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Identification of Chromatin Marks at TERRA Promoter and Encoding Biochemical and Biophysical Research Communications xxx (2015) 1e6 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Identification of chromatin marks at TERRA promoter and encoding region * Yutaka Negishi a, b, Hideya Kawaji a, b, c, Aki Minoda a, , Kengo Usui a a Division of Genomic Technologies, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan b Department of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan c Preventive Medicine and Diagnosis Innovation Program, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan article info abstract Article history: TERRA is a long non-coding RNA that is essential for telomere integrity. Although it is transcribed from Received 24 September 2015 subtelomeres and telomeres, how it is expressed in heterochromatic region is currently unknown. In this Accepted 30 September 2015 study, we focused our analysis on TERRA-encoding region TelBam3.4 and TelBam3.4-like sequences, and Available online xxx determined their transcription start sites, as well as enrichment of RNA polymerase II and histone modifications. We found that H3K4me3 and H3K9me3 are present at TERRA promoters, whereas Keywords: H3K27ac and H3K9me3 are present at telomeric repeats. Consistently, we show that presence of active TERRA histone modifications H3K4me3 and H3K27ac are correlated to TERRA expression. These results mark an Telomere Chromatin important step towards understanding telomere maintenance and transcription. © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction essential for telomere integrity and loss of histone methyl- transferase Suv3-9h or Suv4-20h cause aberration of telomere Histone modifications play a fundamental role in gene regula- length regulation and recombination frequency [10,11]. Since tion. Characteristic patterns of histone modifications are observed telomeres and its adjacent regions subtelomeres are enriched with at different genomic regions. For example, histone H3 acetylation, transcriptionally silent histone modifications, it had been believed H3K4me3 and H3K4me2 are associated at highly expressed gene for a long time that there is no transcription at subtelomeres or promoters and are involved in transcription initiation [1e3]. telomeres. Recent studies, however, showed that long non-coding H3K36me3 and H3K79me2 are associated with gene bodies of RNA called TERRA is transcribed from subtelomere to telomere of actively transcribed genes and support transcription elongation, as most or all chromosome ends, containing UUAGGG repeats [12,13], well as repressing intragenic transcription initiation [4]. In contrast, and that they are required for heterochromatin formation at telo- H3K9me3, H3K27me3 and H4K20me3 are known as transcrip- meres [14]. Since telomeres and subtelomeres are enriched with tionally silent marks and are observed at inactive genes [5]. transcriptionally silent marks, an intriguing and unanswered Although these active and silent histone modifications are normally question is how TERRA expression is regulated. mutually exclusive, they are found together in some cases, for Terminal sequences on human chromosomes X/Y (Xq/Yq) were example, H3K4me3 and H3K27me3/H3K9me3 at developmental originally identified by sequencing of a clone called TelBam3.4, genes in mouse embryonic cells, and are referred to as bivalent which has been particularly studied for TERRA expression mecha- chromatin [6,7]. nism, since it contains both telomere TTAGGG repeats and se- Telomeres are terminal regions of chromosomes that are quences ~3.5 Kbp upstream of them containing unique sequences essential to prevent chromosome fusion and DNA erosion during (GENBANK accretion#: M57752.1, [15]). It has been shown that replication, thus essential for genome stability [8]. They are het- DNA methylation of a CpG island in TelBam3.4 negatively regulates erochromatic, consist of many TTAGGG repeats, and are enriched TERRA expression and the transcription factor CTCF is located up- for H3K9me3 and H4K20me3 [9]. These histone modifications are stream of TTAGGG repeats that is required for activating TERRA expression [16,17]. However, it is still unknown what chromatin marks are present and required for TERRA expression. The repeti- * Corresponding author. tive sequence nature of subtelomeres and telomeres pose diffi- E-mail addresses: [email protected] (Y. Negishi), [email protected] culties in chromatin marks analysis since it is difficult to align the (H. Kawaji), [email protected] (A. Minoda), [email protected] (K. Usui). http://dx.doi.org/10.1016/j.bbrc.2015.09.176 0006-291X/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Y. Negishi, et al., Identification of chromatin marks at TERRA promoter and encoding region, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.176 2 Y. Negishi et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6 reads to repetitive sequences unambiguously. 100 bp downstream from the most downstream active TSS Here, by generating a custom reference genome, we focused our observed by CAGE (2660e3060 bp on TelBam3.4). Pre-telomere analysis on TERRA-encoding regions on several chromosome arms was defined as a region between TERRA promoter and telomere altogether. We identified precise TSSs1 of TERRA in the CpG island, (3060e3400 bp). Telomere region in TelBam3.4 that was defined and show TERRA promoters are enriched with chromatin marks for previously (3400e4408 bp) [17] was further divided into telomeric active promoters. An active transcription mark H3K27ac is also repeat-poor and -rich regions based on TTAGGG repeat density present at the telomeric repeats. Finally, we show that TERRA (3400e3839 bp and 3839e4408 bp, respectively). For euchromatic expression is positively correlated with these active histone mod- genes, promoters were defined as 300 bp upstream and 100 bp ifications. These results will contribute towards understanding the downstream from TSSs, and gene bodies were defined as 100 bp regulation of TERRA expression and thus telomere maintenance. downstream of TSSs to transcription terminal sites of GENCODE v19 gene model. Transcription activity levels of genes were classified 2. Materials & methods based on gene expression measured by RNA-Seq into active (RPKM 1), weak (0 < RPKM < 1) and inactive promoter 2.1. Generation of a custom reference genome (RPKM ¼ 0) in K562 cells. Alpha satellite repeat and Satellite III repeat were determined based on RepeatMasker track in the UCSC TelBam3.4 sequence was obtained from NCBI (GenBank ID: Genome Browser [25]. M57752.1). TelBam3.4-like sequences were identified by running BLAT implemented in the UCSC genome browser with TelBam3.4 2.5. ChIP-Seq analysis and enrichment calculation sequence as query against the human genome hg19. Top seven TelBam3.4-like regions found in hg19 by BLAT were masked in the ChIP-Seq data were mapped to the custom reference genome by hg19 assembly with ‘N’ by bedtools [18]. TelBam3.4 and masked BWA-backtrack 0.7.9 with the default setting and visualized with hg19 were combined to generate the custom reference genome. IGV. Enrichments of histone modifications were calculated as pro- portion of the number of reads aligned to the target region, which 2.2. NGS2 datasets was subsequently normalized by that of input. The P-value was calculated with Fisher's exact test. All analyzed datasets of ChIP-Seq3, RNA-Seq and CAGE4,were obtained from ENCODE (Supplementary Table 1), with the excep- 2.6. ChIP-Seq for H3K27ac_long tion of the H3K27ac_long, which was generated for this work. ChIP was performed as described previously [25] with following fi  6 fi 2.3. Analysis of transcriptome data modi cations: (i) 6 10 cells were xed by 1% formaldehyde for 5 min in PBS, (ii) LB3 was substituted with SDS lysis buffer (50 mM e Poly(A)þ and whole cell fractions datasets were used for CAGE Tris HCl, pH 8.0, 1% SDS, 10 mM EDTA and Complete Protease In- and RNA-Seq (see Supplementary Table 1 for the list of cells). hibitor Cocktail (Roche, Basel, Switzerland)), (iii) chromatin was e m For CAGE data, linker and EcoP15i recognition site (CAGCAG) in sheared with Covaris S220 to 200 500 bp, (iv) 6 g of anti- CAGE reads were trimmed with FASTX-toolkit (http://hannonlab. H3K27ac (Cosmo bio, Tokyo, Japan; MABI0309) antibody was cshl.edu/fastx_toolkit/). Trimmed CAGE reads were compared used for immunoprecipitation, and (v) washing after antibody with human ribosomal DNA sequences by rRNAdust program and binding were performed with Wash buffer I twice and Wash buffer the rRNA-derived sequences were discarded in MOIRAI system [19]. II three times, with no Wash buffer III. ™ Remaining reads were aligned to the custom reference genome by Sequencing libraries were prepared by Mondrian SP system ® þ BWA-backtrack 0.7.9 with default setting [20]. Frequencies of 50- with Ovation SP Ultralow DR Multiplex System (NuGEN, CA, fi end of CAGE read alignments are counted and displayed along the USA) from 2 ng DNA with 11 cycles of PCR ampli cation, which TelBam3.4 sequences by using
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