ± SD.
(E) Bisulfite sequencing of rDNA in HEK293T cells transfected with control (siCtrl) or Gadd45a-specific siRNAs (siGadd45a). White and black circles represent unmethylated and methylated CpGs. The position of the outermost cytosines and the transcription start site (arrow) are marked.
(F) Gadd45a depletion leads to heterochromatin formation. Crosslinked chromatin from cells transfected with control (C) or Gadd45a-specific siRNA (G45) was immunoprecipitated with the indicated antibodies and analyzed by real-time PCR with primer pair B (see Figure 1A). The results of three independent experiments are plotted ± SD. Light and dark bars represent the association of the respective histones in control siRNA- and siGadd45a-treated cells.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Gadd45a Counteracts NoRC-Dependent rDNA Methylation
(A) Gadd45a counteracts TIP5-mediated repression of rDNA transcription. HEK293T cells were cotransfected with 2 μg of the reporter plasmid pHrP2-BH and the indicated amounts of expression vectors encoding Flag-HA-TIP5 (TIP5) and HA-Gadd45a (G45a). Reporter transcripts were analyzed on northern blots (NB, top panels) and normalized to β-actin mRNA (bar diagram). TIP5 and Gadd45a levels were monitored on immunoblots (WB, bottom panels).
(B) Gadd45a counteracts TIP5-mediated transcription repression of endogenous rRNA genes. HEK293T cells were transfected with expression vectors encoding HA-Gadd45a (G45a) and/or Flag-HA-TIP5 (TIP5). Pre-rRNA was analyzed on northern blots (NB) and normalized to β-actin mRNA (bar diagram). The immunoblots below (WB) show the level of TIP5 and Gadd45a.
(C) Gadd45a counteracts NoRC-mediated methylation. HEK293T cells were transfected like in (B), and DNA methylation was determined by HpaII digestion prior to real-time PCR. Error bars denote standard deviation (n = 3).
(D) Gadd45a and NoRC are associated with epigenetically distinct rDNA repeats. ChIP from HEK293T cells expressing HA-hGadd45a. Precipitated DNA was mock-digested or digested with HpaII, and the ratio of HpaII-resistant (dark bars) versus HpaII-sensitive rDNA repeats (light bars) was determined by qPCR using primer pair B. Error bars denote standard deviation (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Gadd45a-Dependent Demethylation Is Mediated by Nucleotide Excision Repair
(A) DNA damage decreases rDNA transcription. Cells were irradiated with UV light (20 J/m2, 4 hr recovery) or with MMS (1 mM, 2 hr), and pre-rRNA and β-actin mRNA were monitored on northern blots.
(B) DNA damage leads to increased recruitment of Gadd45a to rDNA. Cells overexpressing HA-Gadd45a were irradiated with UV light (20 J/m2, 4 hr recovery) or were treated with MMS (1 mM, 2 hr), rapamycin (rapa, 40 nM, 2 hr), or anisomycin (aniso, 50 mM, 2 hr). rDNA occupancy of Gadd45a in treated versus untreated cells was analyzed by ChIP. Error bars denote standard deviation (n = 3).
(C) DNA damage leads to hypomethylation of the rDNA promoter. HEK293T cells were subjected to UV-irradiation (20 J/m2, 3 hr recovery) or treated with MMS (1 mM, 2 hr), rapamycin (rapa, 40 nM, 2 hr), or anisomycin (aniso, 50 mM, 2 hr). rDNA methylation was analyzed by HpaII digestion followed by qPCR. Error bars denote standard deviation (n = 4).
(D) Demethylation requires components of the NER machinery. HEK293T cells were transfected with either control siRNA (Ctrl), Gadd45a (G45a)-, XPG-, XPA-, XPF- or TDG-specific siRNAs, and methylation of the rDNA promoter was determined by HpaII digestion prior to qPCR. Error bars denote standard deviation (n = 3).
(E) NER-specific agonists or antagonists affect rDNA methylation. HEK293T cells were treated with gemcitabine (gem, 66 nM, 48 h), UCN-01 (UCN, 300 nM, 4 h) or cisplatin (cis, 0.33 mM, 6 h) and rDNA methylation was determined by HpaII digestion and qPCR. Data are expressed as the mean ± SD.
(F) Damage-induced hypomethylation depends on Gadd45a. HEK293T cells were mock-treated (light bars) or treated with gemcitabine (dark bars) in the absence and presence of Gadd45a-specific siRNA (siG45a). rDNA methylation was determined by HpaII digestion and qPCR (left panel); the association of Pol I with the rDNA promoter was analyzed by ChIP (right panel). Data are normalized to untreated cells. Data are expressed as the mean ± SD.
(G) Demethylation requires the endonuclease activity of XPG. DNA from XPG-deficient fibroblasts (XPCS1RO) and from XPCS1RO cells that stably express ectopic XPG-eGFP or mutant XPG/E971A-eGFP was subjected to bisulfite sequencing. Numbers above the bars indicate the position of cytosine relative to the transcription start site. Bars represent the ratio of unmethylated (dark) versus methylated (light) cytosines at individual CpG residues. The numbers below indicate the percentage of methylated CpGs from sequencing 40 independent clones.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Gadd45a Occupancy Correlates with Pol I Binding to rDNA
(A) Actinomycin D treatment and UV radiation cause accumulation of aberrant Pol I transcripts. Northern blot showing rDNA transcripts from HEK293T cells treated with actinomycin D (AMD, 50 ng/ml, 2 hr) or exposed to UV light (20 J/m2, 4 hr recovery). Two sections of the same blot are shown, the upper one showing pre-rRNA, the lower one showing 200–500 nt aberrant transcripts.
(B) Inhibition of Pol I transcription leads to hypomethylation and accumulation of Pol I and Gadd45a at the promoter. ChIP experiments showing rDNA promoter occupancy of Pol I and Gadd45a in HEK293T cells that were treated with actinomycin D (AMD, 50 ng/ml, 2 hr) or cordycepin (Cord, 50 μM, 2 hr). In parallel, DNA methylation was analyzed by HpaII digestion followed by qPCR (right panel).
(C) MMS and actinomycin D induce DNA repair. NIH 3T3 cells arrested at G0/G1 by serum starvation were released for 1 hr in the presence of BrdU and subjected to MMS or AMD treatment for 2 hr. Incorporation of BrdU into the rDNA promoter and the c-fos promoter was analyzed by qPCR. Where indicated, error bars denote standard deviation (n=3).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Inhibition of Pol I Transcription Leads to Hypermethylation of the rDNA Promoter
(A) Pol I and Gadd45a colocalize at rDNA. In the first ChIP, HA-Gadd45a was precipitated and 10% of DNA was analyzed by PCR (−150/+37, lanes 1–5). In the re-ChIP, eluted chromatin was precipitated with antibodies against Pol I (α-Pol I, lanes 6–9).
(B) Knockout of TIF-IA abrogates Pol I transcription. TIF-IA was depleted from TIF-IAfl/fl MEFs expressing estrogen receptor-tagged Cre recombinase (Cre-ER). Cells were labeled with 5-fluorouridine (FUrd) for 10 min, fixed, and stained with anti-BrdU antibody (FUrd), and RNA was visualized by immunofluorescence.
(C) Knockout of TIF-IA leads to de novo methylation of the rDNA promoter. TIF-IAfl/fl MEFs were depleted from TIF-IA by Cre-recombinase (+Cre). pre-rRNA levels were analyzed by RT-PCR (light bars), and CpG methylation was analyzed by HpaII digestion and qPCR (dark bars). Data are represented as mean ± SD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
TAF12 Recruits Gadd45a to Promoters of Class I and Class II Genes
(A) Gadd45a interacts with the Pol I and Pol II transcription machineries. Bead-bound HA-tagged Gadd45a was incubated with nuclear extract from FT210 cells, and Gadd45a-associated proteins were separated by SDS-PAGE. Gadd45a, Pol I, Pol II, TFIIH, and actin were visualized on immunoblots using antibodies against the HA-epitope (Gadd45a), RPA116 (Pol I), the C-terminal domain of Pol II (Pol II), p98 (TFIIH), and actin.
(B) Gadd45a interacts with TAF12. GST-Gadd45a (G45) or GST (C) were incubated with in vitro-translated subunits of SL1/TIF-IB (TBP, TAF12, TAFI48, and TAFI110), and bound proteins (50%) were compared to 20% of input proteins (I).
(C) Gadd45a is associated with SL1/TIF-IB. A fractionated nuclear extract enriched in SL1/TIF-IB was supplemented with immunopurified His-Gadd45a (0.05 μg/μl), SL1/TIF-IB was precipitated with anti-TAFI95 antibodies, and coprecipitated Gadd45a was analyzed on immunoblots.
(D) Gadd45a is associated with TAF12 and TAFI110. HA-Gadd45a was precipitated from HEK293T cells, and coprecipitated Flag-TAF12 and Flag-TAFI110 were analyzed on immunoblots.
(E) Depletion of TAF12 causes DNA hypermethylation. Methylation of the rDNA promoter was monitored in HEK293T cells transfected with either control siRNA (siC) or TAF12-specific siRNAs (siTAF). The values are presented as relative HpaII resistance upon TAF12 knockdown normalized to control siRNA. The immunoblot shows the knockdown of TAF12. Error bars denote standard deviation (n = 2).
(F) Knockdown of TAF12 displaces Pol I and Gadd45a from the rDNA promoter. The bar diagram represents rDNA occupancy of Pol I and Gadd45a in mock- (C) or TAF12-depleted (TAF12) HEK293T cells. Error bars denote standard deviation (n = 3).
(G) Knockdown of Gadd45a and TAF12 leads to hypermethylation of Pol II gene promoters. DNA from mock-transfected (white bars), Gadd45a-depleted (gray bars), and TAF12-depleted cells (black bars) was digested with HpaII, and the promoter region of genes encoding MLH1, H2B, EF4, or U2snRNA was amplified and compared to undigested samples (n = 3). Error bars denote standard deviation (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions