Margaret B. Cervantes

Retrotransposons are genomic parasites frequently reactivated in cancers, in which their mobility can cause genetic alterations. However, it remains unclear whether their gene products contribute to cancer beyond mutagenesis. In this study, we uncover a chromatin-associated function of RNAs from long interspersed element-1 (LINE-1), the only autonomous retrotransposon in the human genome. Subcellular-resolved transcriptomics revealed that LINE-1 RNAs are primarily nascent transcripts produced by full-length, cell type–specific genomic copies of evolutionarily young subfamilies. Using a long-read chromosome conformation assay, we identified a class of highly interactive LINE-1 loci required for gene expression across cancer subtypes, revealing an unexpected regulatory role for LINE-1 locus transcription in oncogenic gene control. LINE-1 RNA depletion disrupted LINE-1–centric chromatin interactions and downregulated associated genes, whereas genomic insertion of an inducible LINE-1 generated de novo chromatin interactions in a transcription-dependent manner. Therefore, beyond their mutagenic potential, retrotransposons also regulate cancer gene expression by nucleating chromatin architecture through their transcriptional activity.Significance:We developed a long-read chromatin conformation assay capable of resolving repetitive DNA, enabling the identification of highly interactive LINE-1 retrotransposon loci that nucleate chromatin architecture in cancer cells. This activity is driven by chromatin-associated LINE-1 transcripts, offering a biological rationale for the frequent reactivation of LINE-1 transcription in cancer.

Figure S1. Subcellular localization of LINE-1 RNAs in cancer cells. Figure S2. Chromatin-associated LINE-1 RNAs are nascent transcripts associated in cis with their sites of transcription. Figure S3. Chromatin RNA-sequencing of human cancer cells and primary tumors to quantify locusspecific LINE-1 expression. Figure S4. Analysis of uniquely and multi-mapped reads by RNA-seq of LINE-1 expression. Figure S5. Identification of the expression cut-off for RNA-seq analysis of LINE-1 locus expression. Figure S6. Identification of cell type-specific LINE-1 locus expression in human cancer cell lines. Figure S7. Lineage-specific transcription factors and chromatin features regulate LINE-1 locus expression. Figure S8. LINE-1 Capture Pore-C for detecting LINE-1-centric chromatin interactions. Figure S9. Identification of LINE-1-centric chromatin interactions by L1CaP-C. Figure S10. Mappability analysis of L1CaP-C sequencing reads associated with chromatin interactions. Figure S11. LINE-1-centric chromatin interactions identified by L1CaP-C. Figure S12. Identification of highly interacting LINE-1 loci. Figure S13. L1CaP-C identifies pairwise and high-order chromatin interactions. Figure S14. Analysis of chromatin marks at HILL LINE-1 loci and super-enhancers (SEs). Figure S15. Correlation of HILL LINE-1 loci with clinical features in TCGA breast cancer samples. Figure S16. LINE-1 locus expression regulates oncogenic gene expression. Figure S17. LINE-1 depletion impairs cell proliferation and oncogenic gene expression.

Table S1. List of genomic datasets used in this study. Table S2. List of oligonucleotides used in this study. Table S3. List of key reagents and resources used in this study. Table S4. List of full-length LINE-1 loci (>5 kb) annotated in the hg38 human reference genome.

Retrotransposons are genomic parasites frequently reactivated in cancers, in which their mobility can cause genetic alterations. However, it remains unclear whether their gene products contribute to cancer beyond mutagenesis. In this study, we uncover a chromatin-associated function of RNAs from long interspersed element-1 (LINE-1), the only autonomous retrotransposon in the human genome. Subcellular-resolved transcriptomics revealed that LINE-1 RNAs are primarily nascent transcripts produced by full-length, cell type–specific genomic copies of evolutionarily young subfamilies. Using a long-read chromosome conformation assay, we identified a class of highly interactive LINE-1 loci required for gene expression across cancer subtypes, revealing an unexpected regulatory role for LINE-1 locus transcription in oncogenic gene control. LINE-1 RNA depletion disrupted LINE-1–centric chromatin interactions and downregulated associated genes, whereas genomic insertion of an inducible LINE-1 generated de novo chromatin interactions in a transcription-dependent manner. Therefore, beyond their mutagenic potential, retrotransposons also regulate cancer gene expression by nucleating chromatin architecture through their transcriptional activity. Significance: We developed a long-read chromatin conformation assay capable of resolving repetitive DNA, enabling the identification of highly interactive LINE-1 retrotransposon loci that nucleate chromatin architecture in cancer cells. This activity is driven by chromatin-associated LINE-1 transcripts, offering a biological rationale for the frequent reactivation of LINE-1 transcription in cancer.

Canonical models of intestinal regeneration emphasize the critical role of the crypt stem cell niche to generate enterocytes that migrate to villus ends. Burmese pythons possess extreme intestinal regenerative capacity yet lack crypts, thus providing opportunities to identify noncanonical but potentially conserved mechanisms that expand our understanding of regenerative capacity in vertebrates, including humans. Here, we leverage single-nucleus RNA sequencing of fasted and postprandial python small intestine to identify the signaling pathways and cell–cell interactions underlying the python’s regenerative response. We find that python intestinal regeneration entails the activation of multiple conserved mechanisms of growth and stress response, including core lipid metabolism pathways and the unfolded protein response in intestinal enterocytes. Our single-cell resolution highlights extensive heterogeneity in mesenchymal cell population signaling and intercellular communication that directs major tissue restructuring and the shift out of a dormant fasted state by activating both embryonic developmental and wound healing pathways. We also identify distinct roles of BEST4+ enterocytes in coordinating key regenerative transitions via NOTCH signaling. Python intestinal regeneration shares key signaling features and molecules with mammalian gastric bypass, indicating that conserved regenerative programs are common to both. Our findings provide different insights into cooperative and conserved regenerative programs and intercellular interactions in vertebrates independent of crypts which have been otherwise obscured in model species where temporal phases of generative growth are limited to embryonic development or recovery from injury.

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.