Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus

Human Molecular Genetics, 2015, Vol. 24, No. 24 6932–6943 doi: 10.1093/hmg/ddv397 Advance Access Publication Date: 23 September 2015 Original Article ORIGINAL ARTICLE Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus 1 Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, 1825 University Blvd., Birmingham, AL 35294, USA, 2Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957, USA, 3 Department of Cell Biology, Poznan University of Medical Sciences, Rokietnicka 5D, Poznan 60-806, Poland, 4 Division of Neurology and Pediatrics, Children’s Hospital of Philadelphia, Abramson Research Center Room 502, Philadelphia, PA 19104, USA, 5Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan 61-704, Poland and 6Institute of Human Genetics, Polish Academy of Science, Strzeszynska 32, Poznan 60-479, Poland *To whom correspondence should be addressed. Tel: +1 2059755320; Fax: +1 2059753335; Email: (M.N.); Tel: +1 2059755335; Email: (J.S.B.) Abstract Friedreich’s ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenineadenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAAinduced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus. Received: July 10, 2015. Revised and Accepted: September 21, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: 6932 Yanjie Li1, Yue Lu2, Urszula Polak2,3, Kevin Lin2, Jianjun Shen2, Jennifer Farmer4, Lauren Seyer4, Angela D. Bhalla1, Natalia Rozwadowska1,6, David R. Lynch4, Jill Sergesketter Butler1, * and Marek Napierala1,5, * Human Molecular Genetics, 2015, Vol. 24, No. 24 Introduction Using next-generation RNA sequencing, we demonstrated a remarkable variability in FXN expression within FRDA as well as control sample groups. Additionally, we showed using RNA-seq and chromatin immunoprecipitation (ChIP) analyses that the epigenetic silencing effect induced by the expanded GAA repeats is confined to the FXN locus and does not affect expression of upstream or downstream neighboring genes. Finally, analysis of FXN pre-mRNA expression between FRDA and control samples combined with ChIP analyses revealed a pronounced transcription elongation defect at the expanded GAA region. Results Frataxin expression and GAA repeat length in FRDA and control fibroblasts We first characterized 35 primary FRDA and control fibroblast lines. Sixteen FRDA lines were obtained from skin biopsies, as described in Materials and Methods. The remaining 2 FRDA lines along with 17 control lines were acquired from Coriell Cell Repositories. The FRDA cohort included 7 females and 11 males with a mean age of onset 17.7 years (4–41) and mean age of sampling 36.3 years (13–70) (Table 1). Clinical data were available for 16/ 18 FRDA patients and 50% of them (8/16) developed cardiomyopathy and hearing loss, and diabetes was confirmed in one patient (Table 1). The control group included nine females and eight males with average age at sampling of 30.1 years (11–64). To minimize variability in our molecular analyses, all fibroblast lines were cultured simultaneously, in the same batch of media to the same cell density. Molecular characterization of FRDA and control fibroblasts included the following: determination of the GAA repeat size by PCR with two different primer sets, analysis of the GAA interruption status using MboII digestion which recognizes and cleaves GAAGA sequence (28) and quantitative analyses of FXN expression using quantitative real-time (qRT)-PCR and western blot (Fig. 1 and Supplementary Material, Figs S1– S3). Results of the GAA repeat PCR showed bands corresponding to two GAA alleles in most cases. However, somatic instability of the expanded GAAs was observed in a few fibroblast lines (e.g. sample 4259, Fig. 1). In such instances, the two longest PCR products were considered parental GAA alleles. The number of GAAs found in FRDA fibroblast samples varied between 110 and 1470 repeats with the average size of the shorter allele (GAA1) being 454 GAAs and longer (GAA2) 898 GAAs (Table 1). No GAA expansions were found in the control lines, thus excluding the possibility of asymptomatic FRDA carriers being included in the control cohort (Supplementary Material, Fig. S1). All PCR reactions were conducted under conditions allowing for simultaneous amplification of short and expanded GAAs (Fig. 1A, lane CR-carrier). The results of MboII digestion consistently showed two bands corresponding to 206- and 242-bp flanking sequences, which remain after complete digestion of the repeat region (Fig. 1B), thus indicating lack of complex interruption of the GAA tracts at the FXN gene in the fibroblast cell lines used in this study. Analyses of FXN mRNA expression in FRDA samples using TaqMan qRT-PCR assays (Fig. 1C and D and Table 1) revealed a strong correlation (correlation coefficient r = −0.75) between the length of the shorter GAA1 allele and FXN mRNA levels (Fig. 1C). FXN mRNA expression as determined by qRT-PCR was normalized to the average FXN expression in all 17 control cell lines. As expected, correlation of FXN expression with the number of repeats in GAA2 was much weaker (r = −0.54 to −0.64; Supplementary Material, Fig. S2A and B). Similarly, steady-state levels of mature frataxin protein correlated very well with GAA1 size (r = −0.75) Friedreich’s ataxia (FRDA, FA, OMIM (...truncated)