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Reciprocal Monoallelic Expression of ASAR Lncrna Genes Controls Replication Timing of Human Chromosome 6

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Reciprocal Monoallelic Expression of ASAR Lncrna Genes Controls Replication Timing of Human Chromosome 6 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 1 Reciprocal monoallelic expression of ASAR lncRNA genes 2 controls replication timing of human chromosome 6. 3 4 Michael Heskett1, Leslie G. Smith2, Paul Spellman1, and Mathew J. Thayer2* 5 6 1Department of Chemical Physiology and Biochemistry 7 Oregon Health & Science University 8 3181 S. W. Sam Jackson Park Road 9 Portland, Oregon 97239, USA 10 11 2Department of Chemical Physiology and Biochemistry 12 Oregon Health & Science University 13 3181 S. W. Sam Jackson Park Road 14 Portland, Oregon 97239, USA 15 16 *Corresponding author. 17 ORCID: 0000-0001-6483-1661 18 TEL: (503) 494-2447 19 FAX: (503) 494-7368 20 Email:[email protected] 21 22 Running Title: ASAR lincRNA genes on chromosome 6 23 Key Words: Replication timing, cis-acting element, non-coding RNA 1 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 24 Abstract 25 DNA replication occurs on mammalian chromosomes in a cell-type distinctive temporal 26 order known as the replication timing program. We previously found that disruption of the 27 noncanonical lncRNA genes ASAR6 and ASAR15 results in delayed replication timing 28 and delayed mitotic chromosome condensation of human chromosomes 6 and 15, 29 respectively. ASAR6 and ASAR15 display random monoallelic expression, and display 30 asynchronous replication between alleles that is coordinated with other random 31 monoallelic genes on their respective chromosomes. Disruption of the expressed allele, 32 but not the silent allele, of ASAR6 leads to delayed replication, activation of the previously 33 silent alleles of linked monoallelic genes, and structural instability of human chromosome 34 6. In this report, we describe a second lncRNA gene (ASAR6-141) on human 35 chromosome 6 that when disrupted results in delayed replication timing in cis. ASAR6- 36 141 is subject to random monoallelic expression and asynchronous replication, and is 37 expressed from the opposite chromosome 6 homolog as ASAR6. ASAR6-141 RNA, like 38 ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the 39 chromosome territory where it is transcribed. Three classes of cis-acting elements control 40 proper chromosome function in mammals: origins of replication, centromeres; and 41 telomeres, which are responsible for replication, segregation and stability of all 42 chromosomes. Our work supports a fourth type of essential chromosomal element, the 43 “Inactivation/Stability Center”, which expresses ASAR lncRNAs responsible for proper 44 replication timing, monoallelic expression, and structural stability of each chromosome. 45 2 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 46 Introduction 47 Numerous reports over the past 50+ years have described an abnormal DNA replication 48 phenotype affecting individual chromosomes in mitotic preparations from mammalian 49 cells [reviewed in (Thayer 2012)]. For example, we found that certain tumor derived 50 chromosome translocations display a delay in replication timing (DRT) that is 51 characterized by a >3 hour delay in the initiation and completion of DNA synthesis along 52 the entire length of individual chromosomes (Smith et al. 2001). Chromosomes with DRT 53 also display a delay in mitotic chromosome condensation (DMC), which is characterized 54 by an under-condensed appearance during mitosis and a concomitant delay in the mitotic 55 phosphorylation of histone H3 (Smith et al. 2001; Chang et al. 2007). We also found that 56 ~5% of chromosome translocations induced by exposing human or mouse cells to 57 ionizing radiation (IR) display DRT/DMC (Breger et al. 2004). To characterize the 58 DRT/DMC phenotype further, we developed a Cre/loxP system that allowed us to create 59 chromosome translocations in a precise and controllable manner (Breger et al. 2004; 60 Breger et al. 2005). Using this Cre/loxP system, we carried out a screen in human cells 61 designed to identify loxP integration sites that generate translocated chromosomes with 62 DRT/DMC (Breger et al. 2004; Breger et al. 2005; Stoffregen et al. 2011; Donley et al. 63 2015). We found that ~5% of Cre/loxP induced translocations display DRT/DMC (Breger 64 et al. 2005). Therefore, ~5% of translocations induced by two different mechanisms (IR 65 or Cre/loxP) result in DRT/DMC. 66 Our Cre/loxP screen identified five cell lines that generate balanced translocations, 67 affecting eight different autosomes, all displaying DRT/DMC (Breger et al. 2005). 68 Characterization of two of these translocations identified discrete cis-acting loci that when 3 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 69 disrupted result in DRT/DMC on human chromosomes 6 or 15 (Stoffregen et al. 2011; 70 Donley et al. 2015). Molecular examination of the disrupted loci identified two lncRNA 71 genes, which we named ASynchronous replication and Autosomal RNA on chromosome 72 6 (ASAR6) and on chromosome 15 (ASAR15) (Stoffregen et al. 2011; Donley et al. 2015). 73 These studies defined the first cis-acting loci that control replication timing, monoallelic 74 gene expression, and structural stability of individual human autosomes (Stoffregen et al. 75 2011; Donley et al. 2015). 76 The vast majority of genes on mammalian autosomes are expressed from both alleles. 77 However, some autosomal genes are expressed preferentially from only one allele, 78 achieving a state of “autosome pair non-equivalence” (Singh et al. 2003; Ensminger and 79 Chess 2004). The most extreme form of differential allelic expression is often referred to 80 as monoallelic expression, where a single allele is expressed exclusively [reviewed in 81 (Gendrel et al. 2016)]. Differential allelic expression can arise from distinct mechanisms. 82 For example, variation in gene expression can be heritable and has been mapped to the 83 genomes of humans and model organisms as expression quantitative trait loci [eQTL; 84 (Petretto et al. 2006)]. eQTL are genetic loci where sequence variation is associated with 85 differential expression of one or more genes. The consequences of eQTL can be 86 observed either in cis or in trans (Bryois et al. 2014; Signor and Nuzhdin 2018). In 87 contrast, differential allelic expression can also occur in the absence of DNA sequence 88 polymorphisms and is connected to situations where there is a “programmed” 89 requirement to regulate gene dosage or to provide exquisite specificity [reviewed in 90 (Alexander et al. 2007; Li et al. 2012; Lin et al. 2012; Gendrel et al. 2014)]. One well 91 established form of programmed monoallelic expression occurs in a parent of origin 4 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 92 specific manner, and is known as genomic imprinting [reviewed in (Bartolomei 2009)]. In 93 addition, monoallelic expression occurring in a random manner was observed from as 94 many as 8% of autosomal genes (Gimelbrant et al. 2007; Chess 2012). One unusual 95 characteristic of all programmed monoallelic genes is asynchronous replication between 96 alleles (Mostoslavsky et al. 2001; Singh et al. 2003; Ensminger and Chess 2004; 97 Schlesinger et al. 2009). This asynchronous replication is present in tissues where the 98 genes are not transcribed, indicating that asynchrony is not dependent on transcription 99 (Singh et al. 2003; Ensminger and Chess 2004; Donley et al. 2013; Donley et al. 2015). 100 Furthermore, asynchronous replication of random monoallelic genes is coordinated with 101 other random monoallelic genes on the same chromosome, indicating that there is a 102 chromosome-wide system that coordinates replication asynchrony of programmed 103 random monoallelic genes (Singh et al. 2003; Ensminger and Chess 2004; Donley et al. 104 2013; Donley et al. 2015). We use the following criteria to classify genes as being subject 105 to Programed Random Monoallelic Expression (PRME): 1) differential allelic expression 106 is detected in multiple unrelated individuals, which rules out rare DNA polymorphisms in 107 promoters or enhancers; 2) differential expression of either allele is detected in single 108 cell-derived subclones from the same individual, which rules out genomic imprinting and 109 eQTL; and 3) asynchronous replication between alleles is present and coordinated with 110 other random monoallelic genes on the same chromosome, indicating that the monoallelic 111 gene is regulated by a chromosome-wide system that coordinates asynchronous 112 replication along the chromosome pair. Using these criteria, we previously found that 113 ASAR6 and ASAR15 are subject to PRME (Stoffregen et al. 2011; Donley et al. 2013; 114 Donley et al. 2015). 5 Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press 115 Recent reports have described very long intergenic non-coding (vlinc) RNAs expressed 116 in numerous human tissues (Kapranov et al. 2010; St Laurent et al. 2012; St Laurent et 117 al. 2016). The vlincRNAs are RNA Pol II products that are nuclear, non-spliced, non- 118 polyadenylated transcripts of >50 kb of contiguously expressed sequence that are not 119 associated with protein coding genes. The initial reports annotated 2,147 human 120 vlincRNAs from 833 samples in the FANTOM5 dataset (St Laurent et al. 2013; St Laurent 121 et al. 2016). A more recent study identified an additional 574 vlincRNAs expressed in 122 childhood acute lymphoblastic leukemia (Caron et al. 2018). Therefore, there are 123 currently >2,700 annotated vlincRNAs that are encoded by >10% of the human genome 124 (St Laurent et al. 2013; St Laurent et al. 2016; Caron et al. 2018). ASAR6 and ASAR15 125 RNAs share several characteristics with the vlincRNAs, including: RNA Pol II products, 126 long contiguous transcripts (>50 kb) that are non-spliced, non-polyadenylated, and are 127 retained in the nucleus (Stoffregen et al.
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