The Treacher Collins Syndrome (TCOF1) Gene Product Is Involved in Ribosomal DNA Gene Transcription by Interacting with Upstream Binding Factor

The Treacher Collins syndrome (TCOF1) gene product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor Benigno C. Valdez*†, Dale Henning*, Rolando B. So*, Jill Dixon‡, and Michael J. Dixon‡ *Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; and ‡School of Biological Sciences and Department of Dental Medicine and Surgery, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 8, 2004 (received for review April 8, 2004) Treacher Collins syndrome (TCS) is an autosomal dominant disorder penetrance and severity of the craniofacial defects observed on characterized by an abnormality of craniofacial development that Tcof1ϩ/Ϫ mice (18). arises during early embryogenesis. TCS is caused by mutations in Despite several genetic studies of TCOF1, the cellular function the gene TCOF1, which encodes the nucleolar phosphoprotein of treacle remains unknown. The localization of treacle to the treacle. Even though the genetic alterations causing TCS have been nucleolus suggests a role in the production of rRNA. A recent uncovered, the mechanism underlying its pathogenesis and the report showed a physical interaction between treacle and function of treacle remain unknown. Here, we show that treacle is pNop56, a component of the ribonucleoprotein complex that involved in ribosomal DNA gene transcription by interacting with 2Ј-O-ribose methylates pre-rRNA (19). Here, we present evi- upstream binding factor (UBF). Immunofluorescence labeling dence that treacle is involved in mammalian ribosomal DNA shows treacle and UBF colocalize to specific nucleolar organizer (rDNA) gene transcription by interacting with upstream binding regions and cosegregate within nucleolar caps of actinomycin factor (UBF), a RNA polymerase I (RNA pol I) transcription D-treated HeLa cells. Biochemical analysis shows the association of factor (20–22). treacle and UBF with chromatin. Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the Materials and Methods two nucleolar phosphoproteins. Down-regulation of treacle ex- Cells and Antibodies. HeLa cells were grown in DMEM with 10% pression using specific short interfering RNA results in inhibition of FBS, penicillin G, and streptomycin sulfate at 37°C, 5% CO2. ribosomal DNA transcription and cell growth. A similar correlation Rabbit anti-treacle antibody (Ab 014) against amino acids 1–55 ؉/؊ is observed in Tcof mouse embryos that exhibit craniofacial of human treacle has been characterized (12). Other anti-treacle defects and growth retardation. Thus, treacle haploinsufficiency in antibodies were produced in rabbits by using recombinant TCS patients might result in abnormal development caused by polypeptides (amino acids 214–290 and 1166–1339) expressed in inadequate ribosomal RNA production in the prefusion neural folds Escherichia coli. Other antibodies included anti-Gu␣ (23), anti- during the early stages of embryogenesis. The elucidation of a nucleolin (24), anti-nucleophosmin (25), anti-UBF (Santa Cruz physiological function of treacle provides important information of Biotechnology), and anti-BrdUrd (Sigma). relevance to the molecular dissection of the biochemical pathology of TCS. Indirect Immunofluorescence. Cells grown on slides were analyzed by indirect immunofluorescence staining as described (26). Cells reacher Collins syndrome (TCS) is an autosomal dominant were examined with a Nikon Eclipse TE2000-U inverted micro- Tdisorder of craniofacial development. The structures af- scope equipped with a Coolsnap digital color camera. fected in TCS patients arise from the first and second branchial MEDICAL SCIENCES arches during early embryogenesis (1). Early studies resulted in Western Blot Analysis. Samples were boiled in Laemmli buffer for mapping of the TCS locus to chromosome 5q31-q34 (2–4). Using 3 min. Protein extracts were electrophoresed on 6% or 9% segregation analysis in affected families, the TCS gene, desig- polyacrylamide-SDS gels and blotted onto Immun-Blot poly(vi- nated TCOF1, was positionally cloned (5). Tcof1 heterozygous nylidene difluoride) membrane (Bio-Rad). Immunochemilumi- mice die perinatally as a result of severe craniofacial anomalies nescence was done by using the ECL-plus Western blotting arising from increased apoptosis in the prefusion neural folds, detection system (Amersham Pharmacia Biosciences). The where the highest level of Tcof1 expression is observed in WT membrane was incubated with one antibody followed by en- mice (6). hanced chemiluminescence to detect the antigen. The membrane Early reports showed that the TCOF1 gene is comprised of 26 was stripped and probed with another antibody. exons (7, 8). Recently, we discovered an additional exon between exons 6 and 7 (9). The encoded protein, treacle, has homology Analysis of Chromosome Spreads and Chromosome-Associated Pro- with Nopp140, a trafficking nucleolar phosphoprotein (10). teins. HeLa cells were blocked in mitosis with 0.1 ␮g͞ml colchi- Although treacle and Nopp140 share common characteristics, cine for 6 h. Chromosome spreads were prepared as described the inability of treacle to colocalize with Nopp140 to Cajal bodies (27). Chromosome-associated proteins were separated from suggests different and distinct functions (11). cytoplasmic proteins, and fractions were analyzed by Western The C-terminal region of treacle is important for localization blot as described above. to the nucleolus (12, 13). It was hypothesized that mutations in the TCOF1 gene (7, 14–17) could result in mislocalization of truncated proteins and, consequently, loss of treacle functions, This paper was submitted directly (Track II) to the PNAS office. suggesting that TCS results from treacle haploinsufficiency. No Abbreviations: TCS, Treacher Collins syndrome; UBF, upstream binding factor; rDNA, genotype–phenotype correlation has been observed, which ribosomal DNA; siRNA, short interfering RNA; BrUTP, bromouridine; RNA pol I, RNA might explain the wide clinical variability observed among TCS polymerase I. patients. This variability may be explained by a recent study that †To whom correspondence should be addressed. E-mail: [email protected]. demonstrated that genetic background has a major effect on the © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402492101 PNAS ͉ July 20, 2004 ͉ vol. 101 ͉ no. 29 ͉ 10709–10714 Downloaded by guest on October 2, 2021 Treatment with Actinomycin D. HeLa cells were grown overnight on RT-PCR product was cloned into the HindIII and BamHI sites slides, treated with 50 ng͞ml actinomycin D for2hat37°C, 5% of pBluescript vector (Stratagene). The construct was linearized 32 CO2, and analyzed by indirect immunofluorescence staining. with HindIII, and a P-labeled riboprobe was synthesized with a T7 Maxiscript kit (Ambion, Austin, TX). The labeled probe Preparation of GFP–Fusion Construct. The human treacle cDNA was was gel-purified and used for the RNase protection assay with an amplified by RT–PCR using BV1049 (5Ј-GGGGCGTGCA- RPA III kit (Ambion). GATCTCCGGCCGGCCGGGGGT-3Ј) and BV1050 (5Ј- A similar procedure was done to analyze the level of pre- GGTGCTGGTGGTACCGCTACAGTCTGCTCTGCTGT- rRNA in mouse embryos. BV1229 (5Ј-GGTACTGACAAGCT- CTTCTT-3Ј). The RT-PCR product was digested with BglII and TCCTTTCCCTATTAACACTAAAGGA-3Ј) and BV1230 (5Ј- KpnI and subcloned into the pEGFP-N1 vector (Clontech). The AGGCTGAAGGGATCCGAAATAAGGTGGCCCTCAAC- resulting clone was sequenced and used to transfect HeLa cells CACA-3Ј) were used to amplify nucleotides ϩ13 to ϩ320 of with Lipofectamine 2000 (Invitrogen) and analyzed 24–48 h mouse pre-rRNA. BV680 (5Ј-CATGCCATCAAGCTTCTGG- after transfection. ACCTGGCTGGC-3Ј) and BV1233 (5Ј-GGCTGGAAAG- GATCCTCAGGGCATCGGAACCGCTCG-3Ј) were used to Immunoprecipitation of Treacle Complex. The cDNA fragment that amplify nucleotides 558–778 of mouse ␤-actin. The amplified coded for amino acids 717-1488 of treacle was PCR-amplified products were cloned, and 32P-labeled probes were prepared as ͞ and subcloned into the BglII XhoI sites of pSG5-KF2M vector. described for the human pre-rRNA probe. HeLa cells were transfected for 48 h. Nuclei were isolated and resuspended in 10 mM Tris⅐HCl, pH 7.6͞1 mM EDTA͞400 mM 32P-Metabolic Labeling of Cells. HeLa cells were transfected with 40 NaCl͞10% glycerol͞0.5% Nonidet P-40͞5mMNaF͞1mM nM siRNA as described above. This transfection was repeated ͞ DTT 0.5 mM Na3VO4 with complete protease inhibitor mix after 24 h. Three days after the second transfection, cells were (Roche Applied Science). Nuclei were lysed by sonication and incubated in phosphate-free medium (Sigma) for 3.5 h. The centrifuged for 20 min at 18,000 ϫ g,4°C. Nuclear extract (1 mg) medium was replaced with fresh phosphate-free medium con- ␮ ͞ ϭ 32 was made to 0.5 ml with lysis buffer and mixed with 0.5 ml of taining 40 Ci ml (1 Ci 37 GBq) [ P] orthophosphate dilution buffer (10 mM Tris⅐HCl, pH 7.6͞1 mM EDTA͞20% (Amersham Pharmacia Biosciences), and 32P-labeled RNA was glycerol͞0.5% Nonidet P-40͞5mMNaF͞1mMDTT͞0.5 mM isolated and analyzed as described (28). ͞ ␮ ͞ Na3VO4 protease inhibitors). RNase A (0.2 g ul) and DNase I (0.5 units͞␮l) were added, kept on ice for 10–15 min, and then Bromouridine (BrUTP) Incorporation Assay. Transfected HeLa cells ͞ centrifuged at 10,000 ϫ g for 10 min. The supernatant was mixed were washed quickly with KHB solution (30 mM KCl 10 mM with anti-FLAG M2-agarose and tumbled overnight at 4°C. The Hepes, pH 7.4) at 37°C as described (29). The cells were then ␮ resin was washed in three consecutive steps: NET-gel buffer (50 overlaid with 150 l of KHB solution containing 10 mM BrUTP mM Tris⅐HCl, pH 7.5͞500 mM NaCl͞0.1% Nonidet P-40͞1mM and incubated at 37°C for 10 min. The BrUTP medium was EDTA͞0.25% gelatin), NET-gel buffer with 0.1% SDS, and a removed, and the cells were washed twice with growth medium ␮ final wash buffer (10 mM Tris⅐HCl, pH 7.6͞0.1% Nonidet P-40). and incubated in 300 l of growth medium at 37°C for 20 min.

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