Non-cycling Cells Research Articles

Acetylation of SAMHD1 at lysine 580 is crucial for blocking HIV-1 infection.

In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies.

Cell-Cycle-Specific Autoencoding Improves Cluster Analysis of Cycling Cardiomyocytes.

Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle." Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle-specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5). The cell-cycle-specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction. The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs.

Abstract 15500: Identifying Cardiomyocytes at Specific Cell-Cycle Stages by a New Cell-Cycle-Specific snRNAseq Analytic Method

Background: Deciphering the cardiomyocyte cell cycle is a critical question in studying heart development and regeneration. We already collected the single-nuclei RNA sequencing (snRNAseq) data from fetal pig hearts and from pigs that underwent apical resection on postnatal day (P) 1 (AR P1 ) followed by myocardial infarction (MI) on P28 (MI P28 ); in these hearts, robust cardiomyocyte proliferation was confirmed. Research questions: Although our previous snRNAseq analyses found cardiomyocyte clusters that primed to proliferate, each of these clusters only appeared at one specific time point; furthermore, these clusters still contain a significant amount of non-cycling cell; and thus, we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Approach: In this report, we designed a new Autoencoder that only utilized 1646 cell-cycle-specific genes to cluster cardiomyocytes and applied the Autoencoder on pig heart GEO datasets number GSE185289. Results: Five cardiomyocyte clusters (pCM1+2, pCM3-pCM6) were identified. Five proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BRCA5) highly co-expressed in pCM1+2; also, the top enriched gene ontologies of pCM1+2 were all related to cell-cycle. PCM1+2 percentages were ~6% in fetal hearts and ~2-3% in hearts from ARP1MIP28 pigs during the two weeks after MI induction but were nearly undetectable in all other experimental groups and at all other time points. We also identified four transcription factors (E2F8, FOXM1, GLI3, and RAD51) that explicitly coexpressed within pCM1. Also, a pseudo-time trajectory was constructed in Fetal cardiomyocyte, which began at pCM3 (G1/S phase, CCND2 localization), passed through pCM2 (S phase, CCNE2 localization), and ended at pCM1 (M phase, CCNB2 localization). Conclusions: Applying the more precise cell-cycle specific Autoencoder, we found three clusters that likely represented cardiomyocytes at three different cell-cycle stages.

DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2β-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

Individual glioblastoma cells harbor both proliferative and invasive capabilities during tumor progression.

Glioblastomas are characterized by aggressive and infiltrative growth, and by striking heterogeneity. The aim of this study was to investigate whether tumor cell proliferation and invasion are interrelated, or rather distinct features of different cell populations. Tumor cell invasion and proliferation were longitudinally determined in real-time using 3D in vivo 2-photon laser scanning microscopy over weeks. Glioblastoma cells expressed fluorescent markers that permitted the identification of their mitotic history or their cycling versus non-cycling cell state. Live reporter systems were established that allowed us to dynamically determine the invasive behavior, and previous or actual proliferation of distinct glioblastoma cells, in different tumor regions and disease stages over time. Particularly invasive tumor cells that migrated far away from the main tumor mass, when followed over weeks, had a history of marked proliferation and maintained their proliferative capacity during brain colonization. Infiltrating cells showed fewer connections to the multicellular tumor cell network, a typical feature of gliomas. Once tumor cells colonized a new brain region, their phenotype progressively transitioned into tumor microtube-rich, interconnected, slower-cycling glioblastoma cells. Analysis of resected human glioblastomas confirmed a higher proliferative potential of tumor cells from the invasion zone. The detection of glioblastoma cells that harbor both particularly high proliferative and invasive capabilities during brain tumor progression provides valuable insights into the interrelatedness of proliferation and migration-2 central traits of malignancy in glioma. This contributes to our understanding of how the brain is efficiently colonized in this disease.

Low CDK Activity and Enhanced Degradation by APC/CCDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells.

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection-a process whereby 3' single-stranded DNA-tails are generated-initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase cells allow modest resection and alt-EJ, as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G0-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.

Insulin-like growth factor binding protein-3 induces senescence by inhibiting telomerase activity in MCF-7 breast cancer cells

Insulin-like growth factor binding protein-3 (IGFBP-3) has been known to inhibit cell proliferation and exert tumor-suppressing effects depending on the cell type. In this study, we aimed to show that IGFBP-3 induces cellular senescence via suppression of the telomerase activity, thereby inhibiting MCF-7 breast cancer cell proliferation. We found that the induction of IGFBP-3 in MCF-7 cells enhanced the loss of cell viability. Flow cytometry revealed a higher percentage of non-cycling cells among IGFBP-3-expressing cells than among controls. IGFBP-3 induction also resulted in morphological alterations, such as a flattened cytoplasm and increased granularity, suggesting that IGFBP-3 induces a senescence-like phenotype. The percentage of IGFBP-3 expressing cells with senescence-associated β-galactosidase activity was 3.4 times higher than control cells. Telomeric repeat amplification and real-time PCR showed that IGFBP-3 decreased telomerase activity by reducing the levels of the RNA component (hTR) and catalytic protein component with reverse transcriptase activity (hTERT) of telomerase in a dose-dependent manner. These results suggest that IGFBP-3 is a negative regulator of MCF-7 breast cancer cell growth by inducing senescence through telomerase suppression.

The Ubiquitin Ligase Itch Skews Light Zone Selection in Germinal Centers.

Ig diversification occurs in peripheral lymphoid organs after establishment of central tolerance during B cell development. In germinal centers (GCs), somatic hypermutation of Ig genes occurs in dark zones, followed by selection of mutated clones in light zones (LZs). This generates high-affinity Ig receptors to pathogens but can also produce autoreactive Ig receptors, which are removed by selection mechanisms that are incompletely understood. The ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop potentially fatal autoimmune diseases; yet, how Itch regulates GC B cells is not well understood. By studying Itch-deficient mice, we have recently shown that Itch directly limits the magnitude of GC responses. Proteomic profiling of GC B cells uncovered that Itch-deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Bone marrow chimera and adoptive transfer experiments revealed that B cell Itch restricts noncycling LZ cells. These results support, to our knowledge, a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and dark zones will aid in understanding how autoreactive clones emerge from GCs in people with autoimmune disease.

Curcumin Nanodiscs Improve Solubility and Serve as Radiological Protectants against Ionizing Radiation Exposures in a Cell-Cycle Dependent Manner.

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into “biomimetic” nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following 137Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors.

Abstract 1598: Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy characterized by robust, however transient, responses to frontline platinum-based therapy that are rapidly followed by refractory relapses. Treatment resistance is a significant concern in SCLC and a major factor regulating resistance is an emergence of unique cellular populations and an increase in intratumoral heterogeneity (ITH). We hypothesize that these emerging populations expressing various resistance-associated pathways, represent multiple, unique drug-resistant persister cell populations that underlie increased ITH and promote therapeutic resistance and, eventually, relapse, despite the initial response to therapy. Persister cells represent a unique population of cancer cells that are resistant to therapeutic pressure. While most persister cells remain arrested under treatment, a rare sub-population exists that can reverse state and re-enter the cell cycle. We performed single cell RNAseq of relapsed, extensive stage SCLC patient paired core biopsies collected 1) following relapse to standard of care (SOC) platinum chemotherapy plus immune checkpoint blockade (ICB) and 2) after six weeks of further therapy. Unlike what we found in platinum-sensitive disease, there is not a meaningful change in ITH score between cancer cells in the first and second biopsy. Presumably, maximum heterogeneity developed along with relapse to SOC treatment. Biopsies represent SCLC across subtypes (SCLC-A/N/P/I) with a modest loss of transcription factor expression following treatment (e.g., 76.8% to 57.9% NEUROD1). Cancer cells were classified as cycling or non-cycling to identify potential persister cell populations in paired patient core biopsies. Molecular subtype marker ASCL1 was reduced in non-cycling cells, but there was no change in NEUROD1 or POU2F3. Cycling cells demonstrate increased expression of NE genes (SYP, INSM1, UCHL1), biomarkers of response (SLFN11 and CDH1), but also genes associated with resistance (EZH2, NFIB). Non-cycling cells exhibit some common resistance mechanisms (e.g., EMT and MYC, AXL, ZFP36L1, and REST overexpression) and increased expression of inflammatory genes (e.g. HLA family) compared to cycling cells, reminiscent of the SCLC-Inflamed (SCLC-I) subtype. These data suggest that non-cycling persister cells may be more sensitive to ICB, AXL and/or AURK inhibition, while cycling cells may be more responsive to platinum chemotherapy, epigenetic modifiers or DNA repair targeted therapies. Clinically, these data underscore the importance of maximizing and maintaining the initial response in platinum-sensitive SCLC tumors, while highlighting the transcriptional complexity underlying SCLC’s profound treatment resistance following SOC and address the major need to develop combination therapies to target these distinct cell populations. Citation Format: C. Allison Stewart, Yuanxin Xi, Runsheng Wang, Michael M. Frumovitz, Jing Wang, Lauren A. Byers, Carl M. Gay. Defining the transcriptional complexity of persister cell populations in relapsed small cell lung cancer patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1598.

MEDB-59. A draft atlas of medulloblastoma cellular evolution under therapy

How standard care shapes the cellular composition of recurrent medulloblastoma (MB), if therapy selects for specific tumor or immune cell types, is unknown. We report the pilot phase of our ongoing effort to profile human longitudinal MB specimens via single-cell transcriptomics and epigenetics. We profiled 11 diagnostic and eight recurrent specimens from 19 subjects via single-nucleus RNA sequencing (snRNA-seq), and four subjects via single-nucleus assay for transposase-accessible chromatin. Specimens from select subjects were also profiled to assess genome-wide enhancer activity via single-nucleus cleavage-under-targets and tagmentation. We found an upregulation of the DNA-damage response, RNA translation, WNT and NOTCH signaling in recurrent specimens. The percentages of stem-like cells increased by over two-fold at recurrence. We found that microglia and oligodendrocyte-lineage cells were the most abundant non-malignant tumor-associated cell types, representing 2%-10% of cells profiled. Microglia abundances were relatively stable across molecular subtypes, and when comparing primary to recurrent tumors. There was a moderate, but statistically significant, increase in oligodendrocyte abundance in SSH and WNT tumors, compared to Group 3/4 tumors. We compared gene expression in tumor cells with public snRNA-seq from developing human cerebella (PCW 9-21). Combined Group-3/4 cell analysis supports a common lineage hierarchy, with an enrichment for unipolar brush-cell and Purkinje-cell phenotypes found in Group-4 tumors. All Group-3/4 cases contained cycling cells expressing markers of PAX2+ interneuron progenitors, most cycling cells had this phenotype. All specimens contained populations of non-cycling granule-cell progenitor-like cells. We performed single-cell co-expression receptor/ligand analysis to infer paracrine signaling between tumor and non-malignant cell types. This identified both tumor cells and microglia as sources of growth factors, pro-inflammatory cytokines, and pro-apoptotic ligands. Non-malignant oligodendrocyte-lineage cells uniquely expressed IL6-family cytokines, pleiotrophin, and class-III semaphorins. These studies shed light on the cellular heterogeneity of MB and the effect of standard therapy in shaping composition at recurrence.

Immuno-senescence and elevated central carbon metabolism in hematopoietic stem cell compartment upon aging.

e24030 Background: Aging is associated with decline of hematopoiesis, compromised innate immunity, and propensity for malignancies. Aging of the hematopoietic stem and progenitor cell (HSPC) compartment is caused by accumulation of senescent cells and elimination of the latter rejuvenates innate immunity in animal models. Methods: HSC (CD34+) were isolated from the bone marrow of healthy human subjects (age range 20 – 74 years; n = 18) and separated by FACS in 3 distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GUhigh, GUinter, GUlow). Single-cell RNA-sequencing (scRNAseq) studies, followed by gene ontology (GO) analysis of biological processes, analysis of cell cycle, and gene set enrichment analysis (GSEA) were performed. Results: While the percentages of GUinter and GUlow subpopulations remain relatively constant with age, the proportion of GUhigh subset increases continuously with age (from 0.2% in adults < 30 years to 6,7% in > 60 years). GO analysis revealed that the GUhigh subset showed a significantly higher expression of genes involved in myeloid development, cell cycle arrest, inflammation and stress response, apoptosis, and significantly lower expressions of genes involved in lymphoid development. GSEA confirmed that the gene sets for cell cycle arrest, senescence associated secretory phenotype (SASP), anti-apoptosis/survival and P53 pathways are significantly up-regulated in the GUhigh subset. The latter shows therefore all deregulations that are inherent hallmarks of senescence. Most of the molecular deregulations are similar to those found in murine models of aging, but there are significant differences. In addition, cell cycle analysis revealed that the proportion of cells in the G1 phase was significantly reduced in the GUhigh subset. Comparing the frequencies of old to young GUhigh, GUlow cells along the cell cycle progression ranks showed a significant depletion of cells in late G1 phase among GUhigh cells but not in the other subsets. The frequencies of non-cycling cells in the GUlowsubset have remained high (> 70%) irrespective of age, thus providing evidence for the quiescence status of the GUlow subset, as well as for persistence of HSPC with “young phenotype”. Conclusions: Our series of proteome and single cell transcriptome analyses have shown that the GUhigh subpopulation is highly enriched in senescent HSPCs. This property may be exploited to enrich, visualize, and trace senescence in human bone marrow. Cell cycle analysis has revealed a significantly shortened G1 phase that is linked to dedifferentiated state in the senescent HSPC. Our results indicate a dependency of senescent HSPCs on elevated glucose metabolism as well as on anti-apoptotic factors for survival. These may represent ideal targets for elimination of senescent HSPC to restore innate immunity as demonstrated in murine models.

Single-cell RNA-seq reveals heterogeneity in hiPSC-derived muscle progenitors and E2F family as a key regulator of proliferation.

Human pluripotent stem cell-derived muscle progenitor cells (hiPSC-MuPCs) resemble fetal-stage muscle progenitor cells and possess in vivo regeneration capacity. However, the heterogeneity of hiPSC-MuPCs is unknown, which could impact the regenerative potential of these cells. Here, we established an hiPSC-MuPC atlas by performing single-cell RNA sequencing of hiPSC-MuPC cultures. Bioinformatic analysis revealed four cell clusters for hiPSC-MuPCs: myocytes, committed, cycling, and noncycling progenitors Using FGFR4 as a marker for noncycling progenitors and cycling cells and CD36 as a marker for committed and myocyte cells, we found that FGFR4+ cells possess a higher regenerative capacity than CD36+ cells. We also identified the family of E2F transcription factors are key regulators of hiPSC-MuPC proliferation. Our study provides insights on the purification of hiPSC-MuPCs with higher regenerative potential and increases the understanding of the transcriptional regulation of hiPSC-MuPCs.

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript.

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.

Abstract 9854: Somatic Mutations in Single Human Cardiomyocytes Reveal Rapid Age-Related DNA Damage and Cardiomyocyte Fusion

Introduction: Advanced age is the most important risk factor for heart disease, yet we have an incomplete understanding of how aging promotes heart disease. Accumulation of somatic DNA mutations has been demonstrated to be a hallmark of aging in many human cell types. The underlying cause of these many aging phenotypes is likely molecular in nature, but its mechanism is not well understood. Methods: To define the effect of aging on cardiomyocytes we performed single-cell whole-genome or panel sequencing from ~1000 single cardiomyocytes from 10 individuals aged 0.4 ~ 82 yrs. Mutational signatures of aging cardiomyocytes were analyzed by non-negative matrix factorization and a cell lineage tree was constructed. Results and Conclusions: Tetraploid cardiomyocytes showed a significant increase of (somatic single nucleotide variants (sSNV) density with age ( p = 0.0027), and this age-dependent increase remained significant even after adjusting for differences in the evenness of amplification. The sSNVs were distributed broadly across the genome. We further confirmed the age-dependent trend in diploid cardiomyocytes. A consistent increase of somatic mutation burden in human heart muscle cells with age, regardless of cardiomyocyte nuclear ploidy was observed. Cardiomyocytes accumulate age-related SNVs at rates higher than those previously described in other noncycling cells like neurons. Signature analysis reveals that cardiomyocyte SNVs display distinctive signatures of mutagenic processes, including increased oxidative stress, defective mismatch repair. A cell lineage tree, constructed from shared somatic single nucleotide variants, revealed that many polyploid cardiomyocytes derive from multiple clonal origins, implicating cell fusion as a major mechanism contributing to polyploidization. Since age-accumulated sSNVs create many damaging mutations, the fusion of clonally distinct cells to form multiploid cardiomyocytes provides a mechanism of genetic compensation that may minimize the complete knockout of essential genes during aging. Age-related accumulation of cardiac mutations provides a paradigm to understand the influence of aging on cardiac dysfunction.

Corrigendum: Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication.

We report that mouse liver cells are highly resistant to extensive telomere dysfunction. In proliferating cells, telomere dysfunction results in chromosome end fusions, a DNA damage signal, and apoptosis or senescence. To determine the consequences of telomere dysfunction in noncycling cells, we used conditional deletion of the telomeric protein TRF2 in hepatocytes. TRF2 loss resulted in telomeric accumulation of gamma-H2AX and frequent telomere fusions, indicating telomere deprotection. However, there was no induction of p53 or apoptosis, and liver function appeared unaffected. Furthermore, the loss of TRF2 did not compromise liver regeneration after partial hepatectomy. Remarkably, liver regeneration occurred without cell division involving endoreduplication and cell growth, thereby circumventing the chromosome segregation problems associated with telomere fusions. We conclude that nondividing hepatocytes can maintain and regenerate liver function despite substantial loss of telomere integrity.

DNA End Joining: G0-ing to the Core.

Humans have evolved a series of DNA double-strand break (DSB) repair pathways to efficiently and accurately rejoin nascently formed pairs of double-stranded DNA ends (DSEs). In G0/G1-phase cells, non-homologous end joining (NHEJ) and alternative end joining (A-EJ) operate to support covalent rejoining of DSEs. While NHEJ is predominantly utilized and collaborates extensively with the DNA damage response (DDR) to support pairing of DSEs, much less is known about A-EJ collaboration with DDR factors when NHEJ is absent. Non-cycling lymphocyte progenitor cells use NHEJ to complete V(D)J recombination of antigen receptor genes, initiated by the RAG1/2 endonuclease which holds its pair of targeted DSBs in a synapse until each specified pair of DSEs is handed off to the NHEJ DSB sensor complex, Ku. Similar to designer endonuclease DSBs, the absence of Ku allows for A-EJ to access RAG1/2 DSEs but with random pairing to complete their repair. Here, we describe recent insights into the major phases of DSB end joining, with an emphasis on synapsis and tethering mechanisms, and bring together new and old concepts of NHEJ vs. A-EJ and on RAG2-mediated repair pathway choice.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

H4K20 Methylation Is Differently Regulated by Dilution and Demethylation in Proliferating and Cell-Cycle-Arrested Xenopus Embryos.

DNA replication during cell division leads to dilution of histone modifications and can thus affect chromatin-mediated gene regulation, raising the question of how the cell-cycle shapes the histone modification landscape, particularly during embryogenesis. We tackled this problem by manipulating the cell cycle during early Xenopus laevis embryogenesis and analyzing invivo histone H4K20 methylation kinetics. The global distribution of un-, mono-, di-, and tri-methylated histone H4K20 was measured by mass spectrometry in normal and cell-cycle-arrested embryos over time. Using multi-start maximum likelihood optimization and quantitative model selection, we found that three specific biological methylation rate constants were required to explain the measured H4K20 methylation state kinetics. While demethylation is essential for regulating H4K20 methylation kinetics in non-cycling cells, demethylation is very likely dispensable in rapidly dividing cells of early embryos, suggesting that cell-cycle-mediated dilution of H4K20 methylation is an essential regulatory component for shaping its epigenetic landscape during early development. A record of this paper's transparent peer review process is included in the Supplemental Information.