7870–7885 Nucleic Acids Research, 2019, Vol. 47, No. 15 Published online 21 June 2019 doi: 10.1093/nar/gkz519 SIRT7 mediates L1 elements transcriptional repression and their association with the nuclear lamina Berta N. Vazquez1,2,† , Joshua K. Thackray 1,† , Nicolas G. Simonet3 , Sanjay Chahar1,4 , Noriko Kane-Goldsmith1 , Simon J. Newkirk5 , Suman Lee5 , Jinchuan Xing1 , Michael P. Verzi1 , Wenfeng An 5 , Alejandro Vaquero2,3 , Jay A. Tischfield1 and Lourdes Serrano1,* 1 Department of Genetics, Human Genetics Institute of New Jersey, Rutgers University, Piscataway, NJ 08854, USA, Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 2 Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute, Badalona, Barcelona 08916, Spain, 3 Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona 08908, Spain, 4 Montpellier Institute of Molecular Genetics (IGMM), CNRS and the University of Montpellier, 34090, France and 5 Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, South Dakota State University, Brookings, SD 57007, USA Received March 13, 2019; Revised May 30, 2019; Editorial Decision June 02, 2019; Accepted June 03, 2019 ABSTRACT INTRODUCTION Long interspersed elements-1 (LINE-1, L1) are Long interspersed elements-1 (LINE-1, L1) are non-LTR retrotransposons that hold the capacity of self- retrotransposons that hold the capacity of self-propagation propagation in the genome with potential mutagenic through retrotransposition in mammalian genomes. L1s outcomes. How somatic cells restrict L1 activity and comprise ∼19% and 17% of the mouse and human genome, how this process becomes dysfunctional during ag- respectively (1,2), and are considered to be a major driv- ing force of human genome evolution (3). A mammalian ing and in cancer cells is poorly understood. L1s are full-length L1 sequence includes an 5 untranslated region enriched at lamin-associated domains, heterochro- (5 UTR) harboring an active promoter, two open read- matic regions of the nuclear periphery. Whether this ing frame proteins (ORF1p and ORF2p) necessary for L1 association is necessary for their repression has retrotransposition and the 3 untranslated region (3 UTR) been elusive. Here we show that the sirtuin family that contains a poly (A) tract. Most mammalian genomic member SIRT7 participates in the epigenetic tran- L1 sequences are incapable of self-propagation due to mu- scriptional repression of L1 genome-wide in both tations and truncations (1). L1 transcriptional activation mouse and human cells. SIRT7 depletion leads to in- has been reported during mammalian early embryogene- creased L1 expression and retrotransposition. Mech- sis and in germ cells (4,5,6), which led to the concept that anistically, we identify a novel interplay between L1 de novo retrotransposition might lead to germline and SIRT7 and Lamin A/C in L1 repression. Our results somatic mosaicism, reviewed in (7). Indeed, distinct L1 in- sertions have been reported among individual somatic cells demonstrate that SIRT7-mediated H3K18 deacetyla- such as in neurons (8,9,10,11). Whether this somatic genetic tion regulates L1 expression and promotes L1 as- variability contributes to cell-specific functional determina- sociation with elements of the nuclear lamina. The tion or disease is still controversial. Undoubtedly, L1 ac- failure of such activity might contribute to the ob- tivity has profound effects on gene expression. L1s partic- served genome instability and compromised viabil- ipate in the formation of chimeric repeat-genic transcripts ity in SIRT7 knockout mice. Overall, our results reveal (12). L1s inserted in gene bodies might affect the quality and a novel function of SIRT7 on chromatin organization quantity of the mRNA transcript (13), and in a more global by mediating the anchoring of L1 to the nuclear enve- scale L1 transcriptional activation can affect the overall cel- lope, and a new functional link of the nuclear lamina lular pool of non-coding and coding RNAs (14). In ad- with transcriptional repression. dition, L1 retrotransposition often involves the transduc- tion of 3 nearby sequences (15,16), and the mobilization of other non-autonomous retrotransposons such as human * To whom correspondence should be addressed. Tel: +1 848 445 9656; Email: serrano@biology.rutgers.edu † The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Research, 2019, Vol. 47, No. 15 7871 Alu sequences and murine B1 and B2 elements (17,18) po- matin are compartmentalized in the eukaryotic nucleus. tentially shuffling transcriptional regulatory regions. More- Heterochromatin is predominantly found at the nuclear ex- over, active L1s can compromise genome integrity. The ac- terior associated with elements of the nuclear envelope such tivity of the L1-encoded endonuclease generates DNA dam- as components of the nuclear lamina (42,43,44,45). Indeed, age far above their retrotransposition potential promoting the anchoring of chromatin to the nuclear lamina is im- cell cycle arrest (19,20), induction of apoptosis (21) and cell portant for gene silencing and the maintenance of expres- senescence (22). Consistently, L1 activity is observed in can- sion programs (46). Transcriptional activation disrupts as- cer and aging cells (23,24). Whether L1 activity is the cause sociations with the nuclear lamina (47,48) and alternatively, or the effect of these cellular degenerative processes is not the tethering of genes to the nuclear lamina promotes their clear. There are a few reported examples of L1 retrotranspo- repression (49,50). L1 elements are enriched in Lamini- sition promoting insertional mutagenesis at tumor suppres- associated domains (43,51). However, whether L1-Lamin sor genes, which might precede oncogenic transformation association is necessary for L1 transcriptional repression is (25,26,27). not known. Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 The cell has developed multiple mechanisms of de- Here we report that SIRT7, a member of the mam- fense against L1 activity, ranging from small RNA-related malian sirtuin family of deacylases and ADP ribosylases, machineries to restriction proteins acting at the post- participates in the epigenetic regulation of L1 repression translational level such as AID/APOBEC proteins re- genome-wide in both mouse and human cells. ChIP-seq viewed in (28). Transcriptional silencing is predominantly analysis shows that the majority of chromatin-associated mediated by epigenetic mechanisms involving the deposi- SIRT7 binds L1 elements where it regulates H3K18Ac lev- tion of repressive histone post-translational modifications els. Mechanistically, SIRT7-mediated H3K18 deacetylation and by DNA methylation. These chromatin modifications promotes the association of L1 with lamin proteins. Impor- vary depending on cell type and cell developmental stage. tantly, association of L1 sequences to the nuclear lamina is DNA methylation at L1 is established in early embryogene- indispensable for their transcriptional repression. Accord- sis by de novo DNA methyltransferases (29) and is believed ingly, SIRT7 depletion is correlated with increased L1 ex- to be the main mechanism of L1 silencing in somatic tis- pression and retrotransposition. sues. Trimethylation of lysine 9 on histone H3 (H3K9me3), a characteristic epigenetic mark of constitutive heterochro- matin, is present at the 5 UTR of intact L1 in both differen- MATERIALS AND METHODS tiated and undifferentiated cells (30). H3K9me3-mediated Cell lines and treatments repression seems to be the primary L1 silencing mecha- nism in ESCs (31,32) and germ cells (30), which is medi- Mouse embryonic fibroblasts (MEFs) were isolated from ated by the H3K9me3 methyltransferases Suv39h1/2 (31). WT and SirT7−/− mice as previously described (52). K562 In male germ lines, L1 repression is facilitated by P element- cells were obtained from ATCC (CCL-121) and HT1080 induced wimpy testis (Piwi)-interacting RNAs (piRNAs), were kindly provided by Dr Brenemann. Cells were trans- which target L1 transcripts and facilitate the recruitment fected by electroporation as described previously (53). of the DNA and histone methyltransferases (30,33). For Briefly, 1 × 107 cells were resuspended in 250 ␮l of Ingenio more ancient L1s, with plausibly limited transcriptional buffer (Mirus) and were electroporated with 1 ␮M of SCR, activity, H3K9me3 deposition is mediated by the DNA SIRT7 or Lamin A/C siRNA using a BioRad device (Gene binding Krüppel-associated box domain-containing zinc Pulser Xcell) with 4 mm gap cuvette (BioRad) at 200 V and finger proteins (KRAB-ZFPs) and their cofactor KAP1 950 ␮F. Electroporated cells were then transferred to com- (KRAB-associated protein 1) by promoting the recruitment plete medium and incubated at 37◦ C for 48 h before harvest- of the methyltransferase SETDB1 (34). Other repressive ing. MEF cells were transfected with an Amaxa Nuclefactor marks characteristic of facultative heterochromatin such as System (for details, see ‘Retrotransposition Assay’ section) trimethylated H4K20 and H3K27 found at different sub- or infected with retroviral particles as described previously families of endogenous retrovirus did not show a uniform (54). pattern of enrichment on L1s in ESCs (35). Analysis of L1 repression in somatic tissues from aging and cancer cells re- Plasmids veal additional epigenetic mechanisms of L1 silencing such as histone lysine deacetylation, which is correlated with a SIRT7-, SirT7H188Y-, H3K18WT-, H3K18Q- and closer or inaccessible repressive chromatin state. In can- H3K18R-pMSCV vectors were established as described cer cells, deacetylated histones decorate the chromatin of previously (53). Human HA-Lamin A was cloned into newly L1 insertions (36) and the use of histone deacetylases the pcDNA4/TO vector (Invitrogen). The pWA125 and (HDACs) inhibitors is sufficient to induce L1 reactivation pWA126 retrotransposition plasmids were kindly provided (36,37). The specific HDACs responsible for L1-associated by Dr Ann, and are both based on a synthetic mouse L1 chromatin deacetylation are not known. element (55) (i.e. ORFeus-Mm) and contain a GFP-based Overall, L1s are vulnerable to the progressive loss of het- retrotransposition reporter. The pWA126 vector contains erochromatin that characterize cancer and aging cells, such two mutations that abolish the endonuclease and reverse as progressive CpG island hypomethylation and loss of his- transcriptase activities and is used as a control for back- tone methylation and deacetylation (38,39,40,41). Indeed, ground GFP signal. pMSCV-GFP was generated in (56). L1 sequences are an important part of the mammalian pMD7-5 UTR-Fluc described in detail in Luciferase assay cell heterochromatic content. Euchromatin and heterochro- section of ‘Materials and Methods’. SCR siRNA was 7872 Nucleic Acids Research, 2019, Vol. 47, No. 15 obtained from SIGMA and SIRT7 and Lamin siRNA were performed using MACS version1.4.2 (60), and RPKM- obtained from Dharmacon. normalized signal files were generated from BAM data us- ing DeepTools version 2.4.2 (61). Repetitive element anno- tations for the mm9 and hg19 reference genomes were ob- Chromatin immunoprecipitation tained from repeatmasker (RepeatMasker Open-4.0. 2013– ChIP was carried out as previously described (53). Briefly, 2015 ), and were processed samples were crosslinked in 1% formaldehyde for 10 min to defragment elements based on their ID field. Intersection at 37◦ C, washed twice with PBS and then collected from of ChIP peaks with repetitive elements was performed with dishes. For SIRT7, ChIP was carried out with nuclear ex- the Bedtools package version 2.17.0 (62). Classification of tracts. First, cells were resuspended in Lysis Buffer 1 (5 mM ChIP peak genomic context was performed with CEAS ver- HEPES, 85 mM KCl, 0.5% NP-40) and incubated for 5 sion 0.9.9.7 (63). Heat map and average profile plots were min on ice to disrupt cellular membranes. After centrifu- generated using in-house scripts utilizing metaseq version gation, nuclei were resuspended in Lysis Buffer 2 (1% SDS, 0.5.6 (64), pybedtools version 0.6.9 (65) and Matplotlib ver- Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 10 mM EDTA, 50 mM Tris and protease inhibitors) and in- sion 2.0.2 (Zenodo. http://doi.org/10.5281/zenodo.573577). cubated on ice for 40 min. For H3K18Ac and Lamin A/C Individual profile plots were generated using IGV version ChIP, whole cell extracts were obtained by directly lysing 2.3.98 (66). PhastCon scores (30-way vertebrate) against the cells in Lysis Buffer 2 for 40 min on ice. Chromatin was mm9 reference genome were obtained from UCSC (67,68), then sonicated to an average fragment size of 150–300 bp and average profiles were generated with in-house scripts. as analyzed by agarose gel electrophoresis, and subjected to SIRT7 peak proximity to TSS elements was computed with immunoprecipitation with anti-SIRT7, anti-H3K18Ac or GREAT v2.0.2 (69) using the mm9 reference genome and anti-LaminA/C antibodies (see Supplementary Table S1 for default settings. Alignment of ChIP-seq reads to L1 consen- details). ChIPs were washed five times with ice-cold RIPA sus sequences was performed using bowtie2 v2.2.6. L1 con- buffer (1% NP-40, 0.7% Na deoxycholate, 50 mM Tris, 1 sensus sequences obtained from repbase (70,71) were first mM EDTA, 500 mM LiCl2 , pH 8.0), crosslinks were re- indexed with bowtie2-build using default options followed versed (1% SDS, 0.1 M NaHCO3 ; 6 h at 65◦ C) and DNA by alignment of ChIP-seq reads using bowtie2 with options was purified (minElute PCR Purification Kit, Qiagen). Fi- –N 1 –local. ChIP signal over the length of the consensus nally, the concentration of ChIP and INPUT DNA was sequence was then plotted using in-house scripts, with data measured using PicoGreen (Life Technologies). For Quan- expressed as the log2 ratio of ChIP divided by Input after titative PCR, GAPDH and LINE elements were analyzed adjustment for library sizes. Data shown are from replicate with 1 ng and 50 pgr of ChIP and INPUT DNA, respec- #1 from two independent concordant replicates for both tively. See Supplementary Table S2 for primer sequences. human and mouse. Library preparation and ChIP-sequencing RNA extraction and quantitative RT-PCR ChIP libraries were prepared using a ThruPlex DNA-seq Cultured cells and tissues were homogenized in library preparation Kit (Rubicon Genomics) following the TriReagent® (Invitrogen) and RNA was isolated with a manufacturer’s protocol. ChIP libraries were then size se- Direct-zol™ RNA Kit (Zymo Research) according to the lected with PippinPrep (Sage Science) for a final library manufacturer’s instructions. About 1.5 ␮g of RNA was fragment size of 350–400 bp, and quantified with KAPPA then digested with DNase I (Invitrogen), purified with an qPCR. ChIP libraries were then sequenced on a HiSeq 2500 RNAeasy kit (Qiagen) and subjected to cDNA synthesis (Illumina) with 2 × 100 bp paired-end reads to a depth using the High Capacity cDNA Reverse Transcription Kit of >20 × 106 fragments per sample at the RUCDR, Infi- (Applied Biosystems) with random hexamers and poly-dT nite Biologics sequencing center (Rutgers University, NJ). according to manufacturer’s instructions. For 5 RACE Reads were de-multiplexed and concatenated, and delivered qPCR, cDNA was generated using a SMARTer RACE as fastq files. Kit (Clontech Laboratories). cDNA generated without the SMARTer II Oligonucleotide was used as a negative control. PCR products were cloned into the pGEM-TEasy ChIP-seq analysis vector (Promega). DNA sequencing was performed on Raw read quality was first assessed with fastqc (ver- an ABI PRISM 377 DNA sequencer (Applied Biosys- sion 0.11.3, Babraham Bioinformatics). FastQC: a qual- tems). Real-Time PCR was performed using SYBR Green ity control tool for high-throughput sequence data (avail- (Applied Biosystems) in an ABI 7900HT real-time PCR able online at: http://www.bioinformatics.babraham.ac.uk/ machine. Reactions were run in triplicate with an initial projects/fastqc), followed by adapter clipping and quality denaturation step at 95◦ C for 10 min, followed by 40 cycles trimming with Trimmomatic version 0.36 (57). Reads were of denaturation at 95◦ C for 15 s and annealing/elongation then aligned to the mouse (UCSC mm9) or human (UCSC at 60◦ C for 1 min and a final melting curve analysis to verify hg19) reference genomes with Bowtie with options –n 2 –k amplification specificity. The housekeeping gene ␤-Actin 10 (58), and converted to BAM format with Samtools (59). was used as an internal control to normalize expression ChIP and alignment quality was evaluated computing li- levels and reactions without the addition of reverse tran- brary complexity (PBC), FRiP, insert size and strand cross- scriptase were performed in parallel as negative controls. correlation analysis. All samples passed the ENCODE The primer sequences are provided in Supplementary Table quality standards for ChIP-seq studies. Peak calling was S2. Nucleic Acids Research, 2019, Vol. 47, No. 15 7873 Retrotransposition assays Thermo Fischer) and digested at 37◦ C for 0, 1.5, 3, 5, 10 and 15 min, respectively. Then 3 ␮l of 0.5 M EDTA was WT and SirT7−/− primary MEFs cells were transfected added to stop digestion, and DNA was purified by us- with pWA125 or pWA126 retrotransposition vectors (see ing the NucleoSpin® Gel and PCR Clean-up procedure Supplementary Figure S9A for more details), and with (Macherey-Nagel). About 500 pgr of DNA was used for the pMSCV-GFP transfection efficiency control vector qPCR analysis using SybrGreen (ThermoFisher). Primers using the Amaxa P4 Primary Cell 96-well Nucleofector were designed to target the promoters of the L1MdA con- Kit (Lonza) according to the manufacturer’s instructions. sensus sequence and the B-globin gene to obtain PCR Briefly, 105 cells were resuspended in 20 ␮l of the Nucleo- amplicons longer than the length of a single nucleosome. fector Solution together with 500 ng of plasmid DNA and Specifically, amplicons were 248 and 350 bp long for the were electroporated in a 96-well Shuttle Nucleofector with B-globin and L1Mda promoters, respectively. Mononucle- the program setting 96-CZ-167. Transfected cells were then osomes formed as the result of MNase digestion cannot transferred to cell culture dishes with pre-warmed complete be amplified by qPCR. Therefore, reduced amplification in- DMEM and treated 24 h post-transfection with puromycin Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 dicates more MNase digestion, which is interpreted as the (Sigma-Aldrich) at a final concentration of 1 ␮g/ml. Cells presence of more accessible/open chromatin at the assayed were harvested 7 days post-transfection. The number of loci. EGFP positive cells and the mean fluorescence intensity of GFP+ cells was quantified by flow cytometry using FAC- Scalibur (Beckman Coulter) and analyzed with Flow Jo 7/8 Luciferase assay (Tree Star). We generated a stable HCT-116-5 UTR-Fluc cell line carrying an integrated 5 UTR-Fluc transgene (pMD7), Protein immunoprecipitation which expresses the firefly luciferase (Fluc) reporter un- Lamin A/C antibody (#abcam, ab8984) was first conju- der the human L1RP 5 UTR promoter (Genebank acces- gated to protein A and protein G magnetic beads (Life Tech- sion #AF148856.1). pMD7 was constructed through three nologies) according to manufacturer’s instructions. Chro- cloning steps. First, pMD2 was derived from the Sleeping matin fractions from 293 cells were obtained as previously Beauty transposon cloning vector pT2BH (72) by double described (54). Briefly, cells were first lysed in Hypotonic digestion with SalI and XhoI and subsequent religation. Buffer (10 mM Tris:HCl pH 7.9, 1.5 mM MgCl2 , 10 mM Second, the Fluc reporter under SV40 promoter was re- KCl, 0.1 mM PMSF, 0.5 mM DTT and protease inhibitors) leased from pGL4.13 (Promega) as a EcoRV/BamHI frag- for 10 min at 4 ◦ C to break the cellular membrane. Af- ment, and ligated into EcoRV/BglII digested pMD2, mak- ter centrifugation, protein supernatant was discarded, and ing pMD3. Third, the SV40 promoter in pMD3 was re- cellular pellet was resuspended in RIPA buffer (50 mM placed with L1RP 5 UTR promoter by ligating the 5 UTR Tris:HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, from pJCC5-RPS (73) as a EcoRV/NcoI fragment with 0.1% SDS; 0.1 mM PMSF, 0.5 mM DTT and protease in- EcoRV/NcoI digested pMD3, making pMD7. To make the hibitors) to obtain nuclear extracts. Nuclear extracts were stable cell line, 90 000 HCT-116 cells were seeded in a 24- treated with benzonase (SIGMA) according to manufac- well plate, and 24 h later cotransfected with 100 ng of pMD turer’s instructions, and the protein supernatant was incu- and 20 ng of SB100X (72) using FuGENE HD reagent. bated with anti-Lamin A/C beads overnight at 4 ◦ C. Pro- Cells were harvested 24 h post transfection and reseeded tein complexes were washed three times with ice-cold BC- into 96-well plates at the density of 0.8 cells/well. Single cell 100 washing buffer (20 mM Tris:HCl, pH 8.0, 100 mM KCl, clones showing Fluc activity were retained for downstream 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P- analysis. For luciferase assays, a total of 10.000 HCT-116- 40; 0.1 mM PMSF and Protease inhibitors), and elution 5 UTR-Fluc cells were plated in triplicate into a 96-well was performed by incubating the beads 10 min with 25 ␮l plate and transfected with 200 mM Scr or SirT7 siRNA of 0.1 M glycine, pH 2.8. About 25% of the eluted pro- (Dharmacon) using Fugene reagent (Promega). Seventy- tein complexes and 1% of the INPUT were then resolved four hours after transfection, cell viability was measured us- on NuPAGE 4–12% Bis-Tris gel (Invitrogen) and analyzed ing Sensilite (PerkinElmer) and luciferase activity with Lu- by western blot using anti-SIRT7 and anti-LaminA/C (Cell ciferase Assay System (Promega). Signaling, 2032) antibodies. Pull-down assays Micrococcal nuclease assay The following histones peptides were synthesized by Three 10-cm dishes of SirT7−/- and WT MEFs were grown AnaSpec (San Jose, CA): Histone H3 (1–21) (Cat# to 80–90%confleunce. About 10–15 × 106 cells of each AS-61702) [ARTKQTARKSTGGKAPRKQLA-GG- genotype were resuspended in 1 ml of lysis buffer (10 mM K(BIOTIN)-NH2], Histone H3K18ac (1–21) (Cat#AS- Tris, 10 mM NaCl, 3 mM MgCl2 , 1% of NP-40, pH 7.5) 64638) [ARTKQTARKSTGGKAPR-K(Ac)-QLA- and incubated on ice for 10 min. Nuclei were collected by GGK(Biotin)-NH2], Histone H3K18me2 (1–21) (Cat#AS- centrifugation at 300 × g for 5 min at 4◦ C. The nuclear pel- 64620) [ARTKQTARKSTGGKAPR-K(Me2)-QLA- let was then resuspended in 400 ␮l of nuclear lysis buffer GGK(Biotin)-NH2]. Pull-down assays were performed (20 mM Tris, 20 mM KCl, 70 mM NaCl, 3 mM CaCl2 and with Streptavidin agarose beads purchased from Millipore protease inhibitor cocktail, pH 7.5). Aliquots of 60 ␮L were (#16-126). Preparation of the peptide-bound resin and then treated with 4 Units of micrococcal nuclease (MNase, peptide pull-down from nuclear extracts were performed 7874 Nucleic Acids Research, 2019, Vol. 47, No. 15 as described (54). Briefly, HA-Lamin A/C purified from of an arbitrary point within a given nucleus, we used the HEK-293F cells was incubated with the respective strep- library geometry3Sharp (https://github.com/gradientspace/ tavidin bound biotin labeled peptide for 12 h at 4◦ C (salt geometry3Sharp) to generate 3D AABBTrees representing concentration adjusted to 150 mM of KCl in presence of the isosurface mesh used to segment each nucleus. We then 0.1% Triton X-100). The streptavidin agarose beads were query this tree to find a point on the surface that lies on washed five times with 10 volumes of PBS/0.1%Triton the ray originating at the center of the nucleus and pass- X-100 and finally eluted with 1 volume of 2× Laemmli ing through the point of interest. We define the radial posi- buffer. The amount of HA-Lamin A/C pulled-down was tion for this point as the ratio of distances: distance(center, detected by western blot using anti-HA antibody, while a point) / distance(center, surface). All images among all dot blot with anti-biotin was performed as loading control. samples within a given experiment were acquired and quan- See Supplementary Table S1 for antibodies sources. tified using the same microscope and analysis parameters. Correlation metrics were computed as previously described (56). Additional reagents details can be found on Supple- Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 FISH and imaging mentary Tables S1 and S2. For single locus FISH, BACs were obtained from CHORI (RP24-286G19 and RP24-184M19) and were amplified and RESULTS isolated using standard techniques. Probes were generated by incorporation of modified nucleotides (aa-dUTP, Sigma) SIRT7 preferentially binds to active families of LINE-1 ele- via nick translation and coupled to fluorophores with the ments ARES kit (Invitrogen) following manufacture’s instruc- We used chromatin immunoprecipitation (ChIP) followed tions. For L1 probe FISH, probes were generated via PCR by high-throughput DNA sequencing (ChIP-seq) to iden- incorporating biotin-dUTP using primers designed against tify SIRT7-binding sites throughout the genome in pri- the L1MdT family of L1 elements. Specifically, primers for mary MEFs. Bioinformatic analysis of the data revealed this probe were designed against the L1MdT consensus se- that SIRT7 binds 72 622 loci scattered across the genome quence (RepBase) with amplicons generated starting from (Figure 1), and that these binding sites are highly conserved the internal 5 UTR through the length of the ORF2 re- between species (Supplementary Figure S1A). The major- gion. PCR reactions were run partially substituting dTTP ity of SIRT7 binding sites are located within intergenic re- with biotin-dUTP (1:3 ratio), and using WT MEF genomic gions and only ∼2% are found within promoter and ex- DNA as template. All PCR reactions were subjected to size onic regions (Figures 1A and Supplementary S1B). Strik- selection via agarose gel electrophoresis with a target win- ingly, 80% of SIRT7-binding sites were found at L1 elements dow of 200–500 bp (see Supplementary Table S2 for primer (Figure 1B). The binding of SIRT7 at L1 elements was sequences). Probes were combined in an equimolar ratio, in further confirmed by ChIP coupled to quantitative PCR addition to mouse Cot-1 (BAC probes only) and sheared (ChIP-qPCR) and compared with SIRT7 occupancy at se- salmon sperm DNA (Invitrogen) for blocking, precipitated lected genomic regions (Figure 1C). SIRT7 is enriched at and resuspended in hybridization buffer. Probes were ap- the ribosomal gene 28S consistent with the role of SIRT7 plied to cells grown on coverslips that were fixed and pro- in rDNA transcription regulation (74), but dramatically in- cessed for FISH. Slides were denatured for 3.5 min at 75◦ C, creased (15-fold) at L1 loci relative to a negative control lo- followed by incubation at 37◦ C for 1–2 days. Slides were cus (GAPDH). The alignment of the SIRT7 ChIP-seq reads then washed 3 × 10 min with 2X SSC at 37◦ C, then 3 × 10 to the consensus mouse L1 sequence indicates that SIRT7 min with 0.1× SSC at 62◦ C. For L1 probe FISH, biotin was binding is present throughout the L1 (Supplementary Fig- detected using anti-biotin primary antibody and visualized ure S1C). Importantly, our results reveal that SIRT7 specif- with secondary antibody coupled to fluorophore (see Sup- ically binds the most recently evolved and therefore, pu- plementary Table S1 for antibodies sources). Finally, slides tatively active families of L1 elements (Figure 1D). More- were counterstained with DAPI and mounted with slow- over, SIRT7 occupancy at L1 elements genome-wide was fade gold mounting media (Invitrogen). also present in K562 human cell line but to a lesser ex- For all FISH experiments, images were acquired using a tent (Supplementary Figure S2A). Similar to mouse cells, LSM510 META confocal microscope (Carl Zeiss) equipped SIRT7 in this human cell line preferentially binds the most with a Plan-Apochromat 100×/1.46 NA oil immersion lens. recently evolved families of L1 elements (Supplementary 3D reconstruction and image analysis were performed using Figure S2B). Imaris software (Bitplane, A.G.). Nuclei were segmented based on DAPI staining intensity using the standard Sur- SIRT7 promotes the association of L1 elements to Lamin faces function of Imaris software. For single locus FISH, A/C hybridization signals were located and modeled using the Spots module, which detects the hybridization signal cen- Previous reports have shown that L1 elements are en- ter of mass. These coordinates were used to compute the riched at Lamin-associated domains (LADs) (44,51), a radial position of each hybridization signal relative to the transcriptional repressive nuclear compartment (46). Strik- nucleus. For L1 probe FISH (see Supplementary Figure ingly, analysis based on our own and previously published S5), the relative radial position of each voxel within each data (GSE36132 (44)) showed an enrichment of lamin pro- nucleus was computed and the average L1 probe inten- teins to all L1 elements as indicated by a positive Lamin- sity along the nuclear radius, which was divided into 20 DamID score, which was dramatically increased in the sub- bins, was measured. To compute the relative radial position set of L1 elements bound by SIRT7 (Figure 2A). Consis- Nucleic Acids Research, 2019, Vol. 47, No. 15 7875 A 0.8% 1.3% B All LINEs (701,130) SIRT7 Peaks (72,622) SIRT7 ∩ LINEs (58,537) 80.61% of 21.5% SIRT7 Peaks 76.4% 99.40% L1 0.48% L2 Distal Intergenic 0.60% Intron CR1 0.10% Promoter 0.02% Exon, UTR and Downstream RTE Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 C D *** 80% 20 L1Md_F2 % of SIRT7-bound L1s L1Md_F3 L1Md_T 10 60% L1Md_F L1Md_A 3 SIRT7 ChIP L1Md_Gf L1_Mus1 ** L1_Mus2 40% L1_Mus3 L1_Mus4 L1VL1 2 1 20% Lx 0 0% 11.89 11.69 10.50 8.74 7.28 5.57 4.35 4.12 3.77 1.45 1.33 0.58 S e H 1 E llit E- 28 N D SI AP te N LI Sa G α- L1 Age (Myr) Figure 1. SIRT7 binds L1 elements genome-wide. (A) Genomic distribution of SIRT7-binding sites in MEFs categorized by genomic features. (B) Venn diagram (top) showing the number of SIRT7 peaks that intersect with all LINEs genome-wide. Pie chart (bottom) showing the family classification of LINEs occupied by SIRT7 in MEFs. Data indicate the percentage of family in the set of all LINEs occupied by SIRT7. Genomic LINE annotations were obtained from RepeatMasker, and intersections computed with BEDTools. (C) ChIP-qPCR analysis showing SIRT7 occupancy in MEFs at the indicated genomic regions. All samples were normalized to input DNA (**P < 0.01; ***P < 0.001 by ANOVA single factor). (D) Relative occupancy of SIRT7 at the indicated L1 subfamilies in MEFs. Data represent the percentage of L1 elements in the indicated L1 subfamilies that are occupied by SIRT7. L1 subfamilies were organized according to their evolutionary age, from the oldest to the youngest, as reported in (89). tently, endogenous SIRT7 and Lamin A/C are able to be co- signal, were used to calculate the relative radial distance purified by immunoprecipitation in MEFs cells (Figure 2B to the nuclear surface. Although the overall distribution and Supplementary Figure S3). Importantly, we observed was not statistically different according with Kolmogorov– that Lamin A/C association with L1 elements is reduced Smirnov (KS) testing, we computed the correlation coef- in SIRT7-depleted cells (Figure 2C), despite similar levels ficient between the WT and SirT7−/− radial distributions of Lamin A/C between SirT7−/− and WT cells (Figure for both probes (LAD rho = 0.9549 with 95% CI [0.816, 2D). In addition, the reduced presence of Lamin A/C at 0.989] versus Non-LAD rho = 0.9932 with 95% CI [0.971, L1 loci in SIRT7-deficient cells was directly dependent on 0.998]), which shows a decreased correlation of the relative SIRT7 deacetylase activity. Viral-mediated transduction of positional frequency in the LAD locus as compared to the SirT7−/− MEFs with an active SIRT7, but not with a cat- Non-LAD locus. In addition, we observed a significant de- alytically inactive SIRT7 point mutant, rescued Lamin A/C crease in the frequency of the most peripheral positioning levels at L1 elements (Figure 2C). Collectively, these data of the LAD locus in SirT7−/− cells compared to WT cells, suggest a functional interplay between SIRT7 and Lamin while no significant changes in the nuclear localization of A/C in L1 regulation. the adjacent non-LAD probe were observed between both It is well documented that the anchoring of chromatin cell types. To further discern the impact of SIRT7 in L1 to the nuclear lamina is important for gene transcriptional nuclear spatial distribution, we designed a L1 FISH probe repression (46). Similarly, SIRT7-mediated L1 repression spanning the consensus sequence of the L1MdT family of might involve the anchoring of L1 elements to the nuclear L1 elements with the rationale that the vast majority of lamina. To test this possibility, we performed fluorescence these L1 family members are occupied by SIRT7 (Figure in situ hybridization (FISH) targeting selected L1 loci in 1D). After hybridization of this probe on primary WT and WT and SirT7−/− cells (Figure 2E and F). We first used SirT7−/− MEFs followed by confocal 3D imaging analy- two BACs probes: one within a LAD enriched for SIRT7- sis (Supplementary Figure S5), we observed that the ma- occupied L1 elements and an adjacent non-LAD control lo- jority of the FISH signal in WT cells was found at the nu- cus devoid of SIRT7-bound L1 elements (Figure 2E). Probe clear periphery (Figure 2F, left and Supplementary Figure coordinates, based on weighted centers of each fluorescence S6). This is consistent with previous reports placing ∼60% 7876 Nucleic Acids Research, 2019, Vol. 47, No. 15 A B C D E F Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 Figure 2. SIRT7 is required for the tethering of L1 elements to the nuclear lamina. (A) SIRT7 peaks are located in regions with higher LaminB1 DamID score. Lamin B1 DamID score tracks in MEFs were obtained from (43). Average score profiled over SIRT7-associated L1 elements (black) and at all L1 elements (red). (B) Endogenous Lamin A/C was immunoprecipitated from 293 whole cell extracts and probed for Lamin A/C and SIRT7. (C) ChIP-qPCR of L1 in WT and SirT7−/- MEFs, and upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in SirT7−/− MEFs, using an anti-Lamin A/C antibody. Data are normalized to input DNA and GAPDH. (D) Detection of Lamin A/C and SIRT7 from WT and SirT7−/− MEFs by western blot. H3 was used as a loading control. (E) (Top) Schematic showing genomic BAC probe placement and its association with genes, LINEs, Lamin B1, and SIRT7 signal. (Bottom) FISH analysis showing nuclear localization of probes targeting a LAD and a non-LAD region in WT and SirT7−/- cells. (Bottom left) 3D reconstruction and rendering of the nuclear volume based on DAPI staining (blue), and BAC fluorescence signal was performed using IMARIS software from confocal Z stacks (scale bar = 2 ␮m). (Bottom right) BAC coordinates based on weighted centers of each fluorescence signal were used to calculate radial distance to the nuclear surface. (F) FISH analysis of the nuclear distribution of L1MdT family. (Top) Schematic representation of L1MdT consensus sequence annotated with location of FISH probes. (Left) Representative images (scale bar = 2 ␮m). (Right) L1MdT probe intensity plotted against nuclear radial position, where 0 indicates the nucleus center point and 1 indicates the nuclear periphery (n = 50 cells per genotype). Data represent mean ± SEM of three independent experiments per condition except otherwise indicated; *P < 0.05 by ANOVA single factor. of LINE elements in LAD regions (44). Quantification of and promoters of active genes (75). Consistently, SIRT7- the L1 probe intensity per voxel in relation to their rela- mediated H3K18 deacetylation at the promoters of a spe- tive nuclear radial position revealed a mild but significant cific set of genes has been shown to lead to their silenc- decrease in the mean intensity at peripheral locations (0.8– ing (76,77). We hypothesized that SIRT7-mediated H3K18 1.0) and an increase in the central compartment (0.0–0.8) in deacetylation has a broader role in transcriptional regula- SirT7−/− cells compared to WT (Figure 2F, right). In ad- tion considering the predominant occupancy of SIRT7 at dition, WT nuclei showed a higher correlation of radial po- L1 elements (Figure 1B). This hypothesis is further sup- sition and L1 probe intensity compared to SirT7−/− nuclei ported as SIRT7 catalytic activity is necessary to restore (rho = 0.907 ± 0.022 versus rho = 0.776 ± 0.045, respec- L1 association with the nuclear lamina in SirT7−/- MEFs tively. P-value of 0.0108 by ANOVA). Taken together, our (Figure 1C). To test this hypothesis, we performed ChIP- results indicate that L1 nuclear distribution becomes dis- seq to determine H3K18Ac levels genome-wide in WT and rupted upon SIRT7 depletion and support a model where SirT7−/− MEFs. ChIP-seq analysis of H3K18Ac enriched SIRT7 regulates the anchoring of L1 elements to the nuclear chromatin from WT MEFs corroborated previous reports periphery. of H3K18Ac occupancy. H3K18Ac is found at the peaks of H3K27Ac and H3K4me1 histone modifications that are enriched enhancers, and at promoters marked by the pres- SIRT7 regulates H3K18 acetylation levels at L1 loci ence of H3K4me3 and the binding of RNA Polymerase II Subunit A (Supplementary Figure S4). Importantly, the SIRT7 is known to be a NAD+ -dependent deacetylase of ly- levels of H3K18Ac at enhancers and promoters of canon- sine 18 of histone H3 (H3K18). Acetylation of this residue ical genes did not change significantly between WT and has been previously described to be enriched at enhancers Nucleic Acids Research, 2019, Vol. 47, No. 15 7877 SirT7−/− cells, consistent with our observation that SIRT7 purified Lamin A protein (Figure 4D and E). Our results is generally not enriched at these sites (Figure 1A). How- indicate that Lamin A is able to directly bind the H3 his- ever, we observed that H3K18Ac is enriched at L1 elements tone tail peptide, and importantly, that acetylation of K18 occupied by SIRT7, and that SIRT7 deficiency resulted in reduces this interaction. Intriguingly, H3K18me2 peptides a significant increase of H3K18Ac levels at these loci (Fig- (peptide control) were also able to reduce Lamin A interac- ure 3). SIRT7 specifically binds the most recently evolved tion. However, it is difficult to evaluate the biological rele- and DNA sequence-intact families of L1 elements, such as vance of this result as the role of H3K18 methylation has not the L1MdA family (Figure 1D). Consistently, the effect of been defined yet. Nevertheless, this observation strength- SIRT7 on H3K18ac levels is bigger in Figure 3B, in which ens the idea that modification of the H3K18 residue alters the average enrichment profile is over L1MdAL1 elements, Lamin binding to chromatin. Overall, these results suggest as compared with Figure 3A that represents the H3K18ac that in the absence of SIRT7, H3K18 hyperacetylation pre- average levels over all L1 elements. Moreover, knockdown vents the association of L1 elements with the nuclear lamina of SIRT7 in HT1080 cells also resulted in a significant in- by interfering with Lamin A/C binding to chromatin. Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 crease of H3K18Ac levels at L1 elements (Figure 4A) in- dicating that SIRT7 modulation of H3K18Ac levels at L1 Increased LINE-1 expression and retrotransposition in the elements also holds in human cells. absence of SIRT7 Collectively, our results reveal an unanticipated and novel Histone H3K18 acetylation levels modulate Lamin A/C in- functional interplay between SIRT7 and Lamin A/C and teraction with chromatin suggest that SIRT7 may promote L1 repression via its inter- Previous reports indicate that the association of sirtuins action with the nuclear lamina. We first determined whether with components of the nuclear lamina impacts their enzy- SIRT7 activity promotes L1 transcriptional silencing by matic activities. Lamin A interaction with both SIRT1 and measuring L1 expression levels in MEFs isolated from WT SIRT6 activates, albeit by an unknown mechanism, their and SirT7−/− sibling mouse embryos. We observed a sig- deacetylase activity (78,79). To test whether SIRT7-Lamin nificant upregulation of L1 transcripts in SirT7−/− -derived A/C interplay could impact SIRT7 deacetylase activity at MEFs compared to WT cells (Figure 5A). Moreover, sev- L1 elements, we measured H3K18Ac levels at L1 sequences eral tissues from SirT7−/− mice also displayed L1 deregu- in control and Lamin A/C knockdown cells by ChIP-qPCR lation (Figure 5A), being more predominant in those tissues (Figure 4A). We observed that, relative to control cells, de- where SIRT7 is highly expressed and become dysfunctional pletion of Lamin A/C did not cause H3K18Ac levels at L1 upon SIRT7 depletion (53,74,77,81,82). Importantly, the loci to change significantly. However, depletion of SIRT7 single copy gene TBP, which is devoid of any transcription- led to a significant increase in H3K18Ac enrichment, con- ally competent L1 element or SIRT7-binding sites, had sim- sistent with our observations in WT and SirT7−/- -derived ilar levels of L1 expression between WT and KO cells from mouse cells (Figure 3). all tissues that we investigated. These results indicate that To directly test whether SIRT7-mediated H3K18 SIRT7 maintains the repression of more recently evolved, deacetylation was necessary for Lamin A/C recruitment and therefore putatively active, L1 elements, consistent with to L1 elements, we transduced WT and SirT7−/− cells the relative enrichment of SIRT7 at different L1 families with histone H3 mutants in which K18 was mutated either (Figure 1D and Supplementary Figure S2B). Most impor- to glutamine (K18Q) or to arginine (K18R) to mimic tantly, transcriptional silencing of L1 elements was directly an acetylated or deacetylated lysine residue, respectively. dependent on SIRT7 activity because viral-mediated trans- Overexpression of the H3K18Q mutant in WT cells duction of SirT7−/− MEFs with an active SIRT7 was suffi- resulted in a significant reduction of Lamin A/C levels cient to repress L1 expression by 65% (Figure 5B). In addi- at L1 elements measured by ChIP-qPCR. Conversely, tion, we generated a human colon carcinoma cell line with overexpression of the H3K18R mutant in SirT7−/− cells integrated firefly luciferase reporter cassette driven by the increased Lamin A/C levels (Figure 4B). Interestingly, we by a sense 5 UTR promoter derived from the human L1RP were able to detect via western blot a dramatic reduction instance. Our results showed increased expression of the of Lamin A/C levels in the chromatin fraction of WT cells L1 construct in siRNA-mediated SirT7 knockdown cells as overexpressing the H3K18Q mutant, suggesting an effect compared with cells transfected with scramble siRNA (Fig- of this histone acetylation status in other chromatin loci ure 5C). Taken together, these results indicate that SIRT7 outside L1 (Figure 4C). Altogether these results indicate deficiency results in increased L1 expression across multi- that SIRT7-mediated H3K18 deacetylation at L1 is not ple mice tissue types and in human cells. Moreover, overex- affected by SIRT7–Lamin A/C association but seems to be pression of the H3K18Q mutant histone resulted in a signif- necessary for Lamin A/C recruitment to L1. icant increase of L1 expression (Figure 5D) indicating that Taken together, these results support that SIRT7- modulation of H3K18Ac levels at L1 elements regulates L1 mediated H3K18 deacetylation is critical for Lamin A/C- transcription. Importantly, knockdown of Lamin A/C re- L1 element association. Structural analysis of Lamin A/C sulted in the transcriptional upregulation of L1 loci occu- protein indicates that Lamin A/C is able to directly pied by SIRT7 (Figure 5E). Although overexpression of the bind chromatin (80). To test whether the association of deacetylated K18 mutant histone (H3K18R) was sufficient Lamin A/C with chromatin is impaired by the presence of to reduce L1 expression as compared to control cells, this H3K18Ac, we used biotinylated H3 peptides with K18 in an reduction did not occur in the absence of Lamin A/C (Fig- unmodified, acetylated or di-methylated state to pull-down ure 5F). 7878 Nucleic Acids Research, 2019, Vol. 47, No. 15 A H3K18Ac B H3K18Ac at L1MdA L1 Elements SIRT7 WT SirT7 -/- WT 30 SirT7-/- Average Signal 25 20 L1 Elements 15 10 5 −10 −5 0 5 10 Distance from the Center of L1 (Kb) C Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 37 SIRT7 20 40 Average Signal SIRT7 WT 0 154 WT H3K18Ac SirT7-/- 0 154 SirT7-/- H3K18Ac 2 5 0 -10 -5 0 5 10 -10 -5 0 5 10 LINEs chr7:58,328,843-58,337,327 Distance from the Center of L1 (Kb) Figure 3. SIRT7 regulates H3K18Ac levels at L1 elements. (A, Top) Heat maps showing enrichment of SIRT7 and H3K18Ac in WT and SirT7−/− MEFs at genomic L1 elements. Left heat map (L1 elements) shows Smith–Waterman alignment score of genomic L1 elements as reported by RepeatMasker. (A, Bottom) Average enrichment profiles for (left) SIRT7 and (right) H3K18Ac in WT (blue) and SirT7−/− (red). (B) Average enrichment profiles for H3K18Ac in WT (blue) and SirT7−/− (red) in L1MdA family members. (C) Visualization of representative signal profiles for SIRT7 and H3K18Ac in WT and SirT7−/- MEFs at an L1MdA locus. Thus far, we employed standard RT-qPCR to quantify served change in chromatin structure in SirT7−/− cells (Fig- the effect of SIRT7 on L1 expression. According to a recent ure 6A) could affect the insertion site of the retrotranspo- report (83), this approach could not distinguish authentic sition reporter. However, when analyzing the mean GFP L1 transcripts (i.e. those transcribed from L1 s own pro- fluorescence intensity in the GFP+ population (which cor- moter) from L1 co-transcripts (i.e. those transcribed from a responds to cells in which the construct has successfully neighboring gene promoter). To determine whether SIRT7 retrotransposed) and in cells transfected with our GFP- regulates L1 expression from its own promoter, we devised a based control vector we did not observe differences between 5 RACE-qPCR strategy to specifically quantify full-length WT and SirT7−/− cells (Supplementary Figure S9C and D, L1 transcripts in total cellular RNA (Supplementary Fig- respectively), supporting our interpretation. This said, we ure S7). Results were highly similar to those obtained with cannot rule out either that the increased retrotransposition standard RT-qPCR (Supplementary Figure S8). in SirT7−/− cells is solely the result of increased L1 expres- Overall, our results suggest that Lamin A/C is a down- sion. In addition, SIRT7 might modulate the retrotranspo- stream factor of H3K18 deacetylation but still crucial for sition lifecycle at other levels. Indeed, L1 elements require L1 repression. In agreement with the common vision of the host DNA repair machinery for integration (86) and we the nuclear periphery as a heterochromatic compartment, have previously published that SIRT7 regulates DNA repair and the known association of histone deacetylation with (53). chromatin compaction (84), SIRT7 depletion was corre- Taken together, our results indicate that SIRT7 partic- lated with increased chromatin accessibility. Genomic DNA ipates in L1 repression genome-wide in mouse and hu- from SirT7−/− cells was more sensitive to Micrococcal Nu- man cells. Mechanistically, our results support that SIRT7- clease (MNase) treatment as compared with WT cells (Fig- mediated deacetylation of H3K18 is necessary for the re- ure 6A), especially at L1 loci (Figure 6B). cruitment of L1 to the nuclear periphery by their associa- To further determine the impact of SIRT7 in L1 re- tion with Lamin A/C to promote L1 transcriptional silenc- pression, we questioned whether SIRT7 depletion increases ing and repress retrotransposition (Figure 6D). L1 retrotransposition. To answer this question, WT and SirT7−/− -derived cells were transfected with an L1-GFP retrotransposition reporter (85) (Figure 6C and Supple- DISCUSSION mentary Figure S9). In this system, GFP expression only occurs after a successful retrotransposition event, which Here we provide evidence supporting a new L1 transcrip- is measured by fluorescence-activated cell sorting (FACS). tional silencing mechanism that involves SIRT7 acting as a Importantly, SIRT7 deficiency resulted in a significant in- chromatin modifier and as a tethering factor of chromatin crease in the frequency of de novo retrotransposition events to the nuclear lamina. Sirtuins have multiple enzymatic ac- in SIRT7-depleted cells. We cannot rule out that the ob- tivities including protein deacylation and mono-ADP ri- bosylation (87). Our results reveal that SIRT7-dependent Nucleic Acids Research, 2019, Vol. 47, No. 15 7879 A B C Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 D E Figure 4. H3K18Ac modulates Lamin A/C levels at LINE-1 elements. (A) H3K18Ac ChIP-qPCR in HT1080 cells treated with siRNA Scramble (control), siRNA SirT7 (SirT7 KD) or Lamin A/C siRNA (Lamin A/C KD) using primers specific for human L1Hs consensus sequence and normalized to input DNA (Left). (Right) WB showing SirT7 and Lamin depletion. (B) Lamin ChIP in WT and SirT7−/− MEF cells overexpressing wild-type H3 (H3K18WT), and the histone mutants H3K18Q or H3K18R (acetylated, or deacetylated H3K18 residues, respectively). qPCR performed as in panel (A). (C) Western blot analysis of endogenous chromatin-bound SIRT7 and Lamin A/C from primary WT and SirT7−/− MEFs transduced with the indicated histone H3 vectors. Shown is anti-Myc as control for the ectopic expression of H3 vectors, and H3 for loading control. One representative blot is shown from two independent experiments. (D and E) Pull-down experiments using biotinylated peptides (H3, H3K18Ac and H3K18me2) incubated with purified Lamin A protein from HEK-293F cells transiently transfected with Lamin A HA-tagged. (D) Representative western blot analysis of pulled-down Lamin-HA (upper panel) and ponceau staining of the biotinylated peptides as loading control (bottom panel). Input represents 1% of the Lamin A HA-tagged loaded in the pulldowns. (E) Quantification by densitometry of the experiments in panel (D). The relative binding is expressed as fraction of Lamin A pulled-down normalized to the total biotinylated peptide used. Data represent mean ± SEM of three independent experiments per condition except otherwise indicated. **P < 0.01; ***P < 0.001 by ANOVA single factor. H3K18Ac deacetylation is a novel epigenetic mechanism while more ancient ones are under KAP-1-mediated repres- for the suppression of L1 transcription in mouse embry- sion (34). Separate mechanisms might be also responsible onic fibroblasts and human cell lines. Studies looking at the for the suppression of evolutionary distinct L1s in more breadth of human and mouse L1 variation though a phy- committed/differentiated cells. In support of this notion, logenetic lens have shown that L1 subfamilies can be dis- our ChIP-seq analysis show that SIRT7 specifically binds tinguished by features in the promoter (5 UTR) region and the most recently evolved L1 families in both human and grouped by evolutionary age (88,89). Upon deeper inspec- mouse cells (Figure 1D and Supplementary Figure S2B). tion of these characteristics, it becomes clear that new L1 SIRT7 is recruited to chromatin by its interaction with tran- subfamilies emerge from older, more repressed, subfamilies scription factors (90). Despite SIRT7 associates with the en- and quickly expand via retrotransposition before the cell tire L1 sequence, there is a peak of SIRT7 accumulation at can develop a working response. This classic evolutionary the 5 UTR (Supplementary S1C). Therefore, it is possible arms race has produced an array of cellular defenses against that SIRT7 is recruited through a yet unknown partner at L1 tailored to the features unique to a particular L1 sub- this specific site and it is then extended to the adjacent ORF. family. Indeed, the epigenetic regulation of L1 expression in Previous reports have already linked sirtuins enzymatic mammalian embryonic stem cells can vary among different activities to the regulation of retrotransposon in fission L1 families: young L1 elements seem to be mostly silenced yeast (91). In differentiated mammalian cells, only SIRT6 through SUV39h1/2-dependent H3K9me3 deposition (31), has been associated with L1 transcriptional repression (92). 7880 Nucleic Acids Research, 2019, Vol. 47, No. 15 A MEFs Liver Testis Heart B ** * Relative L1MdA expression * *** ** 1.2 1.6 * 1.8 *** 1.5 5.0 WT *** *** SirT7-/- 1.0 Relative expression 1.5 1.2 4.0 1.2 ** 0.8 1.2 0.9 3.0 0.8 0.9 0.6 0.6 2.0 0.6 0.4 0.4 0.3 1.0 0.3 0.2 0.0 0.0 0.0 0.0 0.0 T - /- T7 dA dA dA dA P P P P f f f f W dG dG dG dG 7 TB TB TB TB rT R M M M M M M M M SI Si L1 L1 L1 L1 L1 L1 L1 L1 + Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 7 -/- rT Si C D H3K18WT H3K18Q E F * *** 1.8 3.0 *** 2.5 *** *** Relative L1Hs expression * 1.6 Relative L1Hs expression Relative Luminescence 1.6 ** Relative expression 2.5 2.0 1.4 1.4 1.2 2.0 1.2 1.0 1.5 1.0 1.5 0.8 0.8 1.0 0.6 1.0 0.6 0.4 0.5 0.4 0.5 0.2 0.2 0.0 0.0 0.0 0.0 dA f f l KD l KD dT dG t ro tro M M on M T on R R 7 C L1 W L1 rT 18 18 L1 C A/ C 18 Si 3K 3K in 3K m H H La H l+ + l+ KD tro tro on C on A/ C C in m La Figure 5. SIRT7 represses L1 expression through regulation of H3K18Ac levels. (A and B) mRNA expression of the indicated L1 families measured by qRT-PCR in MEFs and tissues from WT and SirT7−/− mice (A), and from SirT7−/− derived MEFs overexpressing recombinant SIRT7 protein or empty vector (B). Data represent the mean ± SEM of three biological replicates. P values by ANOVA single Factor (P < 0.05; **P < 0.01). (C) Luciferase activity of a 5’L1 UTR-FLuc reporter construct in HCT116 2C6.3 cells transfected with Scramble (Control) or SirT7 siRNA (SirT7 KD). Data represent the mean luminescence normalized by cell viability ± SEM of four independent transfection replicates. (D) qRT-PCR of the indicated L1 families from WT MEFs overexpressing wild-type histone H3 (H3K18WT) and the histone mutant H3K18Q (acetylated H3K18 residue). (E) qRT-PCR expression of L1Hs transcripts in HT1080 cells transduced with Scramble (Control) or Lamin A/C siRNA (Lamin A/C KD). WB showing Lamin depletion (right). (F) qRT-PCR of L1Hs transcripts in HT1080 cells overexpressing wild-type histone H3 (H3K18WT) and the histone mutant H3K18R (deacetylated H3K18 residue) and transduced with Scramble (Control) or Lamin A/C siRNA (Lamin A/C KD) as indicated. (D and F) Data represent the mean ± SEM of N ≥ 3 independent experiments. P values by paired T-test (*P < 0.05; **P < 0.01; ***P < 0.001). Mechanistically, SIRT6 is able to bind L1 where it mono- H3K18Ac has been previously linked to transcriptional ADP-ribosylates KAP-1, facilitating the formation of the regulation (76,77,93). How H3K18Ac levels mechanisti- KAP-1/HP-1␣ corepressor complex at L1, which has been cally contribute to regulate transcription is only under- linked to the regulation of middle-aged subfamilies of L1 stood in the case of pericentric chromatin, in which SIRT6- (34). Despite that SIRT6 has been shown to deacetylase dependent H3K18 deacetylation stabilizes the transcrip- H3K18Ac in pericentric heterochromatin, loss of SIRT6 tional repressor KAP1 (93). Our results suggest that H3K18 did not change H3K18Ac levels at L1 (93) suggesting that deacetylation is required for the tethering of L1s to the nu- SIRT6 and SIRT7 have developed different mechanisms clear periphery by their association with Lamin A/C (Fig- to mediate L1 transcriptional repression. It is tempting to ure 4), expanding the molecular mechanism of action of speculate that SIRT6 and SIRT7 are specialized in the regu- this histone modification. Consistently, ChIP-seq analyses lation of different L1 families according to their evolution- based on our and previous reports (44) indicate L1 asso- ary age. Future work will be required to examine SIRT6 and ciation with LADs is restricted to SIRT7-bound young L1 SIRT7 occupancy at L1 loci in different cellular context to families, which are still capable of transcription (Figure 1D determine the level of redundancy of these sirtuins in the re- and Supplementary Figure S2B). In addition, we show that pression of L1s. Nevertheless, our results expand the role of depletion of Lamin A/C results in transcriptional upregu- sirtuins in the repression of mobile elements, and strongly lation of these L1 subfamilies (Figure 5E), and that H3K18 support histone acetylation as an important player in L1 deacetylation is not sufficient to repress L1 expression in the transcriptional regulation. absence of Lamin A/C (Figure 5F). These results strongly support that L1-Lamin A/C association is necessary for L1 Nucleic Acids Research, 2019, Vol. 47, No. 15 7881 Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 Figure 6. Increase L1 retrotransposition in the absence of SIRT7. (A and B) Chromatin accessibility assay in WT and SirT7−/- cells. (A) Isolated nuclei from WT and SIRT7 KO cells were digested with 4 units of microccocal nuclease (MNase) for 1.5, 3, 5 and 10 min. Different nucleosomal fractions (mono, di and polynucleosomes) were separated by gel electrophoresis. (Top) Representative western blot. (Bottom) Band density quantification using ImageJ software. Results are expressed as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01 by ANOVA single factor. (B) qPCR showing protection of MNase-digested DNA at the 5 min time point relative to non-treated DNA at indicated loci in WT and SirT7−/- cells. Primers were designed to target the promoters of the L1MdA consensus sequence and the ␤-globin gene to obtain PCR amplicons longer than the length of a single nucleosome. Reduced amplification indicates more MNase digestion, which is interpreted as the presence of more accessible chromatin at the assayed loci. Data represent mean ± SEM of three independent experiments per condition after normalization to actin gene expression. *P < 0.05; **P < 0.01 by ANOVA single factor. (C) FACS analysis showing number of GFP+ events per 105 cells in WT and SirT7−/− MEFs. Quantification (mean ± SEM of 4 samples/genotype. *P = 0.047 by one tail Student’s t-test). (D) Mechanistic model: (Top) SIRT7 is found at L1 loci, where it deacetylates H3K18Ac. Lamin A binds SIRT7 and to deacetylated H3K18 L1 chromatin. (Bottom) In this way, SIRT7 promotes the recruitment of L1 elements to the nuclear periphery resulting in the generation of a repressive chromatin environment that prevents L1 expression. Upon SIRT7 depletion, L1 elements are relocated toward the nuclear interior concomitantly to their activation. repression, which is in agreement with the common vision ner nuclear transmembrane proteins such as LAP2␤ (105) of the nuclear lamina as a transcriptional repressive envi- and emerin (106), along with specific transcription factors ronment and a major player for heterochromatin mainte- such as cKROX (51). However, whether histone acetylation nance (46). status favors the interaction of Lamin A/C to chromatin Mechanistically, Lamin A/C promotes the anchoring of has not directly been tested. We propose that the acetyla- heterochromatin to the nuclear periphery (94,95,96). The tion state at H3K18, which is regulated at L1s by SIRT7, mechanisms by which Lamin A/C tethers heterochromatin promotes the affinity of chromatin to Lamin A/C. Indeed, to the nuclear rim are just starting to be revealed, for review we show that Lamin A/C specifically interact with unmod- see (97). Lamin A/C is able to bind DNA (98), core histones ified histone H3, whereas acetylation on H3K18 disrupts (80) and several inner nuclear membrane proteins (99). this interaction (Figure 4D and E). We cannot rule out However, the effects of histone post-translational modifica- that L1 tethering to the nuclear periphery can be mediated tions in Lamin A/C binding to chromatin are largely un- by SIRT7 interaction with nuclear membrane proteins or known. LADs are enriched in histone marks that are as- other bridge factors. In this regard, our results indicate that sociated with transcriptional repression such as H3K9me3 Lamin A/C and SIRT7 proteins can directly interact (Fig- and H3K27me3 (97). Depletion of the methyltransferases ure 2B and Supplementary Figure S3), and suggest that this responsible for these histones marks results in reduced het- interaction could cooperate with SIRT7-mediated H3K18 erochromatin at the nuclear periphery (100). However, the deacetylation to position L1s at the nuclear periphery. In- methylation status of these histone marks does not affect deed, the interplay between the nuclear lamina and Sirtu- Lamin A interaction with the histone H3 tail (101). His- ins has been previously observed. SIRT1 is recruited to the tone deacetylation is also important for gene positioning at nuclear envelope through direct interaction with Lamin A, the nuclear periphery and gene repression (51,84,102,103). which in turn activates SIRT1 deacetylase activity toward The underlying molecular players are largely unknown and substrates such as p53 (79). Similarly, SIRT6 deacetylase ac- it has mainly been attributed to HDAC3 activity. Cells lack- tivity is enhanced upon SIRT6–Lamin A interaction, which ing HDAC3 have a significant decrease in the amount of regulates SIRT6 recruitment to chromatin upon DNA dam- heterochromatin, especially at the periphery (104). HDAC3 age (78). However, whether this association occurs at chro- is recruited to the nuclear lamina via its interaction with in- matin regions located at the nuclear periphery is unknown. 7882 Nucleic Acids Research, 2019, Vol. 47, No. 15 In our model, the presence of Lamin A/C does not impact DATA AVAILABILITY SIRT7 deacetylase activity as the levels of H3K18Ac at L1 ChIP-sequence data used in this study is available at GEO loci do not change upon Lamin A/C knockdown (Figure Accession Number GSE106964. 4A). However, we do not know if, alternatively, Lamin A/C Lamin B1 DamID performed in MEFs was obtained could be a substrate for SIRT7 enzymatic activity. In this from GEO Series GSE36132. Called peaks for ChIP of regard, total chromatin levels of Lamin A/C are similar in H3K27Ac, H3K4me1, H3K4me3 and POLR2A were ob- WT and SirT7−/− MEFs (Figure 2D). Future work will tained from the ENCODE project under accession num- be required to fully understand the molecular interplay be- bers ENCSR000CDI, ENCSR000CAZ, ENCSR000CBA tween SIRT7 and Lamin A/C in the regulation of L1 ex- and ENCSR000CBX. pression. On top of its role in transcriptional regulation, Lamin is essential to maintain nuclear organization (107). The SUPPLEMENTARY DATA anchoring of specific chromatin domains to the nuclear Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 lamina was one of the first mechanistic explanations for Supplementary Data are available at NAR Online. how nuclear architecture is established and maintained (42). Lamin-mediated regulatory mechanisms become most rel- FUNDING evant considering its disruption in cancer and aging cells (108,109,110). Diseases associated with Lamin mutations, Human Genetics Institute of New Jersey (HGINJ) (to such as progeria, provide evidence that Lamin depletion L.S.); Spanish Ministry of Education, Culture, and Sports causes large scale reorganization of LAD chromatin struc- [EX-2010-278 to B.N.V.]; HGINJ; FPI fellowship [BES- ture, including regions of H3K4me3 and H3K27me3 marks 2012-052200 to N.G.S.]; The Spanish Ministry of Econ- (111). We provide several lines of evidence supporting that omy and Competitiveness (MINECO) [SAF2014-55964-R, L1 association with the nuclear lamina becomes disrupted SAF2017-88975-R to A.V.]; La Marató de TV3, Spain; upon SIRT7 depletion. Considering the abundance of these National Institute of General Medical Sciences (NIGMS) elements in the mammalian genome (1,2), disruption of the Grant [P50GM107632 to W.A.]. Funding for open access anchoring of L1 to Lamin A/C might impact the localiza- charge: Internal Institute grant. tion to or away from the nuclear periphery of adjacent chro- Conflict of interest statement. None declared. matin domains inducing large-scale chromatin organization changes. Interestingly, recent evidence has linked H3K18Ac REFERENCES with early chromatin conformation events (112). The au- thors found H3K18Ac at chromatin nucleation sites, which 1. Lander,E.S., Linton,L.M., Birren,B., Nusbaum,C., Zody,M.C., Baldwin,J., Devon,K., Dewar,K., Doyle,M., FitzHugh,W. et al. dictate the emergence of chromatin architecture that oc- (2001) Initial sequencing and analysis of the human genome. curs at the onset of transcription activation in the zygote. Nature, 409, 860–921. It is known that in this window of time L1 gets reactivated 2. Waterston,R.H., Lindblad-Toh,K., Birney,E., Rogers,J., Abril,J.F., (113). It is possible that L1 activation is a consequence of Agarwal,P., Agarwala,R., Ainscough,R., Alexandersson,M., An,P. the early chromatin conformation events and/or, as recently et al. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature, 420, 520–562. suggested (114), necessary for these events to occur. Never- 3. Kazazian,H.H. Jr. (2004) Mobile elements: drivers of genome theless, inherent in our findings is that the loss of Lamin evolution. Science, 303, 1626–1632. A/C observed in aging and cancer cells will reduce the ca- 4. Branciforte,D. and Martin,S.L. (1994) Developmental and cell type pacity for SIRT7 to anchor L1 elements to nuclear lamina, specificity of LINE-1 expression in mouse testis: implications for transposition. Mol. Cell Biol., 14, 2584–2592. promote L1 activation and its associated phenotypes such 5. Trelogan,S.A. and Martin,S.L. (1995) Tightly regulated, as increased DNA damage and altered gene expression pro- developmentally specific expression of the first open reading frame files. from LINE-1 during mouse embryogenesis. Proc. Natl. Acad. Sci. Recent reports demonstrated that activation of endoge- U.S.A., 92, 1520–1524. nous L1 elements evolves progressively during cell senes- 6. Ergun,S., Buschmann,C., Heukeshoven,J., Dammann,K., Schnieders,F., Lauke,H., Chalajour,F., Kilic,N., Stratling,W.H. and cence resulting in the activation of sterile inflammation Schumann,G.G. (2004) Cell type-specific expression of LINE-1 open (115,116). These reports mechanistically establish L1 tran- reading frames 1 and 2 in fetal and adult human tissues. J. Biol. scriptional dysregulation as a plausible component of the Chem., 279, 27753–27763. type I interferon response activation and the senescence- 7. Rosser,J.M. and An,W. (2012) L1 expression and regulation in humans and rodents. Front. Biosci., 4, 2203–2225. associated secretory phenotype and therefore, with the pro- 8. Baillie,J.K., Barnett,M.W., Upton,K.R., Gerhardt,D.J., cess of aging. We previously provided evidence of an accel- Richmond,T.A., De Sapio,F., Brennan,P.M., Rizzu,P., Smith,S., erated aging phenotype and compromised genome integrity Fell,M. et al. (2011) Somatic retrotransposition alters the genetic in the SIRT7 knockout mouse, which was in part due to im- landscape of the human brain. Nature, 479, 534–537. paired DNA damage repair (53). In this report we unveiled a 9. Muotri,A.R., Chu,V.T., Marchetto,M.C., Deng,W., Moran,J.V. and Gage,F.H. (2005) Somatic mosaicism in neuronal precursor cells novel interplay between SIRT7-mediated chromatin regula- mediated by L1 retrotransposition. Nature, 435, 903–910. tion, Lamin A/C and repression of L1 elements, which adds 10. Coufal,N.G., Garcia-Perez,J.L., Peng,G.E., Yeo,G.W., Mu,Y., to the molecular mechanisms of SIRT7-mediated genome Lovci,M.T., Morell,M., O’Shea,K.S., Moran,J.V. and Gage,F.H. regulation. Taken together, our findings further establish (2009) L1 retrotransposition in human neural progenitor cells. Nature, 460, 1127–1131. SIRT7 as safeguard of genome integrity and a potential 11. Evrony,G.D., Cai,X., Lee,E., Hills,L.B., Elhosary,P.C., therapeutic target to ameliorate age-related pathologies. Lehmann,H.S., Parker,J.J., Atabay,K.D., Gilmore,E.C., Poduri,A. et al. (2012) Single-neuron sequencing analysis of L1 Nucleic Acids Research, 2019, Vol. 47, No. 15 7883 retrotransposition and somatic mutation in the human brain. Cell, 33. Aravin,A.A., Sachidanandam,R., Bourc’his,D., Schaefer,C., 151, 483–496. Pezic,D., Toth,K.F., Bestor,T. and Hannon,G.J. (2008) A piRNA 12. Faulkner,G.J., Kimura,Y., Daub,C.O., Wani,S., Plessy,C., pathway primed by individual transposons is linked to de novo Irvine,K.M., Schroder,K., Cloonan,N., Steptoe,A.L., Lassmann,T. DNA methylation in mice. Mol. Cell, 31, 785–799. et al. (2009) The regulated retrotransposon transcriptome of 34. Castro-Diaz,N., Ecco,G., Coluccio,A., Kapopoulou,A., mammalian cells. Nat. Genet., 41, 563–571. Yazdanpanah,B., Friedli,M., Duc,J., Jang,S.M., Turelli,P. and 13. Han,J.S., Szak,S.T. and Boeke,J.D. (2004) Transcriptional disruption Trono,D. (2014) Evolutionally dynamic L1 regulation in embryonic by the L1 retrotransposon and implications for mammalian stem cells. Genes Dev., 28, 1397–1409. transcriptomes. Nature, 429, 268–274. 35. Day,D.S., Luquette,L.J., Park,P.J. and Kharchenko,P.V. (2010) 14. Ohms,S. and Rangasamy,D. (2014) Silencing of LINE-1 Estimating enrichment of repetitive elements from high-throughput retrotransposons contributes to variation in small noncoding RNA sequence data. Genome Biol., 11, R69. expression in human cancer cells. Oncotarget, 5, 4103–4117. 36. Kannan,M., Li,J., Fritz,S.E., Husarek,K.E., Sanford,J.C., 15. Goodier,J.L., Ostertag,E.M. and Kazazian,H.H. Jr. (2000) Sullivan,T.L., Tiwary,P.K., An,W., Boeke,J.D. and Symer,D.E. Transduction of 3 -flanking sequences is common in L1 (2017) Dynamic silencing of somatic L1 retrotransposon insertions retrotransposition. Hum. Mol. Genet., 9, 653–657. reflects the developmental and cellular contexts of their genomic 16. Pickeral,O.K., Makalowski,W., Boguski,M.S. and Boeke,J.D. (2000) integration. Mob. DNA, 8, 8. Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 Frequent human genomic DNA transduction driven by LINE-1 37. Garcia-Perez,J.L., Morell,M., Scheys,J.O., Kulpa,D.A., Morell,S., retrotransposition. Genome Res., 10, 411–415. Carter,C.C., Hammer,G.D., Collins,K.L., O’Shea,K.S., 17. Dewannieux,M., Esnault,C. and Heidmann,T. (2003) Menendez,P. et al. (2010) Epigenetic silencing of engineered L1 LINE-mediated retrotransposition of marked Alu sequences. Nat. retrotransposition events in human embryonic carcinoma cells. Genet., 35, 41–48. Nature, 466, 769–773. 18. Dewannieux,M. and Heidmann,T. (2005) L1-mediated 38. Pal,S. and Tyler,J.K. (2016) Epigenetics and aging. Sci. Adv., 2, retrotransposition of murine B1 and B2 SINEs recapitulated in e1600584. cultured cells. J. Mol. Biol., 349, 241–247. 39. Lopez-Otin,C., Blasco,M.A., Partridge,L., Serrano,M. and 19. Gasior,S.L., Wakeman,T.P., Xu,B. and Deininger,P.L. (2006) The Kroemer,G. (2013) The hallmarks of aging. Cell, 153, 1194–1217. human LINE-1 retrotransposon creates DNA double-strand breaks. 40. Brunet,A. and Berger,S.L. (2014) Epigenetics of aging and J. Mol. Biol., 357, 1383–1393. aging-related disease. J. Gerontol. A Biol. Sci. Med. Sci., 69(Suppl. 20. Wallace,N.A., Belancio,V.P. and Deininger,P.L. (2008) L1 mobile 1), S17–S20. element expression causes multiple types of toxicity. Gene, 419, 41. Baylin,S.B. and Jones,P.A. (2016) Epigenetic determinants of cancer. 75–81. Cold Spring Harb.Perspect. Biol., 8, a019505. 21. Belgnaoui,S.M., Gosden,R.G., Semmes,O.J. and Haoudi,A. (2006) 42. Guelen,L., Pagie,L., Brasset,E., Meuleman,W., Faza,M.B., Human LINE-1 retrotransposon induces DNA damage and Talhout,W., Eussen,B.H., de Klein,A., Wessels,L., de Laat,W. et al. apoptosis in cancer cells. Cancer Cell Int, 6, 13. (2008) Domain organization of human chromosomes revealed by 22. Belancio,V.P., Roy-Engel,A.M., Pochampally,R.R. and Deininger,P. mapping of nuclear lamina interactions. Nature, 453, 948–951. (2010) Somatic expression of LINE-1 elements in human tissues. 43. Peric-Hupkes,D., Meuleman,W., Pagie,L., Bruggeman,S.W., Nucleic Acids Res., 38, 3909–3922. Solovei,I., Brugman,W., Graf,S., Flicek,P., Kerkhoven,R.M., van 23. De Cecco,M., Criscione,S.W., Peterson,A.L., Neretti,N., Lohuizen,M. et al. (2010) Molecular maps of the reorganization of Sedivy,J.M. and Kreiling,J.A. (2013) Transposable elements become genome-nuclear lamina interactions during differentiation. Mol. active and mobile in the genomes of aging mammalian somatic Cell, 38, 603–613. tissues. Aging (Albany NY), 5, 867–883. 44. Meuleman,W., Peric-Hupkes,D., Kind,J., Beaudry,J.B., Pagie,L., 24. Rodic,N. and Burns,K.H. (2013) Long interspersed element-1 Kellis,M., Reinders,M., Wessels,L. and van Steensel,B. (2013) (LINE-1): passenger or driver in human neoplasms? PLos Genet., 9, Constitutive nuclear lamina-genome interactions are highly e1003402. conserved and associated with A/T-rich sequence. Genome Res., 23, 25. Scott,E.C., Gardner,E.J., Masood,A., Chuang,N.T., Vertino,P.M. 270–280. and Devine,S.E. (2016) A hot L1 retrotransposon evades somatic 45. Kind,J., Pagie,L., de Vries,S.S., Nahidiazar,L., Dey,S.S., Bienko,M., repression and initiates human colorectal cancer. Genome Res., 26, Zhan,Y., Lajoie,B., de Graaf,C.A., Amendola,M. et al. (2015) 745–755. Genome-wide maps of nuclear lamina interactions in single human 26. Shukla,R., Upton,K.R., Munoz-Lopez,M., Gerhardt,D.J., cells. Cell, 163, 134–147. Fisher,M.E., Nguyen,T., Brennan,P.M., Baillie,J.K., Collino,A., 46. van Steensel,B. and Belmont,A.S. (2017) Lamina-associated Ghisletti,S. et al. (2013) Endogenous retrotransposition activates domains: links with chromosome architecture, heterochromatin, and oncogenic pathways in hepatocellular carcinoma. Cell, 153, 101–111. gene repression. Cell, 169, 780–791. 27. Miki,Y., Nishisho,I., Horii,A., Miyoshi,Y., Utsunomiya,J., 47. Tumbar,T., Sudlow,G. and Belmont,A.S. (1999) Large-scale Kinzler,K.W., Vogelstein,B. and Nakamura,Y. (1992) Disruption of chromatin unfolding and remodeling induced by VP16 acidic the APC gene by a retrotransposal insertion of L1 sequence in a activation domain. J. Cell Biol., 145, 1341–1354. colon cancer. Cancer Res., 52, 643–645. 48. Tumbar,T. and Belmont,A.S. (2001) Interphase movements of a 28. Goodier,J.L. (2016) Restricting retrotransposons: a review. Mob. DNA chromosome region modulated by VP16 transcriptional DNA, 7, 16. activator. Nat. Cell Biol., 3, 134–139. 29. Lee,H.J., Hore,T.A. and Reik,W. (2014) Reprogramming the 49. Finlan,L.E., Sproul,D., Thomson,I., Boyle,S., Kerr,E., Perry,P., methylome: erasing memory and creating diversity. Cell Stem Cell, Ylstra,B., Chubb,J.R. and Bickmore,W.A. (2008) Recruitment to the 14, 710–719. nuclear periphery can alter expression of genes in human cells. PLos 30. Pezic,D., Manakov,S.A., Sachidanandam,R. and Aravin,A.A. Genet., 4, e1000039. (2014) piRNA pathway targets active LINE1 elements to establish 50. Reddy,K.L., Zullo,J.M., Bertolino,E. and Singh,H. (2008) the repressive H3K9me3 mark in germ cells. Genes Dev., 28, Transcriptional repression mediated by repositioning of genes to the 1410–1428. nuclear lamina. Nature, 452, 243–247. 31. Bulut-Karslioglu,A., De La Rosa-Velazquez,I.A., Ramirez,F., 51. Zullo,J.M., Demarco,I.A., Pique-Regi,R., Gaffney,D.J., Barenboim,M., Onishi-Seebacher,M., Arand,J., Galan,C., Epstein,C.B., Spooner,C.J., Luperchio,T.R., Bernstein,B.E., Winter,G.E., Engist,B., Gerle,B. et al. (2014) Suv39h-dependent Pritchard,J.K., Reddy,K.L. et al. (2012) DNA sequence-dependent H3K9me3 marks intact retrotransposons and silences LINE compartmentalization and silencing of chromatin at the nuclear elements in mouse embryonic stem cells. Mol. Cell, 55, 277–290. lamina. Cell, 149, 1474–1487. 32. Arand,J., Spieler,D., Karius,T., Branco,M.R., Meilinger,D., 52. David G Brownstein,R.b. (2003) Manipulating the mouse embryo: a Meissner,A., Jenuwein,T., Xu,G., Leonhardt,H., Wolf,V. et al. laboratory manual. third edition. by andras nagy, marina (2012) In vivo control of CpG and non-CpG DNA methylation by gertsenstein, kristina vintersten, and richard behringer. Q. Rev. DNA methyltransferases. PLos Genet., 8, e1002750. Biol., 78, 365–365. 7884 Nucleic Acids Research, 2019, Vol. 47, No. 15 53. Vazquez,B.N., Thackray,J.K., Simonet,N.G., Kane-Goldsmith,N., 74. Ford,E., Voit,R., Liszt,G., Magin,C., Grummt,I. and Guarente,L. Martinez-Redondo,P., Nguyen,T., Bunting,S., Vaquero,A., (2006) Mammalian Sir2 homolog SIRT7 is an activator of RNA Tischfield,J.A. and Serrano,L. (2016) SIRT7 promotes genome polymerase I transcription. Genes Dev., 20, 1075–1080. integrity and modulates non-homologous end joining DNA repair. 75. Wang,Z., Zang,C., Rosenfeld,J.A., Schones,D.E., Barski,A., EMBO J., 35, 1483–1595. Cuddapah,S., Cui,K., Roh,T.Y., Peng,W., Zhang,M.Q. et al. (2008) 54. Serrano,L., Martinez-Redondo,P., Marazuela-Duque,A., Combinatorial patterns of histone acetylations and methylations in Vazquez,B.N., Dooley,S.J., Voigt,P., Beck,D.B., Kane-Goldsmith,N., the human genome. Nat. Genet., 40, 897–903. Tong,Q., Rabanal,R.M. et al. (2013) The tumor suppressor SirT2 76. Barber,M.F., Michishita-Kioi,E., Xi,Y., Tasselli,L., Kioi,M., regulates cell cycle progression and genome stability by modulating Moqtaderi,Z., Tennen,R.I., Paredes,S., Young,N.L., Chen,K. et al. the mitotic deposition of H4K20 methylation. Genes Dev., 27, (2012) SIRT7 links H3K18 deacetylation to maintenance of 639–653. oncogenic transformation. Nature, 487, 114–118. 55. Han,J.S. and Boeke,J.D. (2004) A highly active synthetic 77. Shin,J., He,M., Liu,Y., Paredes,S., Villanova,L., Brown,K., Qiu,X., mammalian retrotransposon. Nature, 429, 314–318. Nabavi,N., Mohrin,M., Wojnoonski,K. et al. (2013) SIRT7 represses 56. Bosch-Presegue,L., Raurell-Vila,H., Thackray,J.K., Gonzalez,J., Myc activity to suppress ER stress and prevent fatty liver disease. Casal,C., Kane-Goldsmith,N., Vizoso,M., Brown,J.P., Gomez,A., Cell Rep., 5, 654–665. Ausio,J. et al. (2017) Mammalian HP1 isoforms have specific roles in 78. Ghosh,S., Liu,B., Wang,Y., Hao,Q. and Zhou,Z. (2015) Lamin a is Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 heterochromatin structure and organization. Cell Rep., 21, an endogenous SIRT6 activator and promotes SIRT6-Mediated 2048–2057. DNA repair. Cell Rep., 13, 1396–1406. 57. Bolger,A.M., Lohse,M. and Usadel,B. (2014) Trimmomatic: a 79. Liu,B., Ghosh,S., Yang,X., Zheng,H., Liu,X., Wang,Z., Jin,G., flexible trimmer for Illumina sequence data. Bioinformatics, 30, Zheng,B., Kennedy,B.K., Suh,Y. et al. (2012) Resveratrol rescues 2114–2120. SIRT1-dependent adult stem cell decline and alleviates progeroid 58. Langmead,B., Trapnell,C., Pop,M. and Salzberg,S.L. (2009) features in laminopathy-based progeria. Cell Metab., 16, 738–750. Ultrafast and memory-efficient alignment of short DNA sequences 80. Taniura,H., Glass,C. and Gerace,L. (1995) A chromatin binding site to the human genome. Genome Biol., 10, R25. in the tail domain of nuclear lamins that interacts with core histones. 59. Li,H., Handsaker,B., Wysoker,A., Fennell,T., Ruan,J., Homer,N., J. Cell Biol., 131, 33–44. Marth,G., Abecasis,G. and Durbin,R. (2009) The Sequence 81. Vakhrusheva,O., Braeuer,D., Liu,Z., Braun,T. and Bober,E. (2008) Alignment/Map format and SAMtools. Bioinformatics, 25, Sirt7-dependent inhibition of cell growth and proliferation might be 2078–2079. instrumental to mediate tissue integrity during aging. J. Physiol. 60. Zhang,Y., Liu,T., Meyer,C.A., Eeckhoute,J., Johnson,D.S., Pharmacol., 59(Suppl. 9), 201–212. Bernstein,B.E., Nusbaum,C., Myers,R.M., Brown,M., Li,W. et al. 82. Yoshizawa,T., Karim,M.F., Sato,Y., Senokuchi,T., Miyata,K., (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol., 9, Fukuda,T., Go,C., Tasaki,M., Uchimura,K., Kadomatsu,T. et al. R137. (2014) SIRT7 controls hepatic lipid metabolism by regulating the 61. Ramirez,F., Ryan,D.P., Gruning,B., Bhardwaj,V., Kilpert,F., ubiquitin-proteasome pathway. Cell Metab., 19, 712–721. Richter,A.S., Heyne,S., Dundar,F. and Manke,T. (2016) 83. Deininger,P., Morales,M.E., White,T.B., Baddoo,M., Hedges,D.J., deepTools2: a next generation web server for deep-sequencing data Servant,G., Srivastav,S., Smither,M.E., Concha,M., DeHaro,D.L. analysis. Nucleic Acids Res., 44, W160–W165. et al. (2017) A comprehensive approach to expression of L1 loci. 62. Quinlan,A.R. and Hall,I.M. (2010) BEDTools: a flexible suite of Nucleic Acids Res., 45, e31. utilities for comparing genomic features. Bioinformatics, 26, 84. Eskeland,R., Freyer,E., Leeb,M., Wutz,A. and Bickmore,W.A. 841–842. (2010) Histone acetylation and the maintenance of chromatin 63. Shin,H., Liu,T., Manrai,A.K. and Liu,X.S. (2009) CEAS: compaction by Polycomb repressive complexes. Cold Spring Harb. cis-regulatory element annotation system. Bioinformatics, 25, Symp. Quant. Biol., 75, 71–78. 2605–2606. 85. Newkirk,S.J., Lee,S., Grandi,F.C., Gaysinskaya,V., Rosser,J.M., 64. Dale,R.K., Matzat,L.H. and Lei,E.P. (2014) metaseq: a Python Vanden Berg,N., Hogarth,C.A., Marchetto,M.C.N., Muotri,A.R., package for integrative genome-wide analysis reveals relationships Griswold,M.D. et al. (2017) Intact piRNA pathway prevents L1 between chromatin insulators and associated nuclear mRNA. mobilization in male meiosis. Proc. Natl. Acad. Sci. U.S.A., 114, Nucleic Acids Res., 42, 9158–9170. E5635–E5644. 65. Dale,R.K., Pedersen,B.S. and Quinlan,A.R. (2011) Pybedtools: a 86. Suzuki,J., Yamaguchi,K., Kajikawa,M., Ichiyanagi,K., Adachi,N., flexible Python library for manipulating genomic datasets and Koyama,H., Takeda,S. and Okada,N. (2009) Genetic evidence that annotations. Bioinformatics, 27, 3423–3424. the non-homologous end-joining repair pathway is involved in 66. Thorvaldsdottir,H., Robinson,J.T. and Mesirov,J.P. (2013) LINE retrotransposition. PLos Genet., 5, e1000461. Integrative Genomics Viewer (IGV): high-performance genomics 87. Houtkooper,R.H., Pirinen,E. and Auwerx,J. (2012) Sirtuins as data visualization and exploration. Brief. Bioinform., 14, 178–192. regulators of metabolism and healthspan. Nat. Rev. Mol. Cell Biol., 67. Siepel,A., Bejerano,G., Pedersen,J.S., Hinrichs,A.S., Hou,M., 13, 225–238. Rosenbloom,K., Clawson,H., Spieth,J., Hillier,L.W., Richards,S. 88. Khan,H., Smit,A. and Boissinot,S. (2006) Molecular evolution and et al. (2005) Evolutionarily conserved elements in vertebrate, insect, tempo of amplification of human LINE-1 retrotransposons since the worm, and yeast genomes. Genome Res., 15, 1034–1050. origin of primates. Genome Res., 16, 78–87. 68. Siepel,A. and Haussler,D. (2004) Combining phylogenetic and 89. Sookdeo,A., Hepp,C.M., McClure,M.A. and Boissinot,S. (2013) hidden Markov models in biosequence analysis. J. Comput. Biol., 11, Revisiting the evolution of mouse LINE-1 in the genomic era. Mob. 413–428. DNA, 4, 3. 69. McLean,C.Y., Bristor,D., Hiller,M., Clarke,S.L., Schaar,B.T., 90. Blank,M.F. and Grummt,I. (2017) The seven faces of SIRT7. Lowe,C.B., Wenger,A.M. and Bejerano,G. (2010) GREAT improves Transcription, 8, 67–74. functional interpretation of cis-regulatory regions. Nat. Biotechnol., 91. Durand-Dubief,M., Sinha,I., Fagerstrom-Billai,F., Bonilla,C., 28, 495–501. Wright,A., Grunstein,M. and Ekwall,K. (2007) Specific functions 70. Jurka,J. (2000) Repbase update: a database and an electronic journal for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and of repetitive elements. Trends Genet., 16, 418–420. retrotransposon silencing. EMBO J., 26, 2477–2488. 71. Jurka,J., Kapitonov,V.V., Pavlicek,A., Klonowski,P., Kohany,O. and 92. Van Meter,M., Kashyap,M., Rezazadeh,S., Geneva,A.J., Walichiewicz,J. (2005) Repbase Update, a database of eukaryotic Morello,T.D., Seluanov,A. and Gorbunova,V. (2014) SIRT6 repetitive elements. Cytogenet. Genome Res., 110, 462–467. represses LINE1 retrotransposons by ribosylating KAP1 but this 72. Mates,L. (2011) Rodent transgenesis mediated by a novel repression fails with stress and age. Nat. Commun., 5, 5011. hyperactive Sleeping Beauty transposon system. Methods Mol. Biol., 93. Tasselli,L., Xi,Y., Zheng,W., Tennen,R.I., Odrowaz,Z., Simeoni,F., 738, 87–99. Li,W. and Chua,K.F. (2016) SIRT6 deacetylates H3K18ac at 73. Ostertag,E.M., Prak,E.T., DeBerardinis,R.J., Moran,J.V. and pericentric chromatin to prevent mitotic errors and cellular Kazazian,H.H. Jr. (2000) Determination of L1 retrotransposition senescence. Nat. Struct. Mol. Biol., 23, 434–440. kinetics in cultured cells. Nucleic Acids Res., 28, 1418–1423. Nucleic Acids Research, 2019, Vol. 47, No. 15 7885 94. Sullivan,T., Escalante-Alcalde,D., Bhatt,H., Anver,M., Bhat,N., 105. Somech,R., Shaklai,S., Geller,O., Amariglio,N., Simon,A.J., Nagashima,K., Stewart,C.L. and Burke,B. (1999) Loss of A-type Rechavi,G. and Gal-Yam,E.N. (2005) The nuclear-envelope protein lamin expression compromises nuclear envelope integrity leading to and transcriptional repressor LAP2beta interacts with HDAC3 at muscular dystrophy. J. Cell Biol., 147, 913–920. the nuclear periphery, and induces histone H4 deacetylation. J. Cell 95. Nikolova,V., Leimena,C., McMahon,A.C., Tan,J.C., Chandar,S., Sci., 118, 4017–4025. Jogia,D., Kesteven,S.H., Michalicek,J., Otway,R., Verheyen,F. et al. 106. Demmerle,J., Koch,A.J. and Holaska,J.M. (2012) The nuclear (2004) Defects in nuclear structure and function promote dilated envelope protein emerin binds directly to histone deacetylase 3 cardiomyopathy in lamin A/C-deficient mice. J. Clin. Invest., 113, (HDAC3) and activates HDAC3 activity. J. Biol. Chem., 287, 357–369. 22080–22088. 96. Solovei,I., Wang,A.S., Thanisch,K., Schmidt,C.S., Krebs,S., 107. Gruenbaum,Y. and Foisner,R. (2015) Lamins: nuclear intermediate Zwerger,M., Cohen,T.V., Devys,D., Foisner,R., Peichl,L. et al. filament proteins with fundamental functions in nuclear mechanics (2013) LBR and lamin A/C sequentially tether peripheral and genome regulation. Annu. Rev. Biochem., 84, 131–164. heterochromatin and inversely regulate differentiation. Cell, 152, 108. Zink,D., Fischer,A.H. and Nickerson,J.A. (2004) Nuclear structure 584–598. in cancer cells. Nat. Rev. Cancer, 4, 677–687. 97. Harr,J.C., Gonzalez-Sandoval,A. and Gasser,S.M. (2016) Histones 109. Sakthivel,K.M. and Sehgal,P. (2016) A novel role of lamins from and histone modifications in perinuclear chromatin anchoring: from genetic disease to cancer biomarkers. Oncol Rev, 10, 309. Downloaded from https://academic.oup.com/nar/article/47/15/7870/5521784 by guest on 20 February 2021 yeast to man. EMBO Rep., 17, 139–155. 110. Ghosh,S. and Zhou,Z. (2014) Genetics of aging, progeria and lamin 98. Stierle,V., Couprie,J., Ostlund,C., Krimm,I., Zinn-Justin,S., disorders. Curr. Opin. Genet. Dev., 26, 41–46. Hossenlopp,P., Worman,H.J., Courvalin,J.C. and Duband-Goulet,I. 111. Shah,P.P., Donahue,G., Otte,G.L., Capell,B.C., Nelson,D.M., (2003) The carboxyl-terminal region common to lamins A and C Cao,K., Aggarwala,V., Cruickshanks,H.A., Rai,T.S., McBryan,T. contains a DNA binding domain. Biochemistry, 42, 4819–4828. et al. (2013) Lamin B1 depletion in senescent cells triggers 99. Burke,B. and Stewart,C.L. (2013) The nuclear lamins: flexibility in large-scale changes in gene expression and the chromatin landscape. function. Nat. Rev. Mol. Cell Biol., 14, 13–24. Genes Dev., 27, 1787–1799. 100. Harr,J.C., Luperchio,T.R., Wong,X., Cohen,E., Wheelan,S.J. and 112. Hug,C.B., Grimaldi,A.G., Kruse,K. and Vaquerizas,J.M. (2017) Reddy,K.L. (2015) Directed targeting of chromatin to the nuclear Chromatin architecture emerges during zygotic genome activation lamina is mediated by chromatin state and A-type lamins. J. Cell independent of transcription. Cell, 169, 216–228. Biol., 208, 33–52. 113. Fadloun,A., Le Gras,S., Jost,B., Ziegler-Birling,C., Takahashi,H., 101. Bruston,F., Delbarre,E., Ostlund,C., Worman,H.J., Buendia,B. and Gorab,E., Carninci,P. and Torres-Padilla,M.E. (2013) Chromatin Duband-Goulet,I. (2010) Loss of a DNA binding site within the tail signatures and retrotransposon profiling in mouse embryos reveal of prelamin A contributes to altered heterochromatin anchorage by regulation of LINE-1 by RNA. Nat. Struct. Mol. Biol., 20, 332–338. progerin. FEBS Lett., 584, 2999–3004. 114. Jachowicz,J.W., Bing,X., Pontabry,J., Boskovic,A., Rando,O.J. and 102. Hendzel,M.J., Delcuve,G.P. and Davie,J.R. (1991) Histone Torres-Padilla,M.E. (2017) LINE-1 activation after fertilization deacetylase is a component of the internal nuclear matrix. J. Biol. regulates global chromatin accessibility in the early mouse embryo. Chem., 266, 21936–21942. Nat. Genet., 49, 1502–1510. 103. Zink,D., Amaral,M.D., Englmann,A., Lang,S., Clarke,L.A., 115. De Cecco,M., Ito,T., Petrashen,A.P., Elias,A.E., Skvir,N.J., Rudolph,C., Alt,F., Luther,K., Braz,C., Sadoni,N. et al. (2004) Criscione,S.W., Caligiana,A., Brocculi,G., Adney,E.M., Boeke,J.D. Transcription-dependent spatial arrangements of CFTR and et al. (2019) L1 drives IFN in senescent cells and promotes adjacent genes in human cell nuclei. J. Cell Biol., 166, 815–825. age-associated inflammation. Nature, 566, 73–78. 104. Bhaskara,S., Knutson,S.K., Jiang,G., Chandrasekharan,M.B., 116. Simon,M., Van Meter,M., Ablaeva,J., Ke,Z., Gonzalez,R.S., Wilson,A.J., Zheng,S., Yenamandra,A., Locke,K., Yuan,J.L., Taguchi,T., De Cecco,M., Leonova,K.I., Kogan,V., Helfand,S.L. Bonine-Summers,A.R. et al. (2010) Hdac3 is essential for the et al. (2019) LINE1 derepression in aged Wild-Type and maintenance of chromatin structure and genome stability. Cancer SIRT6-Deficient mice drives inflammation. Cell Metab., 29, Cell, 18, 436–447. 871–885.