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// Yu Wang 1, 2 , Eric D. Schafler 3 , Phillip A. Thomas 1, 3 , Susan Ha 1 , Gregory David 3 , Emily Adney 4, 6 , Michael J. Garabedian 1, 2 , Peng Lee 1, 5 and Susan K. Logan 1, 3 1 Department of Urology, New York University School of Medicine, New York 10016, NY, USA 2 Department of Microbiology, New York University School of Medicine, New York 10016, NY, USA 3 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York 10016, NY, USA 4 Institute for Systems Genetics, New York University School of Medicine, New York 10016, NY, USA 5 Department of Pathology, New York Harbor Healthcare System, New York 10010, NY, USA 6 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore 21205, MD, USA Correspondence to: Susan K. Logan, email: Susan.Logan@nyumc.org Keywords: UXT; retroelement; prostate cancer Received: April 01, 2018 Accepted: October 06, 2018 Published: January 22, 2019 ABSTRACT Ubiquitously-expressed, prefoldin-like chaperone (UXT) also called Androgen Receptor Trapped clone-27 (ART-27) is widely expressed in human tissues. Our previous studies showed that UXT regulates transcription repression including androgen receptor (AR) signaling in prostate cancer. Here we analyzed a tissue microarray consisting of normal prostate, benign prostatic hyperplasia, high grade prostatic intraepithelial neoplasia (HGPIN) and primary prostate cancer cases for UXT protein expression. We found that HGPIN and malignant tumors have significantly decreased UXT expression compared to the normal prostate. Loss of UXT expression in primary prostate cancer is positively associated with high Gleason grade and poor relapse-free survival. We engineered prostate-specific Uxt KO mice that developed a hyperplastic phenotype with apparent prostate secretion fluid blockage as well as PIN by 4-6 months. Doubly mutant Uxt KO / Pten KO mice developed a more aggressive PIN phenotype. UXT depletion in prostate cancer cells also increased retroelements expression, including LINE-1 and Alu. Consistent with this finding Uxt KO mice have increased LINE-1 protein levels in the prostate compared to control mice. In addition, cancer cells with UXT depletion have increased retrotransposition activity and accumulated DNA damage. Our findings demonstrate that loss of UXT is an early event during prostate cancer progression, which may contribute to genome instability.

Higher-order chromatin structure arises from the combinatorial physical interactions of many genomic loci. To investigate this aspect of genome architecture we developed Pore-C, which couples chromatin conformation capture with Oxford Nanopore Technologies (ONT) long reads to directly sequence multi-way chromatin contacts without amplification. In GM12878, we demonstrate that the pairwise interaction patterns implicit in Pore-C multi-way contacts are consistent with gold standard Hi-C pairwise contact maps at the compartment, TAD, and loop scales. In addition, Pore-C also detects higher-order chromatin structure at 18.5-fold higher efficiency and greater fidelity than SPRITE, a previously published higher-order chromatin profiling technology. We demonstrate Pore-C’s ability to detect and visualize multi-locus hubs associated with histone locus bodies and active / inactive nuclear compartments in GM12878. In the breast cancer cell line HCC1954, Pore-C contacts enable the reconstruction of complex and aneuploid rearranged alleles spanning multiple megabases and chromosomes. Finally, we apply Pore-C to generate a chromosome scalede novoassembly of the HG002 genome. Our results establish Pore-C as the most simple and scalable assay for the genome-wide assessment of combinatorial chromatin interactions, with additional applications for cancer rearrangement reconstruction andde novogenome assembly.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders. During cellular senescence in human and mouse cells, L1 transposons become transcriptionally derepressed and trigger a type-1 interferon response, which contributes to age-associated inflammation and age-related phenotypes.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g. deletion) or complex (e.g. chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are “towers” of low-JCN duplications associated with early replicating regions and superenhancers, enriched in breast and ovarian cancers. Rigma comprise “chasms” of low-JCN deletions at late-replicating fragile sites, enriched in gastrointestinal carcinomas. Tyfonas are “typhoons” of high-JCN junctions and fold-back inversions enriched in expressed protein-coding fusions and breakend hypermutation, frequently found in acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription., eLife digest Our genome consists of about two percent genes, while around 60 to 70 percent are made up of hundreds of thousands of copies of very similar DNA sequences. These repeats have accumulated over time due to specific genetic elements called transposons. Transposons are often referred to as ‘jumping genes’, as they can move within the genome and thereby create mutations that may lead to cancer or other genetic diseases. LINE-1 is the only remaining active transposon in humans, and it expands by copying and pasting itself to new locations. To do so, it is first transcribed into RNA – the molecules that help to make proteins – and then converted back into identical DNA sequences. In a never-ending battle, our cells have been fighting to keep LINE-1 and its ancestors from replicating, and so evolved various defense mechanisms. Yet, LINE-1 has learned to circumvent these barriers, and continues to replicate and cause disease. Our understanding of these defenses and of how LINE-1 evades them is limited. Previous research has shown that the LINE-1 RNA and its two encoded proteins, called ORF1p and ORF2p, interact with a series of other proteins, with which they can form different types of complexes. Now, Taylor, Altukhov, Molloy et al. used human embryonic kidney cells grown in the laboratory with different LINE-1 mutations to identify how they affect the bound proteins and RNAs. The results showed that LINE-1 can form at least two different sets of complexes with other proteins. The complex containing ORF1p and ORF2p and several other proteins was located in the cytoplasm, the fluid that fills the cells. However, the experiments also revealed a new complex in the cell nucleus, which contained ORF2p and proteins involved in DNA replication and repair, but not ORF1p. The results suggest ORF1p delivers RNPs to the nucleus around the time the cell divides. Another group of researchers has looked more closely at what happens during cell division. A next step will be to study how exactly LINE-1 contributes to cancer. In the future, overactive LINE-1 proteins could be targeted to kill cancer cells, to identify cancer early, or to see if the cancer has come back. LINE-1 may also provide clues on how the genome has evolved.

1.SummaryLong Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications and quantitative mass spectrometry, we have characterized the proteins associated with distinctive L1 macromolecular complexes. Our findings support the presence of multiple L1-derived retrotransposition intermediates in vivo. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in template-primed reverse transcription.

SummaryLINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition’s mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H(2)O(2).