LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/ EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.

The histone variant macroH2A occupies large repressive domains throughout the genome, however mechanisms underlying its precise deposition remain poorly understood. Here, we characterized de novo chromatin deposition of macroH2A2 using temporal genomic profiling in murine-derived fibroblasts devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome-wide at both steady state domains and adjacent transcribed regions, the latter of which are subsequently pruned, establishing mature macroH2A2 domains. Pruning of macroH2A2 can be counteracted by chemical inhibition of transcription. Further, CRISPR/Cas9-based locus-specific transcriptional manipulation reveals that gene activation depletes pre-existing macroH2A2, while silencing triggers ectopic macroH2A2 accumulation. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 pruning within transcribed chromatin. Taken together, we have identified active chromatin as a boundary for macroH2A domains through a transcription-associated ‘pruning’ mechanism that establishes and maintains the faithful genomic localization of macroH2A variants.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with roles in inflammation and tumorigenicity. A fraction of STAT3 localizes in mitochondria, where it augments tumorigenesis via regulation of mitochondrial functions, including modulation of respiration and redox status. We show a novel mechanism for mitochondrial STAT3 regulation of redox homeostasis in triple-negative breast cancer cells. Loss of STAT3 diminished complex I dehydrogenase activity and impaired NAD+ regeneration, leading to impaired expression of glutathione biosynthetic genes and other antioxidant genes. Expressing mitochondrially restricted STAT3 or replenishment of the cellular NAD pool restored antioxidant gene expression, as did complementation of the NADH dehydrogenase activity by expression of the STAT3-independent yeast dehydrogenase, NDI1. These NAD-regulated processes contributed to malignant phenotypes by promoting clonal cell growth and migration. Proximity interaction and protein pull-down assays identified three components of complex I that associated with mitochondrial STAT3, providing a potential mechanistic basis for how mitochondrial STAT3 affects complex I activity. Our data document a novel mechanism through which mitochondrial STAT3 indirectly controls antioxidant gene regulation through a retrograde NAD+ signal that is modulated by complex I dehydrogenase activity.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH−/− PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed “q-oxPTPome”, a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTP were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathological PTP oxidation and neoplastic disease.

Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies.

No abstract

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology to aid its proliferation. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and its intragenomic spreading remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.