Yoon Jung Kim

The metabolite α-ketoglutarate (αKG) is required for chromatin demethylation, but mechanisms that control αKG abundance in the nucleus are poorly defined. We designed a biosensor to monitor this metabolite pool in human cells using an αKG-responsive cyanobacterial transcription factor, NtcA, and used it to identify genes that regulate αKG in the nucleus. We defined an interorganelle pathway in which sequential mitochondrial activities of glutamic-pyruvic transaminase 2 (GPT2) and the SLC25A11 transporter supply nuclear αKG. In a mouse model of GPT2 deficiency, an inborn error of metabolism, Gpt2 loss caused histone hypermethylation in the brain and dysregulated neurodevelopmental genes. Restoring αKG counteracted these changes and promoted mouse fitness. Our work provides a tool to directly monitor nuclear αKG and reveals nuclear αKG depletion as a key pathogenic mechanism underlying GPT2 deficiency.

Extrachromosomal DNAs (ecDNAs) attach to chromosomes during mitosis for random segregation and promote cancer heterogeneity. However, the mechanism governing ecDNA–chromosome mitotic interactions remains poorly understood. Here we show that ecDNAs tether to histone H3 lysine 27 acetylation (H3K27ac)-marked chromatin during mitosis. Depleting H3K27ac disrupts this interaction. Diverse bromodomain proteins, as H3K27ac readers, stabilize ecDNA–chromosome binding in a context-dependent and complementary manner. Although disrupting the Mediator complex in asynchronous cells detaches ecDNAs from mitotic chromosomes, Mediator and active Pol II are absent from ecDNAs during mitosis, suggesting that ecDNAs are transcriptionally silent during mitosis. Instead, inactive Pol II mediates ecDNA attachment. Furthermore, CRISPR interference targeting transcriptional regulatory elements on ecDNA impairs ecDNA segregation. Mis-segregated ecDNAs were expelled into the cytosol, leading to diminished oncogene expression and a reversal of therapy resistance. Our research provides universal cis and trans regulatory mechanisms of ecDNA segregation, offering deeper insight into ecDNA-driven oncogenesis.

Adult women are typically exposed to 17β-estradiol (E2) concentrations of ~100 to 200 pM, yet most cell-based studies use 100 nM. We determined the molecular effects of E2 concentrations spanning six orders of magnitude (1 pM to 100 nM) in breast cancer cells. Estrogen receptor α (ERα) enhancers formed at low physiological doses of E2 (1 to 100 pM) are mechanistically distinct from those that form at high pharmacological doses (10 to 100 nM). They (i) form in open chromatin bound by FOXA1, (ii) are clustered within topologically associated domains, (iii) produce enhancer RNAs enriched with functional enhancer RNA regulatory motifs, and (iv) drive expression of proliferation genes with promoter-proximal paused RNA polymerase II and distinct co-regulator enrichment. Low-dose ERα enhancer usage is elevated in patients who have breast cancer with poor responses to aromatase inhibitors, likely as a continued response to low circulating levels of E2. Collectively, our results identify mechanistic differences between low- and high-dose ERα enhancers that specify distinct biological outcomes.

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.

The transcription factor BCL11A is a genetically and clinically validated regulator of the fetal-to-adult hemoglobin switch in human erythroid cells. CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as the first CRISPR-based therapy for β-hemoglobinopathies. However, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation remains elusive. Here, we describe a new genome architecture, an enhancer-dependent chromatin rosette, that is essential for epigenetic insulation and the developmentally regulated, hematopoietic lineage–specific expression of BCL11A. CRISPR-mediated disruption of the BCL11A erythroid enhancer impairs transcription of enhancer-driven RNAs and NIPBL-dependent cohesin loading, leading to the destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion of enhancer RNAs using antisense oligonucleotides silences BCL11A by disrupting epigenetic insulation, causing HbF reactivation in adult erythroid cells. These findings uncover an essential role for enhancer-driven epigenetic insulation in transcriptional control, presenting a new strategy for the therapeutic targeting of BCL11A.

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.

Supplementary Data from Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance

Supplementary Data from Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance