SUPPLEMENTARY INFORMATION

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Hilary Reynolds
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doi: 10.1038/nature05473 SUPPLEMENTARY FIGURES SUPPLEMENTARY INFORMATION Supplementary Figure 1: Association of Runx2 with mitotic chromosomes Mitotic chromosome spreads were prepared for a.

1 doi: /nature05473 SUPPLEMENTARY FIGURES SUPPLEMENTARY INFORMATION Supplementary Figure 1: Association of Runx2 with mitotic chromosomes Mitotic chromosome spreads were prepared for a. Human Saos-2 Osteosarcoma cells, b. Mouse MC3T3 osteoblastic cells, or primary calvarial cells from mice homozygous for c. Wild-type Runx2 or d. the Runx2- C allele, and processed for immunofluorescence microscopy using antibodies directed against the Runx2 protein. The right most column of rows e. through i. shows the relative intensity of the image pixels of Runx2 and the other labeled antibody across the line scan that is shown in the overlay image. The Pearson s correlation coefficient, r was computed to compare the degree of colocalization between the two signals. Human chromosome showing localization of Runx2 with DAPI and DNaseI hypersensitive chromatin (row e, r=0.80), which is labeled by incorporation of FITC-dUTP by DNA Pol I on DNaseI nicked chromosome spreads. DAPI-stained chromosome spreads double labeled for Runx2 and K4-methylated Histone H3 on either human (row f, r=0.81) or mouse (row g, r=0.72) chromosomes reflect open chromatin structure. In contrast, Runx2 does not appreciably co-localize with K9-methylated Histone H3 on human chromosomes (row h, r=0.16), or S10-phosphorylated Histone H3 on mouse chromosomes (row i, r=-0.12) that reflect regions of condensed chromatin. 1

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 2: Colocalization of Runx2 and UBF1 on metaphase chromosome spreads. a-d.

2 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 2: Colocalization of Runx2 and UBF1 on metaphase chromosome spreads. a-d. Mitotic chromosome spreads were prepared for non-transformed human diploid MCF-10A cells and processed for immunofluorescence microscopy using DAPI stain for DNA and antibodies directed against the Runx2 protein (green) and UBF1 (red). Overlay image shows Runx2 and UBF1 colocalization on acrocentric chromosomes. 2

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplmentary Figure 3: Colocalization of Runx2 and UBF1 during mitosis.

3 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplmentary Figure 3: Colocalization of Runx2 and UBF1 during mitosis. Asynchronously growing Saos-2 and primary mouse calvarial cells (data not shown) were fixed and processed for in situ immunofluorescence microscopy using antibodies directed against Runx2 (green) and UBF1 (red). Cells in the stages of mitosis: Prophase, Metaphase, Anaphase, and Telophase were identified by DNA morphology as visualized by DAPI staining. Overlay images of Runx2, UBF1 and DAPI staining are shown. The inset region in overlay images is reflected below with Runx2/DAPI overlay and UBF1/DAPI overlay images. 3

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 4: Ultrastructural localization of Runx2 in nucleoli by immunoelectron gold labeling. a-c.

4 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 4: Ultrastructural localization of Runx2 in nucleoli by immunoelectron gold labeling. a-c. Proliferating Saos-2 cells were permeabilized in 0.5% Triton X-100 in cytoskeletal buffer before fixation in formaldehyde and staining with an anti- Runx2 polyclonal antibody and a second antibody coupled to 5 nm gold beads as described 1. Samples were postfixed in glutaraldehyde, embedded in Epon, sectioned, and sections were counterstained with uranyl acetate-lead citrate. The inset in a. outlining a nucleolus is shown in b. The inner nucleolar region that is surrounded by the inset in b. is also shown (c.). d,e. Nuclear matrix intermediate filament preparations of Saos-2 cells were stained with an anti-runx2 polyclonal antibody and a second antibody coupled to 5 nm gold beads and processed for EM as whole-mount preparations. d. An inner region of the nucleus is shown with a portion of a nucleolus. The inner nucleolar region that is surrounded by the inset in d. is shown in e. Arrowheads in c. and e. indicate 5 nm gold beads. N = Nucleus, Cy = Cytoplasm, Nu=Nucleolus, L = Nuclear Lamina. 1 Nickerson, J. A., He, D. C., Krochmalnic, G., & Penman, S. Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections. Proc. Natl. Acad. Sci. USA 87, (1990). 4

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 5: Runx2 resides at nucleolar sites of active RNA Pol I transcription. a-b.

of the RNA Pol II/III inhibitor α-amanitin, 50 μg/ml.

5 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 5: Runx2 resides at nucleolar sites of active RNA Pol I transcription. a-b. Asynchronously growing Saos-2 cells were subjected to in situ run-on transcription assays using BrUTP to label sites of transcription in the absence a. or presence b. of the RNA Pol II/III inhibitor α-amanitin, 50 μg/ml. Cells were subsequently fixed and processed for in situ immunofluorescence microscopy using antibodies directed against Runx2 (green) and BrUTP (red); DNA was visualized by DAPI staining (blue). Enlarged images of the inset regions are shown with white arrows indicating the regions of signal overlap between nucleolar Runx2 and BrUTP. These single cell images are representative of a larger population of cells examined. 5

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 6: Runx consensus elements within mouse and human rdna loci.

6 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 6: Runx consensus elements within mouse and human rdna loci. The location of Runx consensus elements, TGHGGT, within the human and mouse ribosomal DNA repeats are denoted by red diamonds, see Supplementary Data Excel File for actual locations. Primer locations for chromatin immunoprecipitation assays (ChIP) are shown in blue and primer sequences are outlined in Supplementary Table I. Number below primer positions is according to genomic sequences annotated in Mouse Accession Number BK and Human Accession Number U

doi: 10.1038/nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplemental Figure 7: Cell cycle dependent interactions of Runx2 and UBF1 with rdna loci in vivo. a-d.

7 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplemental Figure 7: Cell cycle dependent interactions of Runx2 and UBF1 with rdna loci in vivo. a-d. MC3T3 cells were synchronized in mitosis by a combination of nocodazole treatment and shake-off. Mitotic cells (0 Hrs) were taken directly for assays after shake-off. Cells in early G1 (2, 4, and 8 h) were obtained by washing and replating mitotic cells in fresh growth media. ChIP was carried out using antibodies directed against Runx2 (a), UBF1 (b), and normal IgG as a control. ChIP DNA was analyzed by qpcr for seven regions (primer mrdna1-7) of the mouse rdna repeat along with a control non-specific genomic DNA reaction for normalization (see Supplementary Table I for sequences). Data are shown as Runx2 or UBF1 versus IgG and normalized to non-specific genomic DNA. (c) Pre-rRNA synthesis was monitored at each time point by qpcr. (d) Cell synchronization was monitored by western blot of cell cycle proteins. The M/G1 transition was defined by monitoring Cyclin D1 levels. The G0/G1 transition was defined by monitoring the phosphorylation status of the Retinoblastoma protein. 7

8 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplemental Figure 7: Cell cycle dependent interactions of Runx2 and UBF1 with rdna loci in vivo. e-i. Cells were synchronized in G0 by serum starvation for 48 hrs. G0 synchronized cells were either processed for assays or restimulated with serum to obtain cells at the G0/G1 transition and the G1/S transition. At all time points in both synchronies, cells were harvested for protein, RNA, and chromatin immunoprecipitation assays. ChIP was carried out using antibodies directed against Runx2 (e), UBF1 (f), and normal IgG as a control. ChIP DNA was analyzed by qpcr for seven regions (primer mrdna1-7) of the mouse rdna repeat along with a control non-specific genomic DNA reaction for normalization (see Supplementary Table I for sequences). Data are shown as Runx2 or UBF1 versus IgG and normalized to non-specific genomic DNA. (g) Pre-rRNA synthesis was monitored at each time point by qpcr. (i) Cell synchronization was monitored by western blot of cell cycle proteins. The M/G1 transition was defined by monitoring Cyclin D1 levels. The G0/G1 transition was defined by monitoring the phosphorylation status of the Retinoblastoma protein. (h) S-phase was determined by monitoring DNA content by FACS analysis along with replication dependent histone H4/n expression by RT-qPCR. ChIP data reflecting Runx2 and UBF occupancy at each rdna primer set location were averaged across the M/G1 (0-8 hrs), G0/G1 (0-9 hrs), and G1/S transitions (12-18 hrs). To establish a standardized spatial occupancy profile for each cell cycle transition, the time averaged ChIP data were then standardized between the seven rdna primer sets. 8

9 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplemental Figure 7: Cell cycle dependent interactions of Runx2 and UBF1 with rdna loci in vivo. j. To illustrate Runx2 and UBF spatial occupancy in the context of the repetitive nature of the repeats, three sets of standardized spatial occupancy profiles are plotted in series. This data analysis synthesizes a multidimensional data set comprised of over 300 ChIP data points for both Runx2 and UBF. k. To determine the degree of spatial dynamics between cell cycle stages, the Pearson s correlation coefficient was computed comparing spatial occupancy profiles between each cell cycle transition. Pearson s correlation coefficients near one are consistent with a temporally static spatial profile. 9

10 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES Supplementary Figure 8: ChIP-Re-ChiP protocol. ChIP-re-ChIP assays were conducted on endogenous proteins in Saos-2 cells that were asynchronously growing or synchronized in mitosis. Primary chromatin immunoprecipitations were conducted using a monoclonal antibody directed against UBF1. Immunoprecipitated protein-dna complexes were eluted in a 10 mm DTT buffer, diluted 1:40 in ChIP lysis buffer and subjected to a second immunoprecipitation (re-chip) using a monoclonal antibody directed against UBF1, a rabbit polyclonal antibody directed against Runx2, a goat polyclonal antibody directed against p48, and normal IgG. 10

11 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTAL TABLE 1: Primer Sequences Name Sequence 5' to 3' Species Description hrdna1 F TGTCAGGCGTTCTCGTCTC R GAGAGCACGACGTCACCAC hrdna2 F GGATGCGTGCATTTATCAGA R GTTGATAGGGCAGACGTTCG hrdna3 F CGCCGGTGAAATACCACTAC R CCAGTCAAACTCCCCACCT hrdna4 F GCCTTATTTGAGTGGCTTCC R CAGATCGGCCAGCTTTTACT hrdna5 F AAGCTGGCCGATCTGAATAA R TTCCCAAGTCTGGTTGATCC hrdna6 F CTCAGCCTCCCAAGTAGCTG R GATCGAGACCATCCTGGCTA hrdna7 F AGGTGTCCGTGTCCGTGT R GGACAGCGTGTCAGCAATAA mrdna1 F GCTTGTTTCTCCCGATTGC R CGCGAACCACTGAGAAAAGT mrdna2 F GTCTCTCGGTCCCTTGTGAG R ACGGGTCAGTCAGAGGAGAG mrdna3 F CGCCGGTGAAATACCACTAC R CTGAGCTCGCCTTAGGACAC mrdna4 F ATCAGGAGGTCCCGCTAGTT R ACGGCTTGACATCCAAACTC mrdna5 F TCCTTCCTTCCTCCCTTTTC R AAGGTCACCCTGGCTTACAA mrdna6 F ACCCTCCTCTTCCACTGCTT R GGCACCCAAAAACGAAAGTA mrdna7 F GCGGTTTTCTTTCATTGACC R ACGACGCCTGGAAGTCATAC hpre-rrna F CCGCGCTCTACCTTACCTAC R GAGCGACCAAAGGAACCATA h28s F GAACTTTGAAGGCCGAAGTG R ATCTGAACCCGACTCCCTTT mpre-rrna F GCTTGTTTCTCCCGATTGC R CGCGAACCACTGAGAAAAGT m28s F GAACTTTGAAGGCCGAAGTG R ATCTGAACCCGACTCCCTTT mhistoneh4n F CCAGCTGGTGTTTCAGATTACA R ACCCTTGCCTAGACCCTTTC mbeta-actin F CAGCTTCTTTGCAGCTCCTT R CACGATGGAGGGGAATACAG hbeta-actin F CGTCTTCCCCTCCATCGT R GAAGGTGTGGTGCCAGATTT mmitcox F ACGAAATCAACAACCCCGTA R GGCAGAACGACTCGGTTATC MR170-BH F ATTCACTGGCCGTCGTTTTA R GGCCTCTTCGCTATTACGC Igγ Enhancer F TGGTGGGGCTGGACAGAGTGTTTC R GCCGATCAGAACCAGAACACC Phox (GP91) F CCAATGATTATTAGCCAATTTCTG ` R CATGGTGGCAGAGGTTGAATGT hcdc6-3 F CAGCGACTTCACTCTCCACA ` R CCTGGACGACAAGAGTGAAAC mcdk2 F ACAGCCGTGGATATCTGGAG ` R TTAGCATGGTGCTGGGTACA Accession Number Reference Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Human Ribosomal DNA Repeating Unit U13369 This work Human Ribosomal DNA Repeating Unit U13369 This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Ribosomal DNA Repeating Unit BK This work Mouse Replication Dependent Histone H4 Gene AY This work Mouse Cytoskeletal protein X03672 This work Human Cytoskeletal protein BC This work Human n/a Mouse Human Mitochondrial cytochrome c oxidase subunit II gene Mouse rdna promoter construct: unique puc9 transcribed sequence Intronic Enhancer upstream of the Igγ constant region CYTOCHROME b(558), BETA SUBUNIT AF This work n/a Budde A 1 V01524 Bergstrom 2 M66390 Khanna- Gupta A 3 Human CDC6 3 Prime UTR Region AY This work Mouse Cell Cycle Regulatory Kinase AJ This work

12 doi: /nature05473 SUPPLEMENTARY INFORMATION REFERENCES Budde, A. & Grummt, I. p53 represses ribosomal gene transcription. Oncogene 18, (1999). Bergstrom, D. A., et al. Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol. Cell 9, (2002). Khanna-Gupta, A., et al. Chromatin immunoprecipitation (ChIP) studies indicate a role for CCAAT enhancer binding proteins alpha and epsilon (C/EBP alpha and C/EBP epsilon ) and CDP/cut in myeloid maturation-induced lactoferrin gene expression. Blood 101, (2003)

13 doi: /nature05473 SUPPLEMENTARY INFORMATION LOCUS HSU13369 DEFINITION Human ribosomal DNA complete repeating unit. ACCESSION U13369 Sequence Start End 1 TGTGGT TATGGT TATGGT TGTGGT TGTGGT TATGGT TATGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TATGGT TATGGT

14 doi: /nature05473 SUPPLEMENTARY INFORMATION LOCUS BK DEFINITION TPA: Mus musculus ribosomal DNA, complete repeating unit. ACCESSION BK Sequence Start End 1 TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TATGGT TGTGGT TGTGGT TATGGT TATGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TATGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TGTGGT TATGGT TGTGGT TATGGT TATGGT TATGGT TATGGT TGTGGT TATGGT TGTGGT TGTGGT TATGGT TGTGGT TGTGGT TATGGT TGTGGT

15 doi: /nature05473 SUPPLEMENTARY INFORMATION 43 TATGGT TGTGGT TGTGGT TGTGGT TATGGT TGTGGT TGTGGT TATGGT TGTGGT TATGGT TGTGGT TATGGT TGTGGT TGTGGT TATGGT TGTGGT TATGGT TGTGGT

16 doi: /nature05473 SUPPLEMENTARY INFORMATION SUPPLEMENTARY PRIMARY DATA The six scans show individual parts of the original western blots used for the generation of Figure 4A. Co-immunoprecipitation assays were conducted with endogenous Runx2 and UBF1 proteins from the following osteoblastic cell lines: human SAOS-2 osteosarcoma cells (Scans #1 and 3), rat ROS17/2.8 osteosarcoma cells (Scans #2 and 4) and mouse MC3T3 calvarial osteoblasts (Scans #5 and 6). Samples in each case represent input (5% lysate; Lane 1), immunoprecipitate obtained using non-specific rabbit IgG control (3 microgram; Lane 2), rabbit polyclonal antibody against Runx2 (3 micrograms, M70 [Santa Cruz Biotechnology], Lane 3), and mouse monoclonal antibody against UBF1 (3 micrograms, Lane 4). Pre-stained protein MW markers of the original blot were aligned with the autoradiogram and indicated as dots. The arrow indicates the band of interest (i.e., UBF1 or Runx2) depending on the antibody used for probing each blot. Scans #1, #4 and #5 represent blots obtained with the UBF1 antibody and Scans #2, #3 and #6 were obtained with the Runx2 antibody. The autoradiograms in all blots show two prominent bands migrating at ~50 and ~25 kd, respectively, that represent the heavy and light chains of the immunoprecipitating antibodies. 16

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9/3/2009 DNA i DNA n euk euk yotes Organizatio Organ izatio n of o f gen ge e n tic Locati t on: In n ucleu e s material mater in e ial

9/3/2009 DNA i DNA n euk euk yotes Organizatio Organ izatio n of o f gen ge e n tic Locati t on: In n ucleu e s material mater in e ial DNA in eukaryotes Organization of genetic material in eukaryotes Location: In nucleus In mitochondria DNA in eukaryotes Nuclear DNA: Long, linear molecules; Chromatin chromosomes; 10% of DNA in genes,