1. Intergenic Transcripts Regulate the Epigenetic State of rRNA Genes
Christine Mayer, Kerstin-Maike Schmitz, Junwei Li, Ingrid Grummt, Raffaella Santoro 
Molecular Cell 
Volume 22, Issue 3, Pages (May 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Nucleolar Localization of NoRC Requires Interaction with RNA
(A) NoRC localization is sensitive to RNase A treatment. Permeabilized NIH3T3 cells were treated with 1 mg/ml RNase A, and localization of NoRC (anti-TIP5) and UBF was visualized by immunofluorescence.
(B) RNase A treatment impairs binding of NoRC to chromatin. Permeabilized NIH3T3 cells were treated with RNase A, crosslinked with formaldehyde, and subjected to ChIP with anti-TIP5 and anti-UBF antibodies.
(C) The TAM domain of TIP5 mediates RNA binding. Labeled MCS-RNA (10 fmol) was incubated with increasing amounts of the TIP5 derivatives depicted above. TIP5-RNA complexes were analyzed by EMSA.
(D) TIP51–598 binds RNA. Filter binding assays using 1 pmol of TIP51–598, 10 fmol radiolabeled MCS-RNA, and increasing amounts of either total cellular RNA, siRNA (21-mer), tRNA, unmethylated and methylated rDNA (−232 to +130), or a 68 nt oligonucleotide (ssDNA). Retained RNA/protein complexes were measured by scintillation counting. Data are duplicates of two independent experiments.
(E) TIP51–598 binds a subfraction of small nuclear RNA. Nuclear RNA was fractionated on a MonoQ column, and 5 ng of size-fractionated RNA was assayed in EMSAs for competition of TIP51–598 binding to labeled MCS-RNA.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Interaction with RNA Is Required for Association of NoRC with rDNA and Heterochromatin Formation
(A) RNA binding of wild-type and mutant TIP5. Increasing amounts of His-tagged wild-type TIP51–598 or the indicated mutants were incubated with labeled rDNA transcripts (from −143 to −39), and RNA/protein complexes were analyzed by EMSA.
(B) Wild-type and mutant TIP5 localize within nucleoli. U2OS cells overexpressing Flag-TIP51–732 and TIP51–732WY531/532 were immunostained with antibodies against the Flag epitope.
(C) The RNA binding activity of TIP5 is required for the association of NoRC with chromatin. Identical cell equivalents from chromatin bound (Chr.) and soluble (Sol.) proteins from HEK293T cells overexpressing Flag-tagged TIP5 and TIP5WY531/532 were analyzed on Western blots with anti- Flag and anti-Pol I antibodies.
(D) RNA binding activity of TIP5 is required for heterochromatin formation. ChIP showing the presence of specific heterochromatic marks at the rDNA promoter after overexpression of Flag-TIP5 (gray bars, wt) or Flag-TIP5WY531/532 (dark bars, WY). Crosslinked chromatin was precipitated with the indicated antibodies, and rDNA promoter occupancy was analyzed by qPCR. The data refer to mock-transfected NIH3T3 cells (light bars). Data are from three independent experiments. Error bars indicate ±SD.
(E) Transcriptional repression by NoRC does not require RNA binding. (Left panel) RNA from HEK293T cells overexpressing Flag-TIP5 (gray bar, wt) or Flag-TIP5WY531/532 (dark bar, WY) was analyzed on Northern blots using a riboprobe that is complementary to nucleotides 1–155 of human pre-rRNA. (Right panel) RNA from NIH3T3 cells harboring the reporter plasmid pMr131-BH and overexpressing wild-type (gray bar, wt) or mutant TIP5 (dark bar, WY) was analyzed on Northern blots using a reporter-specific riboprobe. The bar diagrams indicate the amount of transcripts normalized to β-actin mRNA.
(F) Interaction with RNA is not required for de novo DNA methylation. DNA from NIH3T3 cells harboring the reporter plasmid pMr131-BH and overexpressing wild-type (gray bar, wt) or mutant TIP5 (dark bar, WY) was digested with HpaII prior to PCR amplification. Data represent the ratio of HpaII-resistant rDNA to total rDNA and are normalized to mock-transfected cells (light bar).
(G) ChIP showing different binding of wild-type and mutant TIP5 to rDNA. HEK293T overexpressing HA-tagged wild-type TIP5 (wt) or TIP5WY531/532 (WY) was crosslinked with 1% formaldehyde (left panel) or with 5 mM DTBP followed by 1% formaldehyde (right panel). After precipitation with anti-HA-antibodies, coprecipitated DNA was analyzed by qPCR using promoter-specific primers.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The IGS Is Transcribed by Pol I
(A) IGS transcripts exist in vivo. The diagram shows part of the IGS and the 5′ end of the 45S pre-rRNA coding region. The initiation sites of 45S pre-RNA (+1) and spacer transcripts (−1997) are marked by arrows. Numbers refer to the position of nucleotides with respect to the transcription start site (+1). Cellular RNA from untreated cells (light gray) and cells treated for 3 hr with the Pol II inhibitor DRB, 50 μM (dark gray), was reverse transcribed into cDNA with hexamer primers and subjected to RT-qPCR using specific primer pairs that amplify defined regions of rDNA as indicated (A, −2378/−2175; B, −2175/−2005; C, −1994/−1905; D, −554/−447; E, −105/−1; F, +64/+130).
(B) ChIP showing association of the Pol I preinitiation complex with the spacer promoter and gene promoter. Crosslinked chromatin was precipitated with anti-RPA116 (Pol I), anti-TAF1110 (TIF-IB), and anti-UBF antibodies. Coprecipitated rDNA was analyzed by qPCR using the indicated primer pairs. Error bars indicate ±SD.
(C) TIP51–598 binds Pol I promoter transcripts with high affinity. In vitro transcripts corresponding to selected regions of the murine rDNA transcription unit (designated 1–9 in sense and 10–12 in antisense orientation) were used in EMSA to compete for binding of TIP51–598 to radiolabeled MCS-RNA. Total RNA (1 and 10 ng) was used as a control.
(D) IGS transcripts are present in the nucle(ol)ar fraction. cDNA was synthesized from nuclear and cytoplasmic RNA and analyzed by PCR using primers that amplify rDNA sequences from −105 to −1. Amplifications with 1 ng genomic DNA (g) served as a positive control.
(E) Transcripts that cover the rDNA promoter exist in vivo. Unfractionated RNA and RNA enriched for <500 nt RNAs were transcribed into cDNA using a primer that contains rDNA sequences from −1 to −20. The cDNA was subjected to PCR using the indicated primer pairs.
(F) Primer extension analysis. Unfractionated RNA (lane 1), size-fractionated (<500 nt) cellular RNA (lane 2), and RNA isolated from partially purified NoRC (lane 3) were reverse transcribed using the −20/−1 rDNA primer and linearly amplified in the presence of a 32P-labeled primer (−39/−21).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Ectopic IGS Transcripts Compete for Nucleolar Localization of NoRC
(A) Exogenous IGS transcripts displace NoRC from nucleoli. Permeabilized NIH3T3 cells were incubated with either size-fractionated (<500) nuclear or cytoplasmic RNA or in vitro synthesized promoter RNA (from −160 to +7) in sense and antisense orientation and immunostained with anti-TIP5 and anti-UBF antibodies.
(B) Ectopic promoter transcripts displace NoRC from nucleoli. NIH3T3 cells were transfected with pE3SPMr-142/-40 or the empty vector, and the localization of TIP5 and UBF was monitored by immunofluorescence. The inset shows a 2-fold longer exposure of one cell.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Intergenic Transcripts Are Associated with NoRC
(A) Enrichment of transcripts covering the rDNA promoter in cells overexpressing TIP5. (Left panel) Level of pre-rRNA in NIH3T3 and in 3T3/TIP5 cells. (Right panel) Level of promoter transcripts in NIH3T3 and 3T3/TIP5 cells. cDNA was synthesized from nuclear RNA; transcripts encompassing rDNA sequences from −105 to −1 were monitored by qPCR and normalized to GAPDH mRNA. A Western blot comparing expression of TIP5 in 3T3/TIP5 and NIH3T3 cells (3T3) is shown above. Error bars indicate ±SD.
(B) RNAi-mediated depletion of TIP5 reduces the level of promoter transcripts. (Left panel) The bar diagram shows relative pre-rRNA levels in 3T3/Tet-TER-TIP5 cells (−dox) and after 96 hr treatment with doxycycline (+dox) to induce expression of TIP5-specific shRNA. (Right panel) The level of promoter transcripts in uninduced (−dox) and induced (+dox) cells is shown. The Western blot above shows the knockdown of TIP5 in 3T3/Tet-TER-TIP5 cells after doxycycline-induced expression of TIP5-specific shRNA. Error bars indicate ±SD.
(C) NoRC is associated with promoter transcripts. (Left panel) TAP-tagged TIP5 (gray bar) and TIP5WY531/532 (dark bar) were immunopurified from HEK293T cells, and TIP5-associated RNA was analyzed by RT-qPCR. The amount of promoter transcripts was normalized to rRNA (from to +1435). (Right panel) Flag-tagged TIP5 was immunopurified from 3T3/TIP5 cells using anti-Flag antibodies (gray bar) or control antibodies (light bar). Bead bound promoter RNA was analyzed by RT-qPCR and normalized to rRNA (from +640 to +790).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
LNA-Mediated Knockdown of Promoter Transcripts Impairs NoRC Function
(A) TIP5 fails to bind LNA/RNA hybrids. EMSA showing binding of TIP5510–611 to IGS transcripts (−127/−39) but not to IGS transcripts that are annealed to antisense LNA-DNA gapmers. The electrophoretic mobility of IGS RNA and LNA/RNA hybrids is shown in lanes 1 and 2.
(B) Depletion of promoter transcripts by antisense LNA-DNA gapmers. NIH3T3 cells were transfected with control RNA (light bar, m) 50 nM LNA-DNA gapmers in sense (gray bar, s), and antisense orientation (dark bar, as). The amount of IGS transcripts was determined by RT-qPCR and normalized against GAPDH mRNA. Data are from three independent experiments. Error bars indicate ±SD.
(C) Depletion of promoter transcripts enhances rDNA transcription. 45S pre-rRNA was assayed in mock-transfected NIH3T3 cells (light bar, m) or cells transfected with 50 nM antisense LNA-DNA gapmers (dark bar, as). Bars were derived from Northern blots and represent 45S pre-rRNA levels normalized to β-actin mRNA levels. Data are from three independent experiments. Error bars indicate ±SD.
(D) LNA-mediated depletion of promoter transcripts decreases rDNA promoter methylation. DNA from NIH3T3 cells that were either mock transfected (light bar, m) or transfected with 50 nM antisense LNA-DNA gapmers (dark bar, as) was digested with HpaII prior to PCR amplification. Data represent the ratio of HpaII-resistant rDNA versus total rDNA, and are normalized to mock-transfected cells. Data are from five independent experiments. Error bars indicate ±SD.
(E) Depletion of IGS-promoter transcripts alters the chromatin structure at the rDNA promoter. ChIP assay monitoring histone modification at the rDNA promoter after LNA-mediated depletion of promoter transcripts (dark bars, as). The data refer to mock-transfected NIH3T3 cells (light bars, m).
(F) Depletion of promoter transcripts impairs nucleolar localization of NoRC. NIH3T3 cells were transfected with antisense LNA-DNA gapmers (50 nM), and the localization of TIP5 and UBF was visualized by indirect immunostaining. The inset shows a 3-fold longer exposure of one cell.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions