SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.


Detection of HERV-K (HML-2) in AT/RT tumors. AT/RT tissues derived from 35 of 37 (95%) patients
demonstrated clusters of cells strongly expressing HML-2 env protein as measured by a monoclonal env antibody immunohistochemistry on a tissue microarray (Fig. 1, Supplemental Table S1, Supplemental Fig. S1). No env expression was observed in the normal cerebellum or in non-AT/RT brain samples (Fig. 1C, Supplemental Table S1, Supplemental Fig. S1). All ten of the AT/RT tissues originating from recurring tumors expressed HML-2 env (Supplemental Table S1). Placenta was stained as a positive control 34 due to its mixed composition of mesenchymal and progenitor cells (Supplemental Fig. S1).

Characterization of AT/RT cell lines.
Four AT/RT cell lines CHLA 02, CHLA 04, CHLA 05, and CHLA 06, were used in this study. All four cell lines lacked SMARCB1 expression 35,36 and expressed markers indicative of pluripotent cells, including: HML-2 env and OCT4 (a marker for stem cells), Pax6 (a neuroectodermal marker), NES (a neural stem cell marker, nestin), and ΒIII-tubulin (a neuronal marker) (Fig. 1d-l). Immunocytochemical characterization revealed that the AT/RT cells are a mixed population at various stages of differentiation but they all express HML-2 env in the plasma membrane (Fig. 1). In previous publications, identification of subgroups of AT/RT tumors and tumor cell lines were described with regard to genetic and epigenetic expression and specific therapeutic treatment responses 36 www.nature.com/scientificreports/ all classified as group 1 neurogenic tumors while CHLA 06 cell line was classified as group 2 due to differences in methylation patterns and distinct clinical and genotypic features 36,37 . With an updated analysis of AT/RT subgroups, group 1 neurogenic tumors have been classified as ATRT-SHH, sonic hedgehog, and group 2 tumors fall into either an ATRT-MYC, MYC proto-oncogene, or a ATRT-TYR, tyrosine, subtype 37 . Even though one of the four cell lines was classified as belonging to a different group, all four cell lines expressed HML-2 env suggesting env expression may be a broad marker for AT/RT tumors.
Cellular and extracellular vesicles express of HML-2 env. HML-2 env was identified in the cytoplasm and the plasma membrane by confocal microscopy in the CHLA 02 and CHLA 04 lines (Fig. 2a-c). Env was found to colocalize with CD98, a putative receptor for HML-2 env 35 . We further evaluated the CHLA 02 cells by electron microscopy and found no evidence of typical viral budding in approximately 100 cells. However, membrane-like structures resembling exocytosis of vesicles were noted (Fig. 2d,e). These appear to correspond to vesicles of similar morphology seen on confocal imaging to contain HML-2 env protein (Fig. 2). We confirmed that the atypical membrane structures observed by EM and confocal imaging contain HML-2 env protein by immunoblotting of enriched extracellular vesicles from cell free media via Nanotrap particles (Fig. 2f). (j) The same cell line also expresses βIII tubulin (red), a marker for neurons and HML-2 envelope (green). (k) CHLA 06 cells also express Nestin (red) and Oct4 (green). (l) The same cell line also expresses βIII tubulin (red) and HML-2 Env (green). Nuclei are stained with DAPI (blue). For IHC images, slides were scanned with a OptraScan Digital Pathology Scanner and processed in Adobe Photoshop for incorporation into the figure. For immunofluorescent images, an EVOS fluorescence microscope (AMG) was used. Images were acquired with the native software installed on the EVOS microscope and processed in Microsoft PowerPoint (https:// www. therm ofish er. com/ us/ en/ home/ techn ical-resou rces/ softw are-downl oads/ evos-fl-cell-imagi ng-system. html). and CHLA 04 cells led to significantly reduced transcription of HML-2 genes, suggesting SMARCB1 regulates HML-2 transcription (Fig. 3a). We observed increased binding of SMARCB1 to the HML-2 LTR compared to the housekeeping gene Hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Fig. 3b,c). HML-2 transcription increased following knockdown of SMARCB1 in both 293 T cells (Fig. 3d) and in NSCs (Fig. 3e).
RNA Sequencing Analysis of AT/RT Cell Lines. RNA expression profiles determined by next generation sequencing supported the immunohistochemical findings ( Fig. 1), indicating that each tumor originated from cells at different stages of development/differentiation. We found HML-2 repetitive elements within highly expressed genes in these cell lines. Two groups of highly expressed genes containing either sequences of HML-2 internal coding sequences (HML-2 int) or LTR5_Hs were found by DAVID functional clustering gene ontology analysis 38 . The first group contained genes related to Krüppel associated box (KRAB) and zinc finger proteins   Table S2). The genes in the second group were affiliated with the neuroblastoma breakpoint family (NBPF) (Supplemental Table S2); genes in this family, such as putative tumor suppressor NBPF1, which is highly expressed in the brain, have repetitive structures containing both intragenic and intergenic sequences 39 . Proteomic analysis of these cell lines confirmed the high expression observed by RNA sequencing analysis and the immunohistochemistry (IHC). Two groups of highly expressed proteins were found after analysis with DAVID of the CHLA 02 and CHLA 04 AT/RT cells by liquid chromatography mass spectrometry (LCMS). Both cell lines expressed proteins associated with the cellular immune response to viral proteins, in addition to neural stem cell marker (nestin), neuronal marker (βIII-tubulin), and glial cells (vimentin), which clustered as the first group (Supplemental Table S3). One of the proteins highly expressed in CHLA 02 and CHLA 04 AT/ RT cells is adenovirus early region 1B associated protein 5, a protein known to be activated during adenoviral infection, which has been suggested as a marker for undifferentiated embryonic stem cells 40 . An enrichment of proteins related to cellular adhesion, such as genes affiliated with cadherin cell-cell binding, cell-cell junction, and cell-cell adhesion was detected in the second group (Supplemental Table S3).
Identification of activated HML-2 loci due to SMARCB1 loss. Initial RNA sequencing analysis from all four AT/RT cell lines revealed the expression of several loci from both LTR5_Hs and HML-2 int (Fig. 4a-c). However, while at least 36 loci expressed transcripts of HML-2 internal sequences, a minority of loci encoded potential proteins (Supplemental Table S4). HML-2 elements were actively expressed from all chromosomes except for 14, 15, 18, and 21 ( Fig. 4a,b). The most actively transcribed HML-2 sequences were from chromosomes 1 and 19 each of which had 13 LTR5_Hs transcripts and 3 HML-2 int (Fig. 4a). The expressed transcripts aligning to elements encoding at least one full-length HML-2 gene are listed in Supplemental Table S4. Of these, only the locus on Chr19q11 encodes full length and potentially functional env protein (highlighted in Supplemental Table S4). Using a second analysis method, the TEtranscripts pipeline 41,42 (Supplemental Fig. S3), transcripts were detected from all chromosomes except 21 (Supplemental Fig. S3a). Three loci identified by   Table S4). Transcripts originating from both Chr19q11, Chr7p22.1a, and Chr7p22.1b were confirmed with RT-PCR and Sanger sequencing (Supplemental Table S5). The loci on chromosome 7 are a pair of duplications 24 whose sequences are nearly identical, and both contain an intact env gene. Both methods suggested env expression originated from Chr19q11, but with the TEtranscripts pipeline which uses expectation maximization algorithm also suggested the Chr7 loci. RNA from AT/RT cells was reverse transcribed and then amplified by PCR to confirm the RNA sequencing data (Fig. 4c, Supplemental Table S5). There are two types of HERV-K (HML-2) present in the human genome. Type 1 proviruses possess a 292 bp deletion in the env gene whereas type 2 proviruses have an intact env 14 . Type 1 proviruses express np9, a spliced transcript containing part of the env preceding the deletion, and a sequence 3' of the deletion 14 . Type 2 proviruses can express env as well as rec, another gene spliced from the beginning of the env transcript and an abbreviated 3' sequence of env. The primers used to amplify env were designed to amplify full-length transcript and its smaller spliced products rec, or np9, (Fig. 4c). Rec and np9 sequences were derived from multiple loci including Chr1p31.1, 1q22, 3q12.3, 3q21.2, 7p22.1a, 7p22.1b, 10q24.1, 11q23.3, 19q11, 19q13.12, and 22q11.21 (Supplemental Table S5). After aligning the Sanger sequences to the human genome and consensus env transcript, we compared the data to the expression profile obtained with RNA sequencing (Supplemental Table S4). Only three HML-2 loci which could make a functional full length env protein were identified, namely, Chr19q11, Chr7p22.1a, and Chr7p22.1b.

Effect of down regulation of HML-2 expression on AT/RT cells. HML-2 transcription was down-
regulated 48 h post transfection with a construct containing shRNA targeting HERV-K env or CRISPRi and gRNA targeting the HML-2 LTR and led to a significant decrease in HERV-K env transcription (Fig. 5a) and in the size of cellular aggregates ( Fig. 5a-d). Due to the importance of cellular adhesion for pluripotency [43][44][45] , we also analyzed cell viability following CRISPRi transfections and found a significant decrease at 96 h post transfection in CHLA 02 and CHLA 04 (Fig. 5e). Downregulation of HML-2 env with siRNA (Table 1) Table S6]. www.nature.com/scientificreports/ the shRNA (env) transfected cells (Fig. 5l). Targeting HML-2 env with shRNA or CRISPRi + gRNA resulted in decreased cell proliferation at 48 h and a significant decrease in env protein (Fig. 5m, Supplemental Fig. S2).

HML-2 expression induces cell proliferation through the NRAS pathway.
In previous studies of HML-2 downregulation in tumors, a concurrent decrease in N-Ras protein has been observed suggesting possible regulation of Ras genes by HML-2 29,30,46 . When CHLA 02 cells were treated with shRNA to HML-2 env, there was a significant decrease in N-Ras expression alongside HML-2 expression in the CHLA 02 and CHLA 04 cells (Fig. 6a). Forty-eight hours after CHLA 04 transfection with both the CRISPRi construct (+ gRNA targeting HML-2) and with an NRAS plasmid, higher transcription of HML-2 and NRAS were found by qRT-PCR relative to cells transfected with a pcDNA CAT which served as a control (Fig. 6b). This result indicates that NRAS overexpression is sufficient to restore HML-2 transcription. At 5 days post transfection, we observed a significant difference between both the viability and cell number of the NRAS and CAT transfected cells (Fig. 6c,d). Additionally, we observed the cell aggregates were significantly larger in the cells transfected with both CRISPRi and NRAS (Fig. 6e-g), demonstrating a restoration of the cell proliferation of the AT/RT cells.
The role of C-MYC in HERV-K transcription activation. Many transcription factor binding sites (TFBS) have been reported in the LTR of HERV-K (HML-2) loci; however, not all suggested TFBS have been confirmed 13,14 . In the LTR of Chr7p22.1a and Chr7p221b, both highly expressed loci in AT/RT, there are multiple potential C-MYC binding sites present 47 . Using chromatin immunoprecipitation of crosslinked DNA in 293 T cells, we observed significantly fewer HML-2 LTR5_Hs sequences bound to C-MYC protein when SMARCB1 was overexpressed (Fig. 7a,b). In the CHLA 02 AT/RT cells, the chromatin immunoprecipitation of C-MYC revealed fewer HERV-K LTR sequences bound to C-MYC in the absence of SMARCB1 expression (Fig. 7c,d). There was no significant difference between the SMARCB1 transfected and the control (CAT ) transfected cells when comparing C-MYC bound to a housekeeping gene, HPRT, in both the 293 T and CHLA 02 cells (Fig. 7b,d). We tested binding sites predicted via bioinformatics by PROMO 47 and other manually identified sites, using a pulldown using biotinylated HERV-K (HML-2) LTR sequences combined with purified C-MYC protein (Supplemental methods). C-MYC protein bound to multiple tested HERV-K sequences and was absent in the non-targeting control as well as the scrambled C-MYC sequences tested (Fig. 7e).

Discussion
We report here that the absence of SMARCB1 expression in AT/RT leads to aberrant HML-2 env expression in cells which are meant to differentiate along a neuronal lineage. Our study establishes that aberrant regulation of HML-2 expression at the level of chromatin remodeling is sufficient to drive cellular proliferation. In our study of  www.nature.com/scientificreports/ AT/RT, HML-2 activates cell proliferation through the Ras pathway, and the consequences of a targeted decrease in HML-2 expression can be reversed by an overexpression of NRAS. These results suggest that improper chromatin remodeling during early neural crest migration and neuronal differentiation provide a unique opportunity for HML-2 env expression to be retained via C-MYC binding and thereby maintain an undifferentiated phenotype. We show that HML-2 env downregulation through transcriptional repression reduces functional env protein, resulting in reduced proliferation and cytotoxicity. It has been suggested that some HERVs are selectively maintained in the human genome due to their highly regulated expression patterns during development 25,48 . In cells where HML-2 has been dysregulated during key developmental stages, development cannot proceed 25 . Using tissue arrays we found that 95% of tested AT/RT samples expressed HML-2 env. We confirmed expression of a pluripotency gene, OCT4, and HML-2 env genes in AT/RT cell lines by immunostaining. The cells also expressed neuronal stem cell and neuronal markers suggesting these cells are partially differentiated along a neuronal lineage. Consistent with the cellular heterogeneity and multiple stages of differentiation in AT/RT, HML-2 Env was not uniformly expressed in the tumors. Our results support previous findings in pluripotent stem cells, embryonic stem cells, embryonic carcinoma cells, and many other cancers 10,25,29,30,46,[49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67] .
We observed HML-2 Env expression in two AT/RT cell lines in extracellular vesicles by immunocytochemistry (ICC), Immunoblot, and with electron microscopy. The extracellular vesicles containing HML-2 Env could serve as a continuous signal to adjacent cells to maintain pluripotency thereby supporting tumor survival 68 . Further, the expression of proteins related to viral nucleoproteins, nucleocapsids, virions, cell-cell junctions, Biotinylated nucleotides with a non-targeting sequence, a scrambled canonical C-MYC binding sequence, or varying sequences from the Chr7p22.1a or Chr7p22.1b HML-2 LTR were incubated with recombinant C-MYC protein, washed, and run on an immunoblot to detect specific binding between C-MYC and different sequences. There is a white line between the the final LTR_939_944 and another ladder depicting that a portion of the gel was deleted to simplify the figure. The full gel image can be found in the supplemental information. (f) Contains a diagram of the mechanism by which HML-2 expression is regulated in the absence or presence of SMARCB1 protein. (f1) shows how HML-2 is actively expressed when C-MYC is bound to the LTR (promoter) when SMARCB1 protein is not expressed, while (f2a,b) depict the SMARCB1 mediated inhibition of C-MYC activation of HML-2 transcription. Graphs generated with Prism v9. (f) Generated in Microsoft PowerPoint.
[Error bars represent SEM. Statistics in Supplemental www.nature.com/scientificreports/ cadherin cell-cell binding, and cellular adhesion likely contribute to tumor survival and growth. The enrichment of cellular interaction and attachment proteins reflects the morphology of the cells in vitro and affirms the importance of plasma membrane contact between adjacent cells 44 . Cadherins are calcium dependent transmembrane proteins which are vital for cell adhesion 45 , and E-cadherin expression promotes adhesion between adjacent cells 43,69,70 . Cell-cell communication is key in a tumor to adapt to microenvironmental shifts. In AT/RT, extracellular vesicles with HML-2 env combined with the expression of cellular adhesion proteins may enable the tumor to efficiently adapt to its microenvironment. RNA sequencing showed that there were several active loci from which HML-2 env transcripts were produced. This was confirmed by cloning and sequencing the transcripts. All four cell lines expressed transcripts originating from three loci capable of encoding a full length Env protein on Chr19q11, Chr7p22.1a, or Chr7p22.1b. Targeting HML-2 env with shRNA has been previously shown to lead to reduced cellular proliferation and decreased metastatic potential of breast cancer in vitro through the inhibition of tumor associated genes like Ras, p-RSK, and p-ERK 29 . To further investigate the role of HML-2 env in AT/RT tumorigenesis, we modulated its expression via siRNA and shRNA and found a significant increase in cytotoxicity and decrease in cellular proliferation. After transfecting CHLA 02 with shRNA targeting HML-2 env, we observed a decrease in NRAS expression concurrent with the decrease in proliferation. An overexpression of NRAS was sufficient to rescue AT/RT cells from the loss of HML-2 env expression. Further, C-MYC binding to the HERV-K LTR is significantly increased in the absence of SMARCB1 resulting in high HERV-K expression. These findings suggest that HML-2 env plays a role in cellular viability and proliferation.
Our study establishes a new connection between chromatin remodeling and the regulation of endogenous retroviral elements in disease. We demonstrate that the regulation of HML-2 env expression early in development or differentiation is critical to maintenance of pluripotent stem cell identity in AT/RT. When the SWI/SNF chromatin remodeling complex loses a core subunit, SMARCB1, during development, the aberrant activation of human endogenous retroviral genes can contribute to the maintenance of stem cell features in cells which were meant to differentiate during CNS development. In addition to aberrant HML-2 transcription in the absence of SMARCB1, the transcription factor binding protein C-MYC binds the HML-2 LTR more frequently and upregulates its transcription. This study introduces a new mechanism of endogenous virus-mediated tumorigenesis. Identification of HML-2 expression as a marker of AT/RT following the loss of SMARCB1 is a primary step in establishing the role of endogenous retroelements in this central nervous system tumor development. Based on the findings in our study, HML-2 env expression may have a role as a marker for diagnosis of AT/RT in conjunction with SMARCB1. Further studies are needed to determine if HML-2 expression may be of prognostic significance and if it may be a therapeutic target. Immunofluorescence. The AT/RT cell lines CHLA 02, CHLA 04, CHLA 05, and CHLA 06 all grow in suspension; therefore, the cells were attached to the bottom of the plate with Matrigel for staining and grew overnight at 37 °C. Next, they were fixed with 4% paraformaldehyde (PFA) for 10 min. The cells were washed with 1X phosphate buffered saline (PBS) 2 × for 5 min each and then permeated with PBS with 0.05% triton X 100 (PBST) for 10 min. Cells were incubated with a blocking solution of 5% donkey or goat serum in PBS for 1 h and then washed with PBS 3 × for 5 min each and primary antibodies diluted in blocking solution were applied to the samples. After an overnight incubation at 4 °C, the cells were washed 3 × for 10 min each with PBS and then incubated with the appropriate secondary antibodies at 1:400 dilution for 1 h. Following the secondary antibody, the cells were treated with 4′,6-diamidino-2-phenylindole (DAPI) at 1:10,000 and washed 2 × with PBS before imaging with a fluorescent Zeiss confocal microscope (LSM510) in Fig. 2, images were acquired with Zeiss LSM 5 Image Browser and processed in Microsoft PowerPoint for figure creation. For Fig. 1 immunoflourescence images, an EVOS fluorescence microscope (AMG) was used. Images were acquired with the native software installed on the EVOS microscope and processed in Microsoft PowerPoint. Primary antibodies: AntiCD98 antibody ab108300; anti-Tubulin β3 801213 (Bio-legend), Anti-Oct4 antibody AB3209 (Millipore sigma), Anti-nestin Mab 5326 (Millipore), Anti-Pax6 NBP1 51622 (Novus biologicals). Secondary antibodies: Goat Anti-Rabbit IgG (Alexa Fluor 488, ab150077) Electron microscopy (EM). CHLA 02 cells were pelleted at 2,000 RPM for 5 min and 3 million cells were resuspended in 700 ul of media. 4% room temperature glutaraldehyde was made in a 1% cacodylate buffer. 700 ul of glutaraldehyde solution was added to cell/media mixture and gently mixed in Eppendorf tube. One drop of 22% albumin was added to tube and the sample was incubated at room temperature for 30 min. Following room temperature incubation, sample was stored at 4 °C until tubes are ready to be further processed for. www.nature.com/scientificreports/ EM. When ready for silver enhancement, cells were washed with deionized water thoroughly (5 × 5 min). Silver enhancement was then performed (under safety light) and cells were washed in water (at least 5 times over 10 min). The sample was washed in 0.1 M phosphate buffer, then with 0.2% OsO 4 in 0.1 M phosphate buffer for an additional 30 min. Next, dehydration was performed in ETOH and sample was embedded in resin. Samples were then cut with an ultramicrotome into ultrathin sections and are placed on a copper metal grid.

Methods
Transfections. The CHLA cell lines were transfected using the P3 nucleofector kit (Lonza, catalog no. V4XP-3012) and the CA -137 program at 1 ug of plasmid per million cells. After transfection, the cells were re-suspended in at least 4 mL of media. Sequences for shRNA, siRNA, and gRNAs are included in Table 1. The 293 T cells were transfected with Lipofectamine 3000 following manufacturer's recommendations using 5 ug of plasmid DNA per million cells. SMARCB1 lentiviral plasmid was obtained from Addgene. The pcDNA CAT plasmid contained a bacterial acetylcholine transferase. The CAT construct was used to control for effects of transfection as a plasmid of similar size to the SMARCB1 plasmid 71 Table 1).
SID is a chromatin remodeling protein 73,74 that prevents transcriptional machinery from accessing the DNA resulting in decreased expression of the targeted gene. As a control, cells were transfected with pcDNA and the transfection related toxicity was normalized accordingly in the CRISPRi transfected cells.
Immunoblotting. Immunoblotting was performed as done previously 26 with the exception of the transfer which was performed with the iBlot 2 (ThermoFisher, IB24001). In addition, a new polyclonal antibody to detect HML-2 SU (surface unit) Env was used (additional information and antibody validation can be found in Supplemental Fig. S2 and Supplemental methods.) All images were quantified with ImageJ. The HML-2 Env antibody used in Supplemental Fig. S2 targets the transmembrane portion of the protein. The transmembrane antibody was made using the immunogen CSKRKGGNVGKSKRD, used at a concentration of 1:1000, and was first mentioned in a previous manuscript 75 . Immunohistochemistry. Immunohistochemistry detected cells with Human endogenous retroviral Env expression primary antibody (Austral Biologicals, HERM 1855) at a 1:500 concentration within formalin fixed paraffin embedded tissue samples. Further detail can be found in Supplemental methods.
Enrichment of extracellular vesicles using nanotrap particles. Nanotrap particles, NT82 particles (#CN2010) and NT80 particles (#CN1030) (Ceres Nanosciences, Inc.) have been previously shown to be effective in the enrichment of extracellular vesicles from cell culture supernatant and patient biofluids 76,77 . Equal volumes of these two particles were combined with 1X PBS without calcium or magnesium to create a 30% slurry. For capture of extracellular vesicles (EVs) from cell culture supernatant, 30 µL of the 30% slurry was added to 1 mL of cell-free supernatant and rotated at 4 °C overnight. The following day, nanoparticles were pelleted at 10,000×g for 10 min at room temperature.
The resulting pellets were washed once with 1X PBS without calcium and magnesium and resuspended in 20 µL of Tris-Glycine SDS sample buffer with 10% 2-Mercaptoethanol. Samples were heated at 95 °C for 15 min with vortexing and loaded onto a 4-20% Tris/Glycine gel (Invitrogen). Gels were run at 100 V and transferred at 20 V for 7 min using iBlot 2 Gel Transfer Device. Membranes were blocked in 5% milk in PBST for 2 h at 4 °C. then incubated at 4 °C overnight in PBS-T with HML-2 envelope polyclonal antibody against the transmembrane protein (see Immunoblot section in Supplementary methods).
RT-PCR, PCR, and sequencing of env transcripts. RNA was isolated from iPSC, NSC, neuronal, and AT/RT cell lines with a Qiagen RNeasy kit (Qiagen, 74104) and reverse transcribed with the SuperScript First-Strand Synthesis RT-PCR kit (ThermoFisher Scientific, 11904018). Polymerase chain reactions were used to amplify HML-2 envelope transcripts with primers which target the full length env transcript (Table 1). Q5 high fidelity 2X master mix was used for the PCR with the following cycling conditions: 98 °C for 90 s, [98 °C 10 s, 55 °C 20 s, 72 °C 4 min] repeat 34 times, final extension at 72 °C for 6 min, 4 °C forever. The PCR primers were used to amplify the full length env gene as following: forward primer 5'-cccactagacatttgaagttctaca-3' , and reverse primer 5'-ggagtctcctatgtctacttcttt-3' . One primer aligned to the 3' LTR of the HML-2 element and the other primer was positioned 5' (upstream) of the start of the env gene. For the AT/RT cells, we cloned products into Topo-TA vectors (ThermoFisher Scientific, K203001) and sent them for Sanger sequencing to determine which env loci were actively transcribed in the samples. For the iPSCs, NSCs, and neurons, PCR products were run in a 2% agarose gel with GelStar Nucleic Acid Gel stain (Lonza, 50535) and the gel was imaged using Flourchem Protein Simple imager.
Alamar Blue viability assay. To distinguish the effect of HML-2 downregulation on AT/RT viability the Alamar Blue cell viability reagent (Thermofisher, DAL1100) was used. The fluorescence of the cell media resulting from the reduction of the Resazurin to resorufin indicated the percent of viable cells. Viability was calculated by comparing the fluorescence of each treatment concentration divided by the viability of the control treatment/ transfection. www.nature.com/scientificreports/ Each experiment was performed with at least three technical replicates and three biological replicates. Plates were read on the FlexStation 3 using Softmax Pro with the 'blue fluorescence' at 590 nm viability assay setting. Lentiviral production. Lipofectamine 3000 (Invitrogen L3000008) kit was used to transfect 293 T cells with either a lentiviral construct containing four repressive Sin3 domains, dead Cas9, and four gRNAs designed for HERVK LTR5_Hs or one without gRNA. The aforementioned CRISPRi construct was incorporated into a lentiviral vector, both with and without gRNA targeting the LTR5_Hs, and AT/RT cells were transduced with the lentivirus at MOIs of 0.1 and 0.5. Manufacturer's guidelines were followed for the transfection. Further information can be found in the Supplementary methods. RNA sequencing. Libraries were both prepared and sequenced at NYGC (www. nygen ome. org). For library preparation, the Illumina TruSeq Stranded Total RNA protocol was used (Illumina, San Diego, CA). Per sequencing, each library was paired-end sequenced (125 bp) for a target depth of 40 million reads (HiSeq 2500 Illumina); providing for a pair of .fastq files per library post CASAVA deplexing accessible at NCBI as GSE124210.
For further detail regarding analysis, please see Supplemental methods.
Chromatin immunoprecipitation (ChIP) and qRT-PCR. Mass spectrometry. In-gel digestion of PAGE fractionated cell lysates. The entire lane was excised from the PAGE gel, sectioned into 16 approximately equivalent sections, transferred to 1.5 mL microfuge tubes and subjected to a modified in-gel digestion protocol 78 . Briefly, excised bands were destained by adding 0.5 mL 100 mM NH 4 HCO 3 :CH 3 OH (50:50, v/v) and incubated in a thermomixer (800 RPM, 37 °C) for 60 min. Solvent decanted and discarded, process repeated as necessary. Gel-immobilized proteins were reduced by adding 0.5 mL 100 mM NH 4 HCO 3 , 5 mM DTT, incubated in a thermomixer (800 RPM, 50 °C) for 30 min, and allowed to cool to RT. Reduced proteins were carboxamidomethylated by buffer exchange into 0.5 mL 100 mM NH 4 HCO 3 , 12.5 mM IAM and incubated in the dark for 30 min at RT. Gel bands were washed extensively by sequential buffer exchange using 0.5 mL aliquots of 100 mM NH 4 HCO 3 , 100 mM NH 4 HCO 3 :CH 3 CN (50:50, v/v; 0.5 mL), and neat CH 3 CN (0.25 mL). In each instance, the gel bands were incubated in a thermomixer (800 RPM, 37 °C) and the solvent decanted and discarded.
Peptides were recovered by decanting the supernatant to a new 1.5 mL microfuge tube and extracting the gel bands with an equivalent volume of H 2 O:CH 3 CN:CF 3 CO 2 H (20:80:0.25; v/v/v) and sonicating for 30 min.
Extracts were combined with original supernatants and lyophilized to dryness using a vacuum centrifuge. All samples were desalted by solid phase extraction using TARGA C18 microspin columns according to manufacturer's instructions (The Nest Group). Desalted samples were resolubilized in 10 μL 1% CF 3 CO 2 H transferred to an autosampler vial.
Statistics. Prism 'analyze' tool used for all t-tests and ANOVA calculations. For all biological assays, at least 3 technical replicates and 3 biological replicates were measured and used for calculation of significance unless otherwise noted. P < 0.05 was considered significant for all statistical tests performed and generally * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001, and **** denotes P < 0.0001. See statistics table for further information (Supplemental Table S6).