Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

Comparison of cell response to chromatin and DNA damage.

DNA-targeting drugs are widely used for anti-cancer treatment. Many of these drugs cause different types of DNA damage, i.e. alterations in the chemical structure of DNA molecule. However, molecules binding to DNA may also interfere with DNA packing into chromatin. Interestingly, some molecules do not cause any changes in DNA chemical structure but interfere with DNA binding to histones and nucleosome wrapping. This results in histone loss from chromatin and destabilization of nucleosomes, a phenomenon that we call chromatin damage. Although the cellular response to DNA damage is well-studied, the consequences of chromatin damage are not. Moreover, many drugs used to study DNA damage also cause chromatin damage, therefore there is no clarity on which effects are caused by DNA or chromatin damage. In this study, we aimed to clarify this issue. We treated normal and tumor cells with bleomycin, nuclease mimicking drug which cut predominantly nucleosome-free DNA and therefore causes DNA damage in the form of DNA breaks, and CBL0137, which causes chromatin damage without direct DNA damage. We describe similarities and differences between the consequences of DNA and chromatin damage. Both agents were more toxic for tumor than normal cells, but while DNA damage causes senescence in both normal and tumor cells, chromatin damage does not. Both agents activated p53, but chromatin damage leads to the accumulation of higher levels of unmodified p53, which transcriptional activity was similar to or lower than that of p53 activated by DNA damage. Most importantly, we found that while transcriptional changes caused by DNA damage are limited by p53-dependent activation of a small number of p53 targets, chromatin damage activated many folds more genes in p53 independent manner.

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle.

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

Comparison of cell response to chromatin and DNA damage.

DNA-targeting drugs may damage DNA or chromatin. Many anti-cancer drugs damage both, making it difficult to understand their mechanisms of action. Using molecules causing DNA breaks without altering nucleosome structure (bleomycin) or destabilizing nucleosomes without damaging DNA (curaxin), we investigated the consequences of DNA or chromatin damage in normal and tumor cells. As expected, DNA damage caused p53-dependent growth arrest followed by senescence. Chromatin damage caused higher p53 accumulation than DNA damage; however, growth arrest was p53-independent and did not result in senescence. Chromatin damage activated the transcription of multiple genes, including classical p53 targets, in a p53-independent manner. Although these genes were not highly expressed in basal conditions, they had chromatin organization around the transcription start sites (TSS) characteristic of most highly expressed genes and the highest level of paused RNA polymerase. We hypothesized that nucleosomes around the TSS of these genes were the most sensitive to chromatin damage. Therefore, nucleosome loss upon curaxin treatment would enable transcription without the assistance of sequence-specific transcription factors. We confirmed this hypothesis by showing greater nucleosome loss around the TSS of these genes upon curaxin treatment and activation of a p53-specific reporter in p53-null cells by chromatin-damaging agents but not DNA-damaging agents.

Correction: MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity.

[This corrects the article DOI: 10.1371/journal.pgen.1010171.].

MDM2 E3 ligase activity is essential for p53 regulation and cell cycle integrity.

MDM2 and MDM4 are key regulators of p53 and function as oncogenes when aberrantly expressed. MDM2 and MDM4 partner to suppress p53 transcriptional transactivation and polyubiquitinate p53 for degradation. The importance of MDM2 E3-ligase-mediated p53 regulation remains controversial. To resolve this, we generated mice with an Mdm2 L466A mutation that specifically compromises E2 interaction, abolishing MDM2 E3 ligase activity while preserving its ability to bind MDM4 and suppress p53 transactivation. Mdm2L466A/L466A mice exhibit p53-dependent embryonic lethality, demonstrating MDM2 E3 ligase activity is essential for p53 regulation in vivo. Unexpectedly, cells expressing Mdm2L466A manifest cell cycle G2-M transition defects and increased aneuploidy even in the absence of p53, suggesting MDM2 E3 ligase plays a p53-independent role in cell cycle regulation and genome integrity. Furthermore, cells bearing the E3-dead MDM2 mutant show aberrant cell cycle regulation in response to DNA damage. This study uncovers an uncharacterized role for MDM2's E3 ligase activity in cell cycle beyond its essential role in regulating p53's stability in vivo.

FACT maintains nucleosomes during transcription and stem cell viability in adult mice.

Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT has a role in transcription elongation, although whether it disassembles or assembles nucleosomes during this process is unclear. To elucidate the function of FACT in mammals, we deleted the Ssrp1 subunit of FACT in adult mice. FACT loss is lethal, possibly due to the loss of the earliest progenitors in bone marrow and intestine, while more differentiated cells are not affected. Using cells isolated from several tissues, we show that FACT loss reduces the viability of stem cells but not of cells differentiated in vitro. FACT depletion increases chromatin accessibility in a transcription-dependent manner in adipose mesenchymal stem cells, indicating that nucleosomes are lost in these cells during transcription in the absence of FACT. We also observe activation of interferon (IFN) signaling and the accumulation of immunocytes in organs sensitive to FACT loss. Our data indicate that FACT maintains chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in stem cells and differentiated cells is different.

Induction of cell death in ovarian cancer cells by doxorubicin and oncolytic vaccinia virus is associated with CREB3L1 activation.

We have demonstrated that oncolytic vaccinia virus synergizes with doxorubicin (DOX) in inducing immunogenic cell death in platinum-resistant ovarian cancer cells and increases survival in syngeneic and xenograft tumor models. However, the mechanisms underlying the virus- and doxorubicin-mediated cancer cell death remain unknown. In this study, we investigated the effect of the oncolytic virus and doxorubicin used alone or in combination on activation of the cytoplasmic transcription factor CREB3L1 (cyclic AMP [cAMP] response element-binding protein 3-like 1) in ovarian cancer cell lines and clinical specimens. We demonstrated that doxorubicin-mediated cell death in ovarian cancer cell lines was associated with nuclear translocation of CREB3L1 and that the effect was augmented by infection with oncolytic vaccinia virus or treatment with recombinant interferon (IFN)-ß used as a viral surrogate. This combination treatment was also effective in mediating nuclear translocation of CREB3L1 in cancer cells isolated from ovarian tumor biopsies at different stages of disease progression. The measurement of CREB3L1 expression in clinical specimens of ovarian cancer revealed lack of correlation with the stage of disease progression, suggesting that understanding the mechanisms of nuclear accumulation of CREB3L1 after doxorubicin treatment alone or in combination with oncolytic virotherapy may lead to the development of more effective treatment strategies against ovarian cancer.

Ameliorating the hallmarks of cellular senescence in skeletal muscle myogenic progenitors in vitro and in vivo.

Senescence of myogenic progenitors impedes skeletal muscle regeneration. Here, we show that overexpression of the transcription factor NANOG in senescent myoblasts can overcome the effects of cellular senescence and confer a youthful phenotype to senescent cells. NANOG ameliorated primary hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction. The rejuvenating effects of NANOG included restoration of DNA damage response via up-regulation of DNA repair proteins, recovery of heterochromatin marks via up-regulation of histones, and reactivation of autophagy and mitochondrial energetics via up-regulation of AMP-activated protein kinase (AMPK). Expression of NANOG in the skeletal muscle of a mouse model of premature aging restored the number of myogenic progenitors and induced formation of eMyHC+ myofibers. This work demonstrates the feasibility of reversing the effects of cellular senescence in vitro and in vivo, with no need for reprogramming to the pluripotent state.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

SERINC5 Potently Restricts Retrovirus Infection In Vivo.

The serine incorporator (SERINC) proteins are multipass transmembrane proteins that affect sphingolipid and phosphatidylserine synthesis. Human SERINC5 and SERINC3 were recently shown to possess antiretroviral activity for a number of retroviruses, including human immunodeficiency virus (HIV), murine leukemia virus (MLV), and equine infectious anemia virus (EIAV). In the case of MLV, the glycosylated Gag (glyco-Gag) protein was shown to counteract SERINC5-mediated restriction in in vitro experiments and the viral envelope was found to determine virion sensitivity or resistance to SERINC5. However, nothing is known about the in vivo function of SERINC5. Antiretroviral function of a host factor in vitro is not always associated with antiretroviral function in vivo Using SERINC5-/- mice that we had generated, we showed that mouse SERINC5 (mSERINC5) restriction of MLV infection in vivo is influenced not only by glyco-Gag but also by the retroviral envelope. Finally, we also examined the in vivo function of the other SERINC gene with known antiretroviral functions, SERINC3. By using SERINC3-/- mice, we found that the murine homologue, mSERINC3, had no antiretroviral role either in vivo or in vitro To our knowledge, this report provides the first data showing that SERINC5 restricts retrovirus infection in vivo and that restriction of retrovirus infectivity in vivo is dependent on the presence of both glyco-Gag and the viral envelope.IMPORTANCE This study examined for the first time the in vivo function of the serine incorporator (SERINC) proteins during retrovirus infection. SERINC3 and SERINC5 (SERINC3/5) restrict a number of retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), by blocking their entry into cells. Nevertheless, HIV-1 and MLV encode factors, Nef and glycosylated Gag, respectively, that counteract SERINC3/5 in vitro We recently developed SERINC3 and SERINC5 knockout mice to examine the in vivo function of these genes. We found that SERINC5 restriction is dependent on the absence of glycosylated Gag and the expression of a specific viral envelope glycoprotein. On the other hand, SERINC3 had no antiviral function. Our findings have implications for the development of therapeutics that target SERINC5 during retrovirus infection.

Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos.

It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.

Improved hematopoietic differentiation of mouse embryonic stem cells through manipulation of the RNA binding protein ARS2.

The RNA binding protein ARS2 is highly expressed in hematopoietic progenitor populations and is required for adult hematopoiesis. Recent molecular studies found that ARS2 coordinates interactions between nascent RNA polymerase II transcripts and downstream RNA processing machineries, yet how such interactions influence hematopoiesis remains largely unknown. Techniques to differentiate embryonic stem cells (ESC) to hematopoietic progenitor cells (HPC) and mature blood cells have increased molecular understanding of hematopoiesis. Taking such an in vitro approach to examine the influence of ARS2 on hematopoiesis, we found that ARS2 suppresses expression of some HSC signature genes and differentiation of ESC to a HPC population (CSMD-HPC) identified by markers expressed on bone marrow resident hematopoietic stem cells. In line with ARS2's ability to promote proliferation of cultured cells, ARS2 knockout ESC showed limited expansion and yielded less CSMD-HPC than wild-type ESC. In contrast, transient ARS2 knockdown led to doubling the number of CSMD-HPC generated per ESC without affecting further differentiation into mature T-cells. Overall, data indicate that ARS2 negatively regulates early hematopoietic differentiation of ESC, in stark contrast to its supportive role in adult hematopoiesis. Consequently, manipulation of ARS2 expression and/or function has potential utility in hematopoietic cell engineering and regenerative medicine.

Doxycycline-Dependent Self-Inactivation of CRISPR-Cas9 to Temporally Regulate On- and Off-Target Editing.

Exome and deep sequencing of cells treated with a panel of lentiviral guide RNA demonstrate that both on- and off-target editing proceed in a time-dependent manner. Thus, methods to temporally control Cas9 activity would be beneficial. To address this need, we describe a "self-inactivating CRISPR (SiC)" system consisting of a single guide RNA that deactivates the Streptococcus pyogenes Cas9 nuclease in a doxycycline-dependent manner. This enables defined, temporal control of Cas9 activity in any cell type and also in vivo. Results show that SiC may enable a reduction in off-target editing, with less effect on on-target editing rates. This tool facilitates diverse applications including (1) the timed regulation of genetic knockouts in hard-to-transfect cells using lentivirus, including human leukocytes for the identification of glycogenes regulating leukocyte-endothelial cell adhesion; (2) genome-wide lentiviral sgRNA (single guide RNA) library applications where Cas9 activity is ablated after allowing pre-determined editing times. Thus, stable knockout cell pools are created for functional screens; and (3) temporal control of Cas9-mediated editing of myeloid and lymphoid cells in vivo, both in mouse peripheral blood and bone marrow. Overall, SiC enables temporal control of gene editing and may be applied in diverse application including studies that aim to reduce off-target genome editing.

BORIS Expression in Ovarian Cancer Precursor Cells Alters the CTCF Cistrome and Enhances Invasiveness through GALNT14.

High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecologic cancer-related death. We have previously shown that CTCFL (also known as BORIS, Brother of the Regulator of Imprinted Sites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed BORIS in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC. BORIS-expressing cells exhibited increased motility and invasion, and BORIS expression was associated with alterations in several cancer-associated gene expression networks, including fatty acid metabolism, TNF signaling, cell migration, and ECM-receptor interactions. Importantly, GALNT14, a glycosyltransferase gene implicated in cancer cell migration and invasion, was highly induced by BORIS, and GALNT14 knockdown significantly abrogated BORIS-induced cell motility and invasion. In addition, in silico analyses provided evidence for BORIS and GALNT14 coexpression in several cancers. Finally, ChIP-seq demonstrated that expression of BORIS was associated with de novo and enhanced binding of CTCF at hundreds of loci, many of which correlated with activation of transcription at target genes, including GALNT14. Taken together, our data indicate that BORIS may promote cell motility and invasion in HGSC via upregulation of GALNT14, and suggests BORIS as a potential therapeutic target in this malignancy. IMPLICATIONS: These studies provide evidence that aberrant expression of BORIS may play a role in the progression to HGSC by enhancing the migratory and invasive properties of FTSEC.

Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression.

The FOXM1 transcription factor network is frequently activated in high-grade serous ovarian cancer (HGSOC), the most common and lethal subtype of epithelial ovarian cancer (EOC). We used primary human EOC tissues, HGSOC cell lines, mouse and human ovarian surface epithelial (OSE) cells, and a murine transgenic ovarian cancer model to investigate genetic determinants of FOXM1 overexpression in EOC, and to begin to define its functional contribution to disease pathology. The Cancer Genome Atlas (TCGA) data indicated that the FOXM1 locus is amplified in ~12% of HGSOC, greater than any other tumor type examined, and that FOXM1 amplification correlates with increased expression and poor survival. In an independent set of primary EOC tissues, FOXM1 expression correlated with advanced stage and grade. Of the three known FOXM1 isoforms, FOXM1c showed highest expression in EOC. In murine OSE cells, combined knockout of Rb1 and Trp53 synergistically induced FOXM1. Consistently, human OSE cells immortalized with SV40 Large T antigen (IOSE-SV) had significantly higher FOXM1 expression than OSE immortalized with hTERT (IOSE-T). FOXM1 was overexpressed in murine ovarian tumors driven by combined Rb1/Trp53 disruption. FOXM1 induction in IOSE-SV cells was partially dependent on E2F1, and FOXM1 expression correlated with E2F1 expression in human EOC tissues. Finally, FOXM1 functionally contributed to cell cycle progression and relevant target gene expression in human OSE and HGSOC cell models. In summary, gene amplification, p53 and Rb disruption, and E2F1 activation drive FOXM1 expression in EOC, and FOXM1 promotes cell cycle progression in EOC cell models.

The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage.

Bladder cancer risk is significantly higher in men than in women. 4-Aminobiphenyl (ABP) is a major human bladder carcinogen from tobacco smoke and other sources. In mice, male bladder is more susceptible to ABP-induced carcinogenesis than female bladder, but ABP is more carcinogenic in the livers of female mice than of male mice. Here, we show that castration causes male mice to acquire female phenotype regarding susceptibility of bladder and liver to ABP. However, spaying has little impact on organ susceptibility to ABP. Liver UDP-glucuronosyltransferases (UGTs) are believed to protect liver against but sensitize bladder to ABP, as glucuronidation of ABP and its metabolites generally reduces their toxicity and promotes their elimination via urine, but the metabolites are labile in urine, delivering carcinogenic species to the bladder. Indeed, liver expression of ABP-metabolizing human UGT1A3 transgene in mice increases bladder susceptibility to ABP. However, ABP-specific liver UGT activity is significantly higher in wild-type female mice than in their male counterparts, and castration also significantly increases ABP-specific UGT activity in the liver. Taken together, our data suggest that androgen increases bladder susceptibility to ABP via liver, likely by modulating an ABP-metabolizing liver enzyme, but exclude UGT as an important mediator.

Generation of a novel transgenic mouse model for bioluminescent monitoring of survivin gene activity in vivo at various pathophysiological processes: survivin expression overlaps with stem cell markers.

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker.

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.