Osteosarcoma is the most frequent primary bone tumor with poor prognosis. Through RNA-sequencing of 100,987 individual cells from 7 primary, 2 recurrent, and 2 lung metastatic osteosarcoma lesions, 11 major cell clusters are identified based on unbiased clustering of gene expression profiles and canonical markers. The transcriptomic properties, regulators and dynamics of osteosarcoma malignant cells together with their tumor microenvironment particularly stromal and immune cells are characterized. The transdifferentiation of malignant osteoblastic cells from malignant chondroblastic cells is revealed by analyses of inferred copy-number variation and trajectory. A proinflammatory FABP4+ macrophages infiltration is noticed in lung metastatic osteosarcoma lesions. Lower osteoclasts infiltration is observed in chondroblastic, recurrent and lung metastatic osteosarcoma lesions compared to primary osteoblastic osteosarcoma lesions. Importantly, TIGIT blockade enhances the cytotoxicity effects of the primary CD3+ T cells with high proportion of TIGIT+ cells against osteosarcoma. These results present a single-cell atlas, explore intratumor heterogeneity, and provide potential therapeutic targets for osteosarcoma.

Human telomeres are bound and protected by protein complexes assembled around the six core telomeric proteins RAP1, TRF1, TRF2, TIN2, TPP1, and POT1. The function of these proteins on telomeres has been studied extensively. Recently, increasing evidence has suggested possible roles for these proteins outside of telomeres. However, the non-canonical (extra-telomeric) function of human telomeric proteins remains poorly understood. To this end, we systematically investigated the binding sites of telomeric proteins along human chromosomes, by performing whole-genome chromatin immunoprecipitation (ChIP) for RAP1 and TRF2. ChIP sequencing (ChIP-seq) revealed that RAP1 and TRF2 could be found on a small number of interstitial sites, including regions that are proximal to genes. Some of these binding sites contain short telomere repeats, suggesting that telomeric proteins could directly bind to interstitial sites. Interestingly, only a small fraction of the available interstitial telomere repeat-containing regions were occupied by RAP1 and TRF2. Ectopically expressed TRF2 was able to occupy additional interstitial telomere repeat sites, suggesting that protein concentration may dictate the selective targeting of telomeric proteins to interstitial sites. Reducing RAP1 and TRF2 expression by RNA interference led to altered transcription of RAP1- and TRF2-targeted genes. Our results indicate that human telomeric proteins could occupy a limited number of interstitial sites and regulate gene transcription.

Summary In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors DAXX and ATRX. Using genome-wide binding and transcriptome analysis, we found that DAXX and ATRX have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wildtype mouse ESCs. Global DNA hypomethylation further promoted recruitment of the DAXX/ATRX complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of DAXX/ATRX in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, DAXX/ATRX-mediated repression seems to involve SUV39H recruitment and H3K9 trimethylation. Our data therefore suggest that DAXX and ATRX safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ϳ12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric pro- During mammalian DNA replication, linear chromosomal ends will gradually erode because of the inability of the DNA replication machinery to replicate the extreme 5Ј terminus of a linear DNA sequence (1, 2). This inherent "end replication problem" is circumvented through specialized chromosomal end structures (telomeres) and the action of the RNA-containing DNA polymerase -telomerase (3-9). Telomere homeostasis is essential for genome stability, cell survival, and growth.Telomeres and telomerase help to ensure genome integrity in eukaryotes by enabling complete replication of the ends of linear DNA molecules, and preventing chromosomal rearrangements or fusion. For dividing cells such as stem cells and the majority of cancer cells, the telomerase is an essential positive regulator of their telomere length and ultimately determines the proliferative potential of these cells.Mammalian telomeres consist of a series of (TTAGGG)n sequence repeats and terminate in 3Ј single-stranded DNA overhangs that are extendable by the telomerase (10). Exposed linear chromosome ends or naturally occurring doublestranded breaks pose additional risks including activation of DNA damage responses. The ends of telomeres in mammalian cells appear to fold back in a T-loop structure, with the 3Ј G-rich single-stranded overhang invading into the doublestranded telomere regions to form the D-loop (11). The structure of the telomeres, coupled with the coordinated action of a collection of proteins that protect the ends of chromosomes (12-15), contributes to the maintenance of telomere integrity, genome stability, and proper cell cycle progression.In mammals, the most widely studied telomere-associated proteins include the double-stranded DNA binding proteins TRF1 and TRF2 (16,17), the single-stranded telomeric DNA binding protein POT1 (18), and three associated factors (RAP1, TIN2, and TPP1) (19 -23). Work from our lab and others suggest that TPP1, along with POT1, TIN2, TRF1, TRF2, and RAP1, form a higher order complex (the telosome/ shelterin) at the telomeres (24 -27). Information regarding the state of the telomere ends can be transmitted from TRF1 and TRF2 to POT1, through TPP1 and the other subunits (28). Furthermore, TRF1 and TRF2 function as bona fide protein hubs and interact with a diverse array of factors/complexes that are involved in cell cycle, DNA repair, and recombinat...