Figure 1.
Basonuclin interacts with rDNA promoter in vivo.
A, Western analyses of immunoprecipitated proteins from HaCaT cell lysate. The IP antibodies are shown above the Western blot; B, anti-basonuclin (alpha-hB34), U, anti-UBF, W, anti-WT protein, and G, IgG from a naive rabbit. The detecting antibodies are indicated on the left. The target proteins were monitored in both the pellet and supernatant. Note that no cross-reactivity was observed. B, The resolution of the ChIP assay was assessed by a Southern analysis. A primer was ligated to the immunoprecipitated DNA, which was then amplified by PCR. The amplified DNA was separated by agarose electrophoresis and visualized by ethidium bromide (EtBr). The DNA was then transferred to a nylon membrane and probed with an rDNA promoter probe (Southern). The chromatin immunoprecipitation (ChIP) antibodies are indicated above the gel by letters described in (A). A DNA size marker is shown on the left. The DNA fragments detected by the probe are indicated with an arrow on the right. C, Basonuclin's association with three regions of rDNA was investigated with ChIP-PCR. The top panel depicts a generic rDNA transcription unit. Indicated are the promoters, the rRNA coding sequences (18S and 28S), the external and internal transcribed spacers (ETS and ITS) as well as the intergenic spacer (IGS). Regions tested by PCR are indicated with a (*). Twice-ChIP-precipitated DNAs were used as template for PCR, whose products were analyzed by electrophoresis (lower panel). The ChIP antibodies are indicated above the gel (B, U, G, as in A, Ip, Input DNA). PCR specificity is shown on the right. D, Basonuclin level in HaCaT cells cultured in the presence (+) and absence (-) of serum. Basonuclin was immunoprecipitated from cell lysate and analyzed by Western blot. The precipitation antibodies are indicated above the gel image and the Western detecting antibodies on the left. E, The association of basonuclin and UBF to rDNA promoter in the presence and absence of serum. ChIP-precipitated DNA was used as templates for PCR detection of the rDNA promoter. ChIP-antibodies are listed to the left of gel image.
Figure 2.
Inhibition of basonuclin expression via siRNA reduces pre-rRNA level in the HaCaT cells.
A, HaCaT cells were transfected with siRNA and controls. b, siBnc1 targeting siRNA, c, control siRNA, f, mock transfection (Lipofectamin only). Basonuclin mRNA and protein levels were monitored by PCR and Western blot at four post-transfection time points, 12, 24, 36 and 48 hours. No difference among the experimental groups was detected at 0 h (not shown). A beta-actin RT-PCR served as a control for the quantity and quality (integrity) of RNA. A protein band detected by Brilliant Blue R (B.B.R) was chosen as a loading control because of its consistent quantity in cells receiving different treatments. The protein levels shown in the Western blot were quantified in relation to the loading control (histogram). B, Immunocytochemical staining of transfected cells. HaCaT cells were cultured in chamber slides and transfected with siBnc1-s and siCb-s. Cells were fixed at 48 h post-transfection and stained with anti-basonuclin (anti-Bnc1) and anti-UBF antibodies simultaneously. Microscopic photographs in each column represent the same field, visualized by different secondary antibodies, basonuclin, red (cy5), UBF (FITC) and DNA (DAPI). C, Effect of basonuclin siRNA on the level of 47S pre-rRNA. 47S pre-rRNA was measured by RT-PCR or Northern analysis, with beta-actin and mature rRNA as references, respectively. HaCaT cells were seeded at 1.5×105 cell/35 mm dish and transfected after cells were attached. RNAs were prepared at post-transfection 12, 24, 36 and 48 hours. The treatment groups b, c, and f are as described in (A). The results of Northern analysis was quantified in relation to methylene-green-stained mature rRNA (histogram), n = 3. Note that a reduction of 47S pre-rRNA level was seen only at 48 h post-transfection. M.G., methylene green. D, The effect of basonuclin knock-down could be seen only at high cell density. 47S pre-rRNA level was monitored by Northern analysis. HaCaT cells were seeded at densities: low, 2×104, medium, 5×104 (performed once hence not shown) and high, 1.5×105 and RNA harvested at 48 h post-transfection. The relative level of 47S pre-rRNA to mature rRNA was quantified (histogram) (n = 2). Mock transfection was not performed in every experiment, hence its omission from the quantitative analysis.
Figure 3.
Basonuclin is associated with RPA194 and RPA116, subunits of Pol I.
A, Immunoprecipitations detected a basonuclin-RPA194/RPA116 complex. Immunoprecipitations (IP) were performed with extract of HaCaT cells cultured to a high density and analyzed by Western blots. The IP antibodies are indicated above the gel image and that used for Western blots on the left. B, Basonuclin-deficiency reduces the association of RPA194 and rDNA promoter. Basonuclin was knocked-down in HaCaT cells by siRNA and the cells were analyzed by Western for protein levels and by ChIP for association of various factors to rDNA promoter. The Western (upper panel) showed that basonuclin level was reduced to below detection by treatment of siBnc1-s, but the level of RPA194 and UBF was not affected. The lower panel shows the results of PCR analysis of the amount of rDNA promoter precipitated by ChIP. The siRNA reagents the same as indicated in the upper panel. The Western and ChIP antibodies are indicated on the left. C, A quantification of rDNA promoter precipitated by anti-RPA194 in the presence and absence of basonuclin. Shown are qPCR results normalized to the rDNA quantity in the presence of basonuclin. An asterisk indicates p<0.05 (n = 3).
Figure 4.
Basonuclin-associated rDNA promoter is hypomethylated.
The DNA methylation status of basonuclin-associated rDNA promoter was analyzed by the HpaII/MspI sensitivity assay (A) and by bisulfite sequencing (B). A, A PCR analysis of the integrity of the rDNA promoter after HpaII or MspI digestion. The source of PCR templates is indicated above the gel image, and the treatment received by the templates, below. c, control template (genomic DNA), w, templates were incubated in water, b, in restriction buffer, H, with HpaII, M, with MspI. B, Depicted on top is the region of rDNA amplified by the PCR shown in A. Transcription start site is indicated as a bent arrow and the cis-elements are depicted as gray segments. HpaII/MspI sites are shown as banners and CpG sites, diamond-headed pins. Cytosine at position −132 is marked with a red hallo. The regions of basonuclin and UBF DNase I footprints are indicated by color-coded bars above the DNA. Listed below are DNA sequences from the region in between −180 to −126. The reference (U13369) and HaCaT rDNA promoter consensus sequences (the first two lines, respectively) are shown as they are sequenced after bisulfite treatment; i.e., all CpGs (in red) are assumed to be methylated and the rest of the Cs are converted to Ts due to the bisulfite treatment. The rest nine sequences are actual bisulfite sequencing data from five UBF-associated promoters and four basonuclin-associated promoters. The presence of a T (in blue) in the CpG position signifies that the CpG was not methylated.
Figure 5.
Single nucleotide polymorphism (SNP) in HaCaT rDNA promoters.
A, A consensus DNA sequence of HaCaT cell rDNA promoters. The consensus (in bold letters) was deduced from the sequence of 181 HaCaT rDNA promoter clones, which were obtained from PCR-amplified rDNA promoters. Twenty-one of the clones were obtained directly from genomic DNA, the rest were isolated from ChIP DNA precipitated by anti-basonuclin (49 clones), anti-UBF (44 clones), anti-RFA194 in the presence (44 clones) and absence of basonuclin (23 clones). A human rDNA promoter sequence (U13369) is listed below the HaCaT consensus. The two sequences are aligned according to their transcription start site (+1). B, A tabulation of SNPs in various ChIP groups. A generic rDNA promoter is depicted at the top with transcription start site (bent arrow), and the cis elements (dark gray boxes) indicated. UCE, up-stream control element and CORE, the core element. Nucleotide coordinates are shown below the DNA. The three basonuclin binding sites (light gray boxes labeled A, B, C) are indicated. SNPs are depicted as dots. The horizontal position of each dot indicates its location in the rDNA promoter, and its vertical position, the ChIP groups as indicated on the left. Input, a collection of randomly picked genomic rDNA promoters, Bnc1-, UBF- and RPA194- (Pol I) associated promoters; Pol I/siBnc1, RPA194-associated promoters in basonuclin-deficient cells. Not all clones contained SNP and the percentage of SNP-containing clones in each ChIP group is shown by a histogram on the right. For a statistical analysis of the SNP frequency, see Table 1. C, For analyzing the regional SNP variations, the promoter is divided into seven 50-base non-overlapping regions, which were named alphabetically, as indicated at the bottom of (B), a, −250 to −201, b, 200 to 149, etc. The regional SNP frequencies are plotted for the input and three ChIP groups, RPA194, UBF and Bnc1. Statistically significant differences are indicated (*, p<0.05).
Table 1.
P values of pair-wise comparison of SNP frequency in rDNA promoters obtained from ChIP assays.
Figure 6.
A comparison of the local SNP frequencies in rDNA promoters.
A, Depicted is a generic rDNA promoter, to which graphs in B and C are aligned. B, The frequency of SNP was scored by scanning the promoter sequence with a 50-base window. The scores are expressed as the ratio of SNP/(SNP+non-SNP). Each SNP score is aligned to A, using the center nucleotide of each window. Shown are SNP scores of three promoter sets: Bnc1, UBF and the input. An arrow indicates the low SNP frequency near the transcription start site in basonuclin- and UBF-associated promoters, but not in the input. In C, a series of pair-wise chi-square tests were performed on the corresponding SNP frequencies of basonuclin- and UBF-associated promoters. The resulting p values are plotted in alignment with A and B. As a reference, the positions of cis-elements as well as basonuclin binding sites are shown at the bottom of panel B.
Figure 7.
A statistical pair-wise comparison of local SNP frequency between the randomly picked genomic rDNA promoter and the promoters associated with basonuclin, UBF, or RPA194.
A, a generic rDNA promoter, to which all the other panels (B, C, D) are aligned. The notations on the rDNA promoter are as described in Fig. 5A. In panel B, C, D, p values were calculated by a chi-square test and plotted. The notation and graphic depictions are as described in Fig. 5B. B, input vs. basonuclin, C, input vs. UBF, D, input vs. RPA194.
Figure 8.
A comparison of RPA194-associated promoters in the presence and absence of basonuclin.
A, A generic rDNA promoter is depicted as described in Fig. 5. B, SNP frequencies of RPA194-associated promoters are plotted as described in Fig. 5. Arrows show the region as well as the direction of changes caused by basonuclin-deficiency. C, Pair-wise chi-square tests on SNP frequencies shown in B were performed. The resulting p values are plotted in alignment with panels in A and B. The notations are as described in Fig. 4 and 5.