Endogenous Retroviral Elements Research Articles

Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO): A single-arm, open-label window-of-opportunity trial of neoadjuvant abemaciclib in platinum-ineligible muscle invasive bladder cancer patients.

TPS606 Background: The standard of care for clinically localized muscle-invasive bladder cancer (MIBC) is neoadjuvant platinum-based combination chemotherapy followed by radical cystectomy (RC). Up to 40% of patients (pts) are ineligible to receive cisplatin and proceed to RC without any neoadjuvant therapy. We and others have demonstrated enrichment of molecular alterations in cell cycle genes in MIBC, including copy number losses of CDKN2A in 41% of pts. Abemaciclib is a unique CDK4/6 inhibitor with single agent activity and a target kinome distinct from other CDK4/6 inhibitors. We have demonstrated that CRISPR knockout of CDKN2A increases susceptibility to abemaciclib in bladder cancer cell lines. Beyond tumor-intrinsic effects, abemaciclib also modulates the tumor microenvironment (TME) via upregulating human endogenous retroviral elements and increasing T cell infiltration. Methods: Cell cycLe inhibitiON to target the EVolution of urOthelial cancer (CLONEVO) is a single arm, window-of-opportunity trial of neoadjuvant abemaciclib which will evaluate tumor cell and TME changes in response to abemaciclib. Enrolled pts must be ineligible for platinum-based neoadjuvant therapy for resectable MIBC. Pts receive abemaciclib (200 mg BID PO) for 4 weeks prior to RC. Tumor tissue collected via transurethral resection of bladder tumor (TURBT) and residual tumor at RC undergo single cell RNA sequencing and whole-exome sequencing. Patient-derived organoids and xenografts are generated for a co-clinical trial of abemaciclib alone or in combination. The primary endpoint is the measurement of changes in cell cycle dynamics. Secondary objectives are assessment of toxicity via NCI CTCAE v 5.0 and pathologic downstaging of MIBC. We will perform targeted sequencing of a panel of cell cycle genes in serial plasma and urine cell free DNA to evaluate changes in the variant allele fractions of somatic alterations. The novel design of this trial allows dynamic in vivo assessment of tumor changes and creates a new paradigm for studying tumor evolution in real time. Clinical trial information: NCT03837821.

High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model

Major efforts are underway to identify agents that can potentiate effects of immune checkpoint inhibition. Here, we show that ascorbic acid (AA) treatment caused genomewide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and interleukin 12 production by antigen-presenting cells compared with single-agent anti-PD1. These data indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides compelling rationale for testing combinations of high-dose AA and anti-PD1 agents in patients with aggressive B cell lymphoma as well as in preclinical models of other malignancies.

Chromatin Architecture Modulation in B-Cell Acute Lymphoblastic Leukemia Carrying DUX4 Fusions

B-cell acute lymphoblastic leukemia (B-ALL) carrying DUX4 fusions is a novel cluster of B-ALL. DUX4 fusions are generated from insertions of wild- type (WT) DUX4, mainly into the IGH locus.The translocation replaces the 3′ end of the WT DUX4 coding region with a fragment of IGH or another gene, producing DUX4 out-of-frame fusion proteins devoid of the C terminus of WT DUX4. Usually, WT DUX4 is expressed in germ cells in testis, while its expression is epigenetically repressed in somatic tissues. Recently, it is identified to plays a critical role in transcriptional programs at the cleavage of human fertilized egg. In B-ALL, DUX4-IGH (D-I) is shown to be essential for leukemic transformation; however, little is known about the mechanistic basis. Here in this study, we extensively investigated the biological effects of D-I. First, we assessed the role of D-I using in vitro cell culture assays with human cord blood (CB) CD34+ cells. Introduction of D-I significantly caused retention of the CD34+ cell population compared with the mock vector, even though it failed to preferentially promote differentiation toward B cell lineage in vitro. To analyze the epigenetic and transcription control by D-I, we performed chromatin immunoprecipitation coupled with sequencing (ChIP-seq) using cell lines. In NALM6, a B-ALL cell line carrying D-I, a subset of D-I binding sites is accompanied by H3K4me3 and H3K27ac. We also assessed the histone modification status in Reh cells, a B-ALL cell line without DUX4 fusions, and observed that active histone marks are detected after binding of ectopically expressed D-I. Nevertheless, RNA sequencing of NALM6 and Reh overexpressing D-I showed minimal activation of genes near the D-I binding sites compared with those of NALM6 overexpressing WT DUX4. WT DUX4 is known to preferentially bind and activate repeat elements, especially human endogenous retroviral (HERV) elements in embryonic cells. NALM6 cells overexpressing WT DUX4 showed a drastic increase in the expression of HERV elements, while NALM6 and Reh overexpressing D-I did not. The expression of HERV elements was not altered by D-I in all the genomic regions, and we did not observe increased expression of HERV elements in patient leukemia samples with DUX4 fusions as well. Furthermore, Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) showed that chromatin status was not affected by the binding of D-I at the D-I bound HERV element, indicating that transcriptional and insulating ability of WT DUX4 in these areas are lost in D-I. Next, we performed ATAC-seq using NALM6 cells, comparing the status between pre- and post- D-I knockdown. Genomic areas with decreased ATAC signal after knockdown of D-I are enriched in D-I binding sites, and ATAC signal was increased when we compared the status between pre- and post- induction of D-I in Reh cells. Through the immunoprecipitation of endogenous D-I in NALM6 cells, we identified SWI/SNF complex elements as binding partners of D-I, further highlighting the chromatin opening ability of D-I. Motif analysis of the genomic areas with decreased ATAC signal after knockdown of D-I identified only DUX4 motif as a significant motif, suggesting that D-I is not apparently cooperating with other transcription factors. On the other hand, ATAC signal was increased in substantial genomic areas after knockdown of D-I, and motif analysis identified SPI1, TCF3, and EBF1 motifs. Integrated analysis of transcriptome data also supports the idea that transcription factors related to B cell differentiation are repressed in the presence of D-I, and derepressed after knock down of D-I. Despite the attenuated transcriptional activity, B-ALL carrying DUX4 fusions manifests a characteristic expression pattern. D-I binding sites are not always relevant to the gene areas with increased transcriptions. Therefore, we compared the genomic areas where ATAC signal is raised by D-I, and genes whose expression is affected by D-I. We identified genes with ATAC signal change both in NALM6 cells with D-I knockdown and in Reh cells with D-I induction. We identified D-I binding in some of these genes, and the pharmacological inhibition of one of the genes caused cell death in NALM6 cells in vitro and in vivo, suggesting that this gene is the genuine target of D-I. In summary, our study elucidated the detailed difference of function between WT DUX4 and DUX4-IGH, and demonstrated the ability of DUX4-IGH as a chromatin modulator. Disclosures No relevant conflicts of interest to declare.

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release.

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.

Spliceosomal disruption of the non-canonical BAF complex in cancer.

SF3B1 is the most commonly mutated RNA splicing factor in cancer1–4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here, we integrated pan-cancer splicing analyses with a positive enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, a core component of the recently described GLTSCR1/1L-containing non-canonical BAF (ncBAF) chromatin remodeling complex5–7. Mutant SF3B1 recognizes an aberrant deep intronic branchpoint within BRD9, thereby inducing inclusion of an endogenous retroviral element-derived poison exon and BRD9 mRNA degradation. BRD9 depletion causes loss of ncBAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumor suppressor in uveal melanoma (UVM), such that correcting BRD9 mis-splicing in SF3B1-mutant cells with antisense oligonucleotides (ASOs) or CRISPR-directed mutagenesis suppresses tumor growth. Our results implicate ncBAF disruption in the diverse cancers carrying SF3B1 mutations and suggest a mechanism-based therapeutic for these malignancies.

Comparative Expression Analysis of Human Endogenous Retrovirus Elements in Peripheral Blood of Children with Specific Language Impairment.

Specific language impairment (SLI) is a psychiatric condition with a complex etiology and a substantial genetic basis that affects children's verbal communication abilities. In this study, we examined the expression of five different human endogenous retrovirus elements (HERVs) in a cohort of 25 children with SLI and 25 healthy children in the control group. Human endogenous retrovirus elements, a diverse group of repetitive DNA sequences, can potentially cause considerable genetic heterogeneity. They had been integrated in the genome of our ancestors throughout evolution and now consist of about 8.0% of the human genome. Several HERV loci are transcribed in various cell types. Their expression in peripheral blood and in the brain is altered in many neurological and psychiatric diseases. To date, HERV expression profiles have never been studied in patients with SLI. This study aimed to elucidate differentially regulated human endogenous retroelements in peripheral blood of children with SLI, in comparison with healthy controls, through quantitative reverse tran-scription-polymerase chain reaction (qRT-PCR) methodology. Our results show that two genes: HERV-K (HLM-2) gag and HERV-P env were expressed at lower levels in the blood samples from SLI children in comparison with those in the control group.

An influenza virus-triggered SUMO switch orchestrates co-opted endogenous retroviruses to stimulate host antiviral immunity

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of "non-self" nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of "self" double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.

An endogenous retroviral element exerts an antiviral innate immune function via the derived lncRNA lnc-ALVE1-AS1

Endogenous retroviruses (ERVs) constitute an important component of animal and human genomes and are usually silenced by epigenetic mechanisms in adult cells. Although ERVs were recently reported to be linked to early development, tumorigenesis and autoimmune disease, their impacts on antiviral innate immunity and the underlying mechanisms have not been elucidated. Here, we provide the first direct evidence of an endogenous retroviral element affecting antiviral innate immunity via its derived antisense long non-coding RNA (lncRNA). We found that an antisense lncRNA, which is called lnc-ALVE1-AS1 and is transcribed from the endogenous avian leukosis virus in chromosome 1 (ALVE1), distinctly inhibited the entry and replication of exogenous retroviruses in chicken embryonic fibroblasts (CEFs). This behaviour is at least in part attributed to the induction of an antiviral innate immune pathway by ALVE1 activation, suggesting that an activated endogenous retroviral element may induce antiviral defence responses via its derived antisense lncRNA. We also found that lnc-ALVE1-AS1 mediated these effects by activating the TLR3 signalling in the cytoplasm. Our results provide novel insights into the antiviral innate immune function of ERVs, suggesting that ERVs may play an important role in antiviral defences and provide new strategies for the development of new vaccines.

Abstract CT190: A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR)

Abstract Background Aberrant DNA methylation contributes to cancer initiation and progression. In preclinical models of solid tumors, chronic low dose administration of DNA hypomethylating agents can induce T-cell mediated immune activation responses by stimulating expression of endogenous retroviral elements, culminating in an IFN-mediated response. The addition of physiological levels of vitamin C may potentiate viral mimicry and increase anti-tumor immune priming. Immunologically ‘cold’ tumors were selected to evaluate whether these strategies can enhance their otherwise poor responses to immune checkpoint blockade. Methods PD-L1/PD-1 inhibitor naïve patients (pts) with advanced microsatellite stable colorectal cancer (MSS CRC); platinum resistant ovarian cancer (OC); estrogen receptor positive, HER2 negative breast cancer (ER+HER2- BC) were enrolled in this single institution, multi-cohort, investigator-initiated trial. The initial regimen (regimen A) consisted of oral CC-486 300mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500mg IV Day 15, in 28 day-cycles. A protocol amendment (regimen B) after 19 patients changed CC-486 to 100 mg QD Days 1-21 (cycles 1-3), added oral vitamin C 500mg QD continuously and kept durvalumab 1500mg IV Day 15, in 28-day cycles. Adverse events (AEs) assessed by CTCAEv4.03 grade (G); tumor response by RECIST 1.1 every 2 cycles; paired tumor biopsies at baseline and cycle 2 days 10-14; and serial peripheral blood mononuclear cells (PBMCs) for immune-profiling (IP) and epigenetic analysis. PD-L1 by IHC was assessed using SP263 assay, positivity defined by TC>25%. Results A total of 28 pts with MSS CRC (n=15), OC (n=4), ER+HER2- BC (n=9) were enrolled, 19 pts treated on regimen A, 9 on regimen B. Median age was 56 (range 36-78), ECOG 0:1=7:21, 100% had ≥3 prior lines of therapy, all tumors were PD-L1 negative. Best response was SD (3/28 pts received 3, 3 and 4 cycles respectively) with DCR 7.1%. Median follow-up of 4.7 months, mPFS was 1.9 months (95% CI 1.5-2.3) and mOS was 5 months (95% CI 4.5-10). Three patients (all regimen A) experienced DLTs (2 G4 neutropenia and 1 G3 anemia). Fifteen patients (54%) experienced G1/2 fatigue, 46% experienced G1/2 nausea, vomiting or diarrhea. Toxicity was comparable with addition of vitamin C. Initial analysis of PBMCs by flow cytometry in 3 of 4 OC pts demonstrated an increase in PD-1 and Ki67 expression in CD8 T cells only following administration of durvalumab. EPIC methylation array on 4 pts’ paired tumors and LINE 1 assay on 19 pts’ serial PBMCs (regimen A) demonstrated minimal change in global methylation. Conclusion No meaningful clinical responses to CC-486 plus durvalumab were observed. Tumor tissue and PBMCs both demonstrate minimal global DNA demethylation, with or without physiological dose vitamin C. Incremental immune activation beyond PD-L1 blockade was not demonstrated. Clinical trial information: NCT02811497 Citation Format: Kirsty Taylor, Helen Loo Yau, Ben X. Wang, Philippe L. Bedard, Albiruni R. Razak, Aaron R. Hansen, Anna Spreafico, Dave Cescon, Marcus O. Butler, Amit M. Oza, Stephanie Lheureux, Neda Stjepanovic, Lisa Wang, Brendan Van As, Sarah Boross-Harmer, Trevor Pugh, Lillian L. Siu, Daniel D. De Carvalho. A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT190.

Abstract 5294: The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas

Abstract Progressive age-dependent replacement of canonical H3.1/2 with H3.3 in post-mitotic cells is a crucial mechanism for maintaining genome integrity at heterochromatic sites. The histone chaperone DAXX cooperates with the chromatin remodeller ATRX for the deposition of the non-canonical histone H3.3 at heterochromatin, while it prevents the expression of endogenous retroviral elements in another complex involving the SETDB1 methyltransferases. Loss of function mutations affecting ATRX/DAXX and MEN-1 have been recently identified as a subgroup of pancreatic neuroendocrine tumours (PanNETs) leading to poor prognosis and more aggressive phenotype compared to the ATRX/DAXX and MEN-1 wild-type counterparts. We developed a mouse model for conditional Daxx inactivation in mouse islets to understand the impact of DAXX loss on pancreas homeostasis and PanNETs development. We found that loss of Daxx in the endocrine pancreas, using the Insulin-1Cre driver, leads to aberrant glucose metabolism with the development of hyperglycemia and consequently insulin resistance in mice. We have also shown that loss of DAXX is linked to increased expression of α-cell lineage genes upon glucose stimulation of isolated islets in vitro, resembling the recently reported phenotype of PanNETs harboring ATRX/DAXX or MEN1 mutations. Nonetheless, loss of Daxx was not sufficient to lead to PanNET development. We are currently investigating the chromatin and transcriptional perturbations caused by Daxx loss and how these could be linked to the aberration of cell lineage specification and tissue dysfunction in the pancreas. More generally, this work will provide new insight into how epigenetic alterations contribute to misregulation of developmental programmes and cancer development in the pancreas. Citation Format: Teresa Sposito, Marie-Sophie Nguyen Tu, Guy A. Rutter, Marco Novelli, Chrissie Thirlwell, Paolo Salomoni. The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5294.

Comprehensive transcriptome analysis reveals link between epigenetic dysregulation, endogenous retrovirus expression and immunogenicity in metastatic colorectal carcinoma.

3535 Background: Endogenous retrovirus (ERV) elements represent genomic footprints of ancestral retroviral infections within the human genome. Previous studies have demonstrated increases in ERV mRNA as a result of DNA hypomethylation, and ERV transcription has been associated with increased immunogenicity in metastatic renal cell carcinoma. We performed comprehensive bioinformatics analysis of ERV transcription in metastatic colorectal carcinoma (mCRC), to identify novel links between ERV transcription, epigenetic dysregulation and immunogenicity in metastatic colorectal carcinoma (mCRC). Methods: Tumour samples from 63 patients with mCRC were subjected to RNA sequencing as part of the Personalized OncoGenomics program (POG; NCT02155621) at BC Cancer. Patients were enrolled between 07/2012-07/2017. ERV transcription was quantified across 702,533 distinct loci. Tumors were classified ERV-hi if their total ERV expression (RPKM) was greater than the mean across all samples. High antiviral gene expression tumors (AVG-hi) were designated as having a mean expression of IFIH1, DDX58, TLR3, TANK, TBKBP1, TBK1, IRF3 and IRF7 that was greater than the mean across all samples. All pairwise comparisons of gene expression were subjected to multiple hypothesis correction. Results: Median age was 59 years, with 34 (54%) male and 1 tumor microsatellite unstable. ERV-hi tumors showed increased expression of DNA demethylators TET2 ( q=0.0045) and TET3 ( q<0.0001). Significant overlap existed between ERV-hi and AVG-hi tumors (18/27, p=0.016). Tumors both ERV-hi and AVG-hi trended towards increased PD-L1 expression (p=0.055) and showed a significant increase in survival compared to tumors with high antiviral expression in the absence of high ERV transcription (p=0.0043). Conclusions: Our results suggest DNA demethylation drives increased ERV transcription and ERV-associated immunogenicity in mCRC. Moreover, we provide novel insight into the impact of ERV transcription on the biology of mCRC, highlighting ERV transcription as a potential biomarker and target for precision immunotherapy. Clinical trial information: NCT02155621.

A Dissection of Oligomerization by the TRIM28 Tripartite Motif and the Interaction with Members of the Krab-ZFP Family

TRIM28 (also known as KAP1 or TIF1β) is the universal co-repressor of the Krüppel-associated box-containing zinc finger proteins (Krab-ZFPs), the largest family of transcription factors in mammals. During early embryogenesis, TRIM28 mediates the transcriptional silencing of many endogenous retroviral elements and genomic imprinted sites. Silencing is initiated by the recruitment of TRIM28 to a target locus by members of the Krab-ZFP. Subsequently, TRIM28 functions as a scaffold protein to recruit chromatin modifying effectors featuring SETDB1, HP1 and the NuRD complex. Although many protein partners involved in silencing have been identified, the molecular basis of the protein interactions that mediate silencing remains largely unclear. In the present study, we identified the first Bbox domain (T28_B1 135–203) as a molecular interface responsible for the formation of higher-order oligomers of TRIM28. The structure of this domain reveals a new interface on the surface of the Bbox domain. Mutants disrupting the interface disrupt the formation of oligomers but have no observed effect on transcriptional silencing defining a single TRIM28 dimer as the functional unit for silencing. Using assembly-deficient mutants, we employed small-angle X-ray scattering and biophysical techniques to characterize binding to member of the Krab-ZFP family. This allows us to narrow and define the binding interface to the center of the coiled-coil region (residues 294–321) of TRIM28 and define mutants that abolish binding to the Krab-ZFP proteins.

HERVs, immunity, and autoimmunity: understanding the connection.

Since their discovery in the 1960s, further investigation into endogenous retroviruses (ERVs) has challenged the conventional view of viral sequences as exclusively parasitic elements. Once presumed to be a group of passive genetic relics, it is becoming increasingly clear that this view of ERVs, while generally accurate, is incorrect in many specific cases. Research has identified ERV genes that appear to be co-opted by their mammalian hosts, but the biological function of ERV elements in humans remains a controversial subject. One area that has attracted some attention in this domain is the role of co-opted ERV elements in mammalian immune systems. The relationship between ERVs and human autoimmune diseases has also been investigated, but has historically been treated as a separate topic. This review will summarize the current evidence concerning the phenotypic significance of ERVs, both in the healthy immune system and in manifestations of autoimmunity. Furthermore, it will evaluate the relationship between these fields of study, and propose previously-unexplored molecular mechanisms through which human endogenous retroviruses might contribute to certain autoimmune pathologies. Investigation into these novel mechanisms could further our understanding of the molecular basis of autoimmune disease, and may one day provide new targets for treatment.

Investigation of somatic single nucleotide variations in human endogenous retrovirus elements and their potential association with cancer.

Human endogenous retroviruses (HERVs) have been investigated for potential links with human cancer. However, the distribution of somatic nucleotide variations in HERV elements has not been explored in detail. This study aims to identify HERV elements with an over-representation of somatic mutations (hot spots) in cancer patients. Four HERV elements with mutation hotspots were identified that overlap with exons of four human protein coding genes. These hotspots were identified based on the significant over-representation (p<8.62e-4) of non-synonymous single-nucleotide variations (nsSNVs). These genes are TNN (HERV-9/LTR12), OR4K15 (HERV-IP10F/LTR10F), ZNF99 (HERV-W/HERV17/LTR17), and KIR2DL1 (MST/MaLR). In an effort to identify mutations that effect survival, all nsSNVs were further evaluated and it was found that kidney cancer patients with mutation C2270G in ZNF99 have a significantly lower survival rate (hazard ratio = 2.6) compared to those without it. Among HERV elements in the human non-protein coding regions, we found 788 HERVs with significantly elevated numbers of somatic single-nucleotide variations (SNVs) (p<1.60e-5). From this category the top three HERV elements with significantly over-represented SNVs are HERV-H/LTR7, HERV-9/LTR12 and HERV-L/MLT2. Majority of the SNVs in these 788 HERV elements are located in three DNA functional groups: long non-coding RNAs (lncRNAs) (60%), introns (22.2%) and transcriptional factor binding sites (TFBS) (14.8%). This study provides a list of mutational hotspots in HERVs, which could potentially be used as biomarkers and therapeutic targets.

Identification and characterization of ERV transcripts in goat embryos.

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

Induction of Proinflammatory Multiple Sclerosis-Associated Retrovirus Envelope Protein by Human Herpesvirus-6A and CD46 Receptor Engagement.

The aberrant expression of human endogenous retrovirus (HERV) elements of the HERV-W family has been associated with different diseases, including multiple sclerosis (MS). In particular, the expression of the envelope protein (Env) from the multiple sclerosis-associated retrovirus (MSRV), a member of HERV-W family and known for its potent proinflammatory activity, is repeatedly detected in the brain lesions and blood of MS patients. Furthermore, human herpesvirus 6 (HHV-6) infection has long been suspected to play a role in the pathogenesis of MS and neuroinflammation. We show here that both HHV-6A and stimulation of its receptor, transmembrane glycoprotein CD46, induce the expression of MSRV-Env. The engagement of extracellular domains SCR3 and SCR4 of CD46-Cyt1 isoform was required for MSRV-env transactivation, limiting thus the MSRV-Env induction to the CD46 ligands binding these domains, including C3b component of complement, specific monoclonal antibodies, and both infectious and UV-inactivated HHV-6A, but neither HHV-6B nor measles virus vaccine strain. Induction of MSRV-Env required CD46 Cyt-1 singling and was abolished by the inhibitors of protein kinase C. Finally, both membrane-expressed and secreted MSRV-Env trigger TLR4 signaling, displaying thus a proinflammatory potential, characteristic for this viral protein. These data expand the specter of HHV-6A effects in the modulation of the immune response and support the hypothesis that cross-talks between exogenous and endogenous viruses may contribute to inflammatory diseases and participate in neuroinflammation. Furthermore, they reveal a new function of CD46, known as an inhibitor of complement activation and receptor for several pathogens, in transactivation of HERV env genes, which may play an important role in the pathogenesis of inflammatory diseases.

DNA Methyltransferase 1 Controls Nephron Progenitor Cell Renewal and Differentiation.

Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear. We used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development. We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells (in contrast to deletion of Dnmt3a or Dnm3b) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.

Transcriptional Activity of Human Endogenous Retroviruses in Response to Prenatal Exposure of Maternal Cigarette Smoking.

Transcription of human endogenous retrovirus (HERV) elements is usually suppressed by epigenetic factors such as DNA methylation and heterochromatin silencing by histone modifications. There is an association between maternal smoking during pregnancy and DNA methylation levels in placental tissue and in DNA from cord blood. We assessed the transcriptional activity of HERV-H, HERV-K, and HERV-W in umbilical cord blood from 47 term babies unexposed to tobacco smoke in utero and 23 term babies exposed to tobacco smoke in utero. In our population, the HERV-H, HERV-K, and HERV-W families were always transcriptionally active, and the levels of all HERVs (H, K, W) were significantly higher in unexposed than smoke-exposed babies. This study provides preliminary information about the transcriptional activity of HERV-H, HERV-K, and HERV-W families in human umbilical cord blood.

LTRs activated by Epstein-Barr virus-induced transformation of B cells alter the transcriptome.

Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas. We report here that transformation of primary B cells by EBV leads to genome-wide activation of LTR enhancers and promoters. The activation of LTRs coincides with local DNA hypomethylation and binding by transcription factors such as RUNX3, EBF1, and EBNA2. The set of activated LTRs is unique to transformed B cells compared with other cell lines known to have activated LTRs. Furthermore, we found that LTR activation impacts the B cell transcriptome by up-regulating transcripts driven by cryptic LTR promoters. These transcripts include genes important to oncogenesis of Hodgkin lymphoma and other cancers, such as HUWE1/HECTH9. These data suggest that the activation of LTRs by EBV-induced transformation is important to the pathology of EBV-associated cancers. Altogether, our results indicate that EBV-induced transformation of B cells alters endogenous retroviral element activity, thereby impacting host gene regulatory networks and oncogenic potential.

Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells

Despite their inherent proximity to circulating oxygen and nutrients, endothelial cells (ECs) oxidize only a minor fraction of glucose in mitochondria, a metabolic specialization that is poorly understood. Here we show that the glycolytic enzyme pyruvate kinase M2 (PKM2) limits glucose oxidation, and maintains the growth and epigenetic state of ECs. We find that loss of PKM2 alters mitochondrial substrate utilization and impairs EC proliferation and migration in vivo. Mechanistically, we show that the NF-κB transcription factor RELB is responsive to PKM2 loss, limiting EC growth through the regulation of P53. Furthermore, S-adenosylmethionine synthesis is impaired in the absence of PKM2, resulting in DNA hypomethylation, de-repression of endogenous retroviral elements (ERVs) and activation of antiviral innate immune signalling. This work reveals the metabolic and functional consequences of glucose oxidation in the endothelium, highlights the importance of PKM2 for endothelial growth and links metabolic dysfunction with autoimmune activation in ECs.