DNA Silencing Research Articles

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease I in CHO cells.

While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523-533, 2017.

Use of the DICE (Dual Integrase Cassette Exchange) System.

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

Abstract B01: Integrated genomic analysis identifies two subsets of cervical cancer with potentially distinct therapeutic profiles

Abstract Background and Significance: The continuous expression of HPV oncogenes is responsible for initiating and driving cervical cancer. Current treatments include radiation and Cisplatin-based chemotherapy. Targeted therapies can be directed to specific subset of tumors if they are known. Methods: Molecular genetic data generated by The Cancer Genome Atlas (TCGA) were analyzed. Results: We discovered and characterized a subset of HPV positive cervical cancers that do not express HPV transcripts, including the E6 and E7 oncogenes. These tumors more commonly appear in women with a median age of diagnosis 9 years later (54 vs. 45 years) (p-value = 0.023) and reveal worse survival (p-value 0.06). Gene expression profiles of HPV expressing tumors (HPV-active) are distinct from those tumors that do not express HPV (HPV-inactive). Gene Set Enrichment Analysis of the HPV-active tumors show enrichment for E2F target genes, as expected based on the activity of HPV E7. HPV-inactive tumors show enrichment for WNT/beta-catenin target genes. Additionally, this analysis reveals that the HPV-active tumors have increased AKT/MTOR signaling and HPV-inactive tumors have increased sonic hedgehog signaling indicating different potential therapeutic targets for these two classes of tumors. The majority of differentially expressed genes can be explained by DNA hyper methylation and silencing of expression. Somatic mutation landscapes are significantly different. HPV-inactive tumors are significantly enriched for non-synonymous somatic mutations (p-value <0.0000001) compared to HPV-active tumors. Conclusions: Many of the somatic mutations found in non-expressing tumors mimic the functions of the E6 and E7 viral oncoproteins, such as TP53, CTNNB1, EGFR and KRAS somatic mutations. Unexpectedly, BRCA1 and BRCA2 somatic mutations are more frequent in the HPV-inactive tumors. Therapeutic strategies based on PARP inhibition may be effective against this category of cervical cancers. Application of therapeutics routinely used in other cancer types could be used to exploit the weaknesses we discovered in the HPV-inactive tumors and may improve survival for these cervical cancer patients. Citation Format: Carolyn E. Banister, Lucia Pirisi, Kim E. Creek, Phillip J. Buckhaults. Integrated genomic analysis identifies two subsets of cervical cancer with potentially distinct therapeutic profiles. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B01.

Upregulation of N - myc downstram regulated gene 1 expression by silencing DNA methyltransferases influences the biological behaviors of prostate cancer DU145 cells

Objective To investigate the influence of the upregulation of N-myc downstram regulated gene 1 (NDRG1) expression by silencing DNA methyltransferases (DNMTs) using small interfering RNAs (siRNAs) on the biological behaviors of prostate cancer DU145 cells. Methods SiRNAs were used to inhibit the expression of DNMT1, DNMT3b of DU145 cells; Western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression levels of DNMT1, DNMT3b and NDRG1 of DU145 cells; cell counting kit-8 (CCK-8) assay, muse cell analyzer, wound healing assay and Transwell migration assay were used to detect the proliferation, apoptosis, migration and invasion of DU145 cells; then we inhibited the expression of NDRG1 of DU145 cells which had the most significant changes of cell biological behaviors; and we detected the expression level of NDRG1 and the changes of cell biological behaviors. Results Western blotting and RT-qPCR showed that the mRNA (1.59±0.03, 1.23±0.01, 1.36±0.11 vs.0.90±0.15, 1.00±0.03) and protein (1.07±0.16, 0.49±0.01, 0.92±0.09 vs.0.23±0.04, 0.30±0.07) expression levels of NDRG1 of DNMT1 inhibition group, DNMT3b inhibition group and double inhibition group were significantly higher than these in control groups (P<0.05). CCK-8 assay showed that, at 24, 48 and 72 h, the optical density (A) values of DNMT1 inhibition group (0.189±0.030, 0.266±0.048, 0.419±0.058), DNMT3b inhibition group (0.221±0.023, 0.318±0.072, 0.498±0.064) and double inhibition group (0.199±0.021, 0.284±0.062, 0.488±0.091) were significantly lower than control groups (P<0.05). Apoptosis results showed that the apoptosis rates of DNMT1 inhibition group (22.40±1.36)%, DNMT3b inhibition group (14.20±0.80)% and double inhibition group (17.60±0.74)% were significantly higher than control groups (P<0.05). Wound healing assay showed that the healing rates of DNMT1 inhibition group (53.01±3.22)%, DNMT3b inhibition group (70.97±2.12)% and double inhibition group (63.93±4.42)% were significantly lower than control groups (P<0.05). Transwell migration assay showed that the amount of invasion cells in DNMT1 inhibition group [(16±3) cells], in DNMT3b inhibition group [(32±5) cells] and in double inhibition group [(20±4) cells] were significantly less than control groups (P<0.05). Among these groups, DNMT1 inhibition group had the most significant effect (P<0.05), but the co-transfection had no significant synergistic effect. Compared with control groups, NDRG1-siRNA could significantly reverse the changes of cell biological behaviors of DU145 cells (P<0.05). Conclusion Inhibition of DNMT1, DNMT3b of DU145 cells could partially restore NDRG1 expression, which could inhibit the proliferation, apoptosis, migration and invasion of cancer cells. Moreover, DNMT1 inhibition group had the most significant effect, but the co-transfection had no significant synergistic effect, suggesting that DNMT1 could become an effective target for demethylation treatment of prostate cancer. Key words: N-myc downstram regulated gene 1; DNA methylation; DNA methyltransferase; RNA interference; Prostate cancer

The Oncolytic Measles Virus Preferentially Infects p53 Abnormal Myeloma Cells

P53 plays a pleiotropic role in cell homeostasis by regulating stress-induced adaptive responses leading to survival or cell death. Although it is well demonstrated that the lack of a functional p53 pathway favors cancers, it remains still unclear whether it is related to its genome guardian or in apoptosis inducer role. Leonova and Gudkov demonstrated that, at least in mice, p53 and CpG methylation prevent expression of the so-called junk DNA, expression of which induces suicidal IFN responses (PNAS 2013;110:E89). Cancer cells mostly harbor hypo methylated DNA, reversible or irreversible silencing of p53 pathway and defects in suicidal IFN responses. Although the molecular bases are still not well understood, oncolytic viruses such as the Measles Virus (MV) preferentially infect and kill tumor cells. The aim of this study was to investigate whether del(17p)+/p53 mutant myeloma cells were preferentially sensitive to the Schwarz vaccine MV strain and whether p53 was involved in. Plasma cells from patients with multiple myeloma (MM) or MGUS over express CD46, the main receptor for the vaccine MV strain, as compared to normal plasma cells and to hematopoietic cells. To assess MV infection and replication, we used the GFP modified strain (MV-GFP) and measured GFP expression at day 4 after infection (MOI=1) using flow cytometry. We showed that p53 abnormal MM cell lines (n=25) over expressed CD46 (p=0.039) and were preferentially infected by MV (p=0.03) as compared with p53 wild type MM cell lines (n=12). Expressions of CD46 and GFP were directly correlated (p=0.014, r=0.505) and GFP expression correlated with cell death (n=37, p<0.0001, r=0.82). CD46 inhibition (using CD46 siRNA or anti-CD46 mAb) fully inhibited infection and death. CProduction of alpha IFN was not frequent (11 out of 37 cell lines), was irrespective of TP53 status and not related to MV resistance (no production of beta IFN was found). To assess whether p53 pathway regulates MV infection/replication, we treated cells 24 hours with the MDM2 inhibitor nutlin3a prior to MV infection. In all wild type and in none p53 abnormal cell lines, nutlin3a-pretreatment induced an increase in DR5 expression (Tessoulin B et al, Blood 2014;124:1626) and a decrease in CD46 expression: the mean of nutlin3a/control DR5 ratio of expression was 1.60 ± 0.27 and 1.04 ± 0.05 in wild type (n=10) and p53 abnormal cell lines (n=4), respectively (p=0.001) while that of nutlin3a/control CD46 ratio expression was 0.55 ± 0.08 and 0.97 ± 0.03, respectively, (p=0.0007). At 24 hours, cells were washed to remove nutlin3a and 9 cell lines sensitive to MV (5 wild type and 4 abnormal) were infected with MV (MOI=1). Decrease in CD46 expression was associated with decrease in MV replication in all p53 wild type (mean nutlin3a/control ratio 0.53 ± 0.05, n=5) but in none p53 abnormal cell lines (1.02 ± 0.18, n=4), p=0.016. We assessed infection of primary cells in unpurified mononuclear cells (MOI=1) from bone marrow or peripheral blood harboring various CD138+ infiltrate. Compared with CD138- cells, CD138+ cells over expressed CD46 (MFI ratio 100 versus 22, p=0.0003) and were preferentially infected by MV (GFP+ 62% versus 20%, p=0.0005, GFP MFI ratio 14 versus 5, p=0.0011, n=17). Assessment of TP53 status in primary cells was determined using FISH (del17p) and the functional assay of DR5 increase in response to nutlin3a. Although del(17p)+ myeloma cells over expressed CD46 and were highly infected by MV when compared to del(17p)- myeloma cells, differences didn’t reach statistical significance. We further showed that nutlin3a induced decrease in CD46 expression and MV infection in 3 primary cells without del(17p) but did not in 2 primary CD138+ cells with del(17p): decrease in CD46 expression correlated with decrease in GFP expression (n=5, p=0.0167, r=1.00) and with increase in DR5 expression (p=0.033, r=-0.89). These data showed that p53 abnormal myeloma cells (cell lines and primary cells) were strongly sensitive to the oncolytic MV, which could be of interest for patients with del(17p) refractory to current therapy. The molecular mechanism of p53-induced decrease in CD46 expression and MV infection is under investigation. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria.

Mir-199b, a Candidate Effector of Stress Myelopoiesis and Is Differentially Regulated in Acute Myeloid Leukemia

Recently, we reported the functional role of miR-199b in AML and its prognostic implications where low miR-199b in AML patients correlated with worse overall survival for the FAB-M5 subtype. Herein, we have attempted to define the role of miR-199b in stress myelopoiesis using knockout mice and examine miR-199b’s gene regulation. To define the role of miR-199b and understand its functional role in myeloid proliferation and AML, we generated miR-199b knockout mice via CRISPR approach. The deleted region was confirmed by DNA sequencing and silencing of miR-199b transcripts in nucleated peripheral blood cells was verified via RT-PCR. At steady state, except for significantly elevated neutrophil levels in miR-199b KO mice (n=5) compared to control mice (n=5) at 6 weeks of age, there were no significant differences in peripheral blood profiles. Bone marrow derived HSPCs (hematopoietic stem and progenitor cells) from miR-199b and wild type mice were examined for LT-HSC, KL, LSK, MPP, GMP and MEP populations. At steady state no significant differences were observed in all the cohorts except for KL (Lin-Kit+Sca-) population, where miR-199b KO mice exhibited significantly decreased levels compared to wild type mice (p<0.0093). Next, we examined the role of miR-199b under stress myelopoiesis by using 5-Fluouracil (5FU) induced myeloablation model. miR-199b KO mice exhibited significantly elevated levels of CFU-GEMM and CFU-GM compared to wild type mice. miR-199b levels in C57Bl/6 wild type mice were significantly decreased in HSCs post 5FU treatment. Gene profiling studies in HSCs post 5FU in wild type C57BL/6 mice identified three putative highly conserved miR-199b targets. They were Itga8 (Intergrin alpha 8), Zmpste 24 (metalloproteinase) and Scamp1 (secretory carrier membrane protein 1), whose roles are being investigated using miR-199b KO mice. Though we recently showed silencing of miR-199b in AML, the regulation of miR-199b gene expression in normal and leukemic cells is yet to be understood. Here, for the first time, using bioinformatics approach in integrated Genomics Viewer, we mined genomic database ENCODE (Encyclopedia of DNA elements) to identify both transcriptional and epigenetic landscape for miR-199b. Compared to CD34 and CD14, AML lines such as NB4 and HL-60 had relatively more regions of DNAase hypersensitivity. For DNA methylation, via reverse bisulphite sequencing data sets, no aberrant methylation was present between normal CD34 and HL-60, NB4 leukemic cells, which corroborates with our earlier finding of decitabine’s ineffectiveness in inducing miR-199b expression in AML cells. Previously we had demonstrated that HDAC inhibitors significantly induced miR-199b expression in THP-1 cells. Here, for CD34 cells, we observed a significant presence of H3K4 and H3K9 methylation in miR-199b promoter via CHiP-seq data sets, implying an active transcriptional status. In CD14+ monocyte progenitors, a similar pattern for H3K4 methylation was observed and as well as significant levels of H3K9 acetylation. However, in CD15+ cells (neutrophil) H3K9 methylation was absent. Interestingly, in HL-60 and NB4 AML cells, H3K4me3 histone modifications peaked directly above the miR-199b coding region, whose significance is yet to be understood. For transcription factors, in HL-60 cells, via Chip-seq data sets, we found significant levels of GABP, NRSF and PU.1 binding activity in the upstream region of miR-199b. In NB4 (APL) cells, the Chip-seq data revealed a significant co-presence of cMyc and MAX binding in the upstream region of miR-199b. Further, the binding sites for the respective factors were also tested and confirmed using Genomatix-MatInspector. To experimentally validate these genomics derived data, we treated AML lines (HL-60, NB4 and THP-1) with inhibitors for cMyc (10058-F4), Myc-Max heterodimer (10074-G5), Sp1 (Mithramycin A) and NRSF/REST (X5050) for 24 and 48 hours. Exposure to Myc-Max heterodimer and NRSF/REST inhibitors significantly elevated miR-199b levels for HL-60 cells. For THP-1 cells, treatment with Myc-Max and Sp1 inhibitors significantly elevated miR-199b levels. Further functional assays are required to identify the biological roles of miR-199b in AML. In terms of miR-199b gene expression, we observe a differential regulation pattern in normal and leukemic cells, which in part may reflect the molecular heterogeneity in AML. DisclosuresNo relevant conflicts of interest to declare.

RNAi soothes the path to quiescence

Cell Cycle Cells, such as those in stem cell niches and immunity memory cells, can exist in a nondividing, quiescent state, from which they can be aroused with the appropriate signal. RNA interference (RNAi) is an important epigenetic pathway in many organisms. Roche et al. found that in fission yeast, RNAi was an essential regulator of the quiescent state. RNAi promoted proper chromosome segregation during entry into quiescence. It also prevented inappropriate silencing of the ribosomal DNA during quiescence. Science , this issue p. [10.1126/science.aah5651][1] [1]: /lookup/doi/10.1126/science.aah5651

Methylation of Notch3 modulates chemoresistance via P-glycoprotein

The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells (MCF-7/PTX) and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells.

RNA interference is essential for cellular quiescence.

Quiescent cells play a predominant role in most organisms. Here we identify RNA interference (RNAi) as a major requirement for quiescence (G0 phase of the cell cycle) in Schizosaccharomyces pombe RNAi mutants lose viability at G0 entry and are unable to maintain long-term quiescence. We identified suppressors of G0 defects in cells lacking Dicer (dcr1Δ), which mapped to genes involved in chromosome segregation, RNA polymerase-associated factors, and heterochromatin formation. We propose a model in which RNAi promotes the release of RNA polymerase in cycling and quiescent cells: (i) RNA polymerase II release mediates heterochromatin formation at centromeres, allowing proper chromosome segregation during mitotic growth and G0 entry, and (ii) RNA polymerase I release prevents heterochromatin formation at ribosomal DNA during quiescence maintenance. Our model may account for the codependency of RNAi and histone H3 lysine 9 methylation throughout eukaryotic evolution.

Suffocation of gene expression

If a tumour outgrows its blood supply, oxygen levels in its cells decrease. It emerges that this change can alter gene expression by limiting the activity of TET enzymes, which remove methyl groups from DNA. See Article p.63 Tumours are epigenetically distinct from their tissue of origin, frequently showing increased DNA methylation of tumour suppressor gene promoters, but how these changes arise is poorly understood. Here, Diether Lambrechts and colleagues report that tumour hypoxia, pervasive in many solid tumours, reduces the activity of the oxygen-dependent ten-eleven translocation (TET) enzymes, which catalyse DNA demethylation through 5-methylcytosine oxidation. They show that oxygen is an important co-factor for TET activity, and hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro and at tumour suppressor genes in hypoxic tumours. The authors propose that tumour hypoxia directly reduces TET activity, leading to changes in DNA methylation and silencing gene expression. Countering hypermethylation by inhibiting DNA methylation or by normalizing tumour blood supply may therefore be of therapeutic benefit.

Abstract 4055: Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors

Abstract Ultraviolet (UV) radiation has variety of deleterious effects on mammalian skin, and triggers several causative factors such as DNA damage, inflammation, immune suppression and genetic and epigenetic alterations which leads to the development of skin cancer. Earlier, we have shown that the deficiency of interleukin (IL)-12, an immunoregulatory cytokine, enhances photocarcinogenesis in mice through inhibition of repair of damaged DNA. In further studies we have tried to explore and examine the effect of IL-12 on epigenetic regulators in UV-exposed skin and UV-induced skin tumors using IL-12 knock out (IL-12 KO) mice as an in vivo animal model. The exposure of IL-12 KO mice with UVB (200 mJ/cm2) radiation resulted in higher levels of DNA methylation, DNA methyltransferase (Dnmt) activity, Ten-eleven translocations (TETs), a family of methylcytosine dioxygenase, histone deacetylases (HDAC), and lower levels of histone acetylase (HAT) activity than their wild type counterparts. To verify that IL-12 deficiency is associated with DNA hypermethylation and higher HDAC activity in UVB exposed skin, IL-12-KO mice were treated with endotoxin-free murine rIL-12 (50 ng/mouse/100 μl PBS; subcutaneous injection) 3 h before UVB exposure. Our results indicate that significant reduction in the levels of global DNA methylation, Dnmt and HDAC activity were observed compared with non-rIL-12 treated mice. Conversely, UVB exposed IL-12 wild type mice were treated with anti-IL-12 antibody (500 ng/mouse/100 μl PBS; subcutaneous injection). This treatment resulted in increased levels of DNA methylation, Dnmt and HDAC activities. These observations were also noted and compared between skin tumors from IL-12 KO mice and their wild type counterparts. To further explore the role of IL-12 deficiency on epigenetic modification-mediated photocarcinogenesis, we determined its effect on methylated and unmethylated levels of tumor suppressor genes, such as p16INK4a and RASSF1A, in UV-exposed skin and UV-induced skin tumors. It was observed that the levels of methylated tumor suppressor genes were higher in IL-12 KO mice skin than their wild type counterparts. Together, these findings indicate that IL-12 deficiency promotes: (i) DNA hypermethylation, (ii) HDAC activity and (iii) silencing of tumor suppressor genes in UV exposed mice and that makes the mice more susceptible to UV radiation-induced skin cancer risk. Together, these observations suggest that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of UV radiation-induced skin cancer risk. Citation Format: Ram Prasad, Tripti Singh, Santosh K. Katiyar. Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4055.

Abstract 53: Epigenetic silencing of krebs cycle metabolism in kidney cancer

Abstract Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC) including recent large-scale transcriptomic data demonstrating loss of tricarboxylic acid (TCA) cycle enzyme expression with aggressive tumors. Using a metabolomics/transcriptomics approach, we identified loss of mRNA expression of PGC-1α and ERR-γ is associated with tumor progression and worsened outcomes in RCC patients. PGC-1α both induces and cooperates with ERR-γ to promote TCA cycle enzyme expression. Additionally, PGC-1α reconstitution in RCC cells reverses the glycolytic phenotype and suppresses invasive phenotypes. Loss of PGC-1α and ERR-γ is through DNA and histone repressive silencing of the transcription factor PRDM16. Loss of PRDM16 is among the most prominent transcriptional changes in metastatic tumor sites. PRDM16 expression in RCC cells restorers TCA cycle enzyme expression, mitigates the Warburg phenotype, and suppresses in vivo tumorigenesis. Collectively, our data uncover a transcription factor network epigenetically silenced in renal cancer resulting in suppression of mitochondrial metabolism. Citation Format: Eun-Young Kho, Carolina B. Livi, Phillip Buckhaults, Francesca Carobbio, Hye-Young Nam, Richard Kirkman, David Crossman, Praveen K. Vayalil, Shi Wei, Ralph J. DeBerardinis, Sunil Sudarshan. Epigenetic silencing of krebs cycle metabolism in kidney cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 53.

Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial trans-acting small interfering RNA.

TAS atasiRNA-producing region swapping used one-step, high efficiency, and high fidelity directional TC-cloning. Uniform silencing was achieved without lethality using miRNA trigger- TAS overexpression fusion cassettes to generate 21-nt atasiRNA. Plant transgenic technologies are very important for basic plant research and biotechnology. Artificial trans-acting small interfering RNA (atasiRNA) represents an attractive platform with certain advantages over other silencing approaches, such as hairpin RNA, artificial microRNA (amiRNA), and virus-induced gene silencing (VIGS). In this study, we developed two types of constructs for atasiRNA-mediated gene silencing in plants. To functionally validate our constructs, we chose TAS1a as a test model. Type 1 constructs had miR173-precursor sequence fused with TAS1a locus driven by single promoter-terminator cassette, which simplified the expression cassette and resulted in uniform gene silencing. Type 2 constructs contained two separate cassettes for miR173 andTAS1aco-expression. The constructs in each typewere further improved by deploying the XcmI-based TC-cloning system for highly efficient directional cloning of short DNA fragments encoding atasiRNAs into TAS1a locus. The effectiveness of the constructs was demonstrated by cloning anatasiRNA DNA into the TC site of engineered TAS1a and silencing of CHLORINA 42 (CH42) gene in Arabidopsis. Our results show that the directional TC-cloning of the atasiRNA DNA into the engineered TAS1a is highly efficient and the miR173-TAS1a fusion system provides an attractive alternative to achieve moderate but more uniform gene silencing without lethality, as compared to conventional two separate cassettes for miR173 and TAS locus co-expression system. The design principles described here should be applicable to other TAS loci such as TAS1b, TAS1c, TAS2, or TAS3, and cloning of amiRNA into amiRNA stem-loop.

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

Silencing DNA methyltransferase 1 (DNMT1) inhibits proliferation, metastasis and invasion in ESCC by suppressing methylation of RASSF1A and DAPK.

Our previous study showed DNMT1 is up-regulated in esophageal squamous cell carcinoma (ESCC), which is associated with methylation of tumor suppressors. In the current study, we investigate the role of DNMT1 in ESCC. We found silencing DNMT1 inhibited proliferation, metastasis and invasion of three different ESCC cells, K150, K410 and K450. We also found silencing DNMT1 induced G1 arrest and cell apoptosis in K150, K410 and K450 cells. In vivo study showed silencing DNMT1 suppressed tumor growth in nude mice. In addition, silencing DNMT1 increased expression of tumor suppressor genes, RASSF1A and DAPK, in ESCC cells and ESCC xenograft in nude mice. Moreover, silencing DNMT1 decreased methylation in promoter of RASSF1A and DAPK. In conclusion, our data demonstrated that silencing DNMT1 inhibits proliferation, metastasis and invasion in ESCC by suppressing methylation of RASSF1A and DAPK.

Rab11 and Lysotracker Markers Reveal Correlation between Endosomal Pathways and Transfection Efficiency of Surface-Functionalized Cationic Liposome-DNA Nanoparticles.

Cationic liposomes (CLs) are widely studied as carriers of DNA and short-interfering RNA for gene delivery and silencing, and related clinical trials are ongoing. Optimization of transfection efficiency (TE) requires understanding of CL-nucleic acid nanoparticle (NP) interactions with cells, NP endosomal pathways, endosomal escape, and events leading to release of active nucleic acid from the lipid carrier. Here, we studied endosomal pathways and TE of surface-functionalized CL-DNA NPs in PC-3 prostate cancer cells displaying overexpressed integrin and neuropilin-1 receptors. The NPs contained RGD-PEG-lipid or RPARPAR-PEG-lipid, targeting integrin, and neuropilin-1 receptors, respectively, or control PEG-lipid. Fluorescence colocalization using Rab11-GFP and Lysotracker enabled simultaneous colocalization of NPs with recycling endosome (Rab11) and late endosome/lysosome (Rab7/Lysotracker) pathways at increasing mole fractions of pentavalent MVL5 (+5 e) at low (10 mol %), high (50 mol %), and very high (70 mol %) membrane charge density (σM). For these cationic NPs (lipid/DNA molar charge ratio, ρchg = 5), the influence of membrane charge density on pathway selection and transfection efficiency is similar for both peptide-PEG NPs, although, quantitatively, the effect is larger for RGD-PEG compared to RPARPAR-PEG NPs. At low σM, peptide-PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in σM, from low to high, lead to decreases in colocalization with recycling endosomes and simultaneous increases in colocalization with the late endosome/lysosome pathway. Combining colocalization and functional TE data at low and high σM shows that higher TE correlates with a larger fraction of NPs colocalized with the late endosome/lysosome pathway while lower TE correlates with a larger fraction of NPs colocalized with the Rab11 recycling pathway. The findings lead to a hypothesis that increases in σM, leading to enhanced late endosome/lysosome pathway selection and higher TE, result from increased nonspecific electrostatic attractions between NPs and endosome luminal membranes, and conversely, enhanced recycling pathway for NPs and lower TE are due to weaker attractions. Surprisingly, at very high σM, the inverse relation between the two pathways observed at low and high σM breaks down, pointing to a more complex NP pathway behavior.

The Epigenetic Pathways to Ribosomal DNA Silencing.

Heterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing in Saccharomyces cerevisiae and in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases.

The SNF2 family ATPase LSH promotes cell-autonomous de novo DNA methylation in somatic cells.

Methylation of DNA at carbon 5 of cytosine is essential for mammalian development and implicated in transcriptional repression of genes and transposons. New patterns of DNA methylation characteristic of lineage-committed cells are established at the exit from pluripotency by de novo DNA methyltransferases enzymes, DNMT3A and DNMT3B, which are regulated by developmental signaling and require access to chromatin-organized DNA. Whether or not the capacity for de novo DNA methylation of developmentally regulated loci is preserved in differentiated somatic cells and can occur in the absence of exogenous signals is currently unknown. Here, we demonstrate that fibroblasts derived from chromatin remodeling ATPase LSH (HELLS)-null mouse embryos, which lack DNA methylation from centromeric repeats, transposons and a number of gene promoters, are capable of reestablishing DNA methylation and silencing of misregulated genes upon re-expression of LSH. We also show that the ability of LSH to bind ATP and the cellular concentration of DNMT3B are critical for cell-autonomous de novo DNA methylation in somatic cells. These data suggest the existence of cellular memory that persists in differentiated cells through many cell generations and changes in transcriptional state.

Arabidopsis AtMORC4 and AtMORC7 Form Nuclear Bodies and Repress a Large Number of Protein-Coding Genes.

The MORC family of GHKL ATPases are an enigmatic class of proteins with diverse chromatin related functions. In Arabidopsis, AtMORC1, AtMORC2, and AtMORC6 act together in heterodimeric complexes to mediate transcriptional silencing of methylated DNA elements. Here, we studied Arabidopsis AtMORC4 and AtMORC7. We found that, in contrast to AtMORC1,2,6, they act to suppress a wide set of non-methylated protein-coding genes that are enriched for those involved in pathogen response. Furthermore, atmorc4 atmorc7 double mutants show a pathogen response phenotype. We found that AtMORC4 and AtMORC7 form homomeric complexes in vivo and are concentrated in discrete nuclear bodies adjacent to chromocenters. Analysis of an atmorc1,2,4,5,6,7 hextuple mutant demonstrates that transcriptional de-repression is largely uncoupled from changes in DNA methylation in plants devoid of MORC function. However, we also uncover a requirement for MORC in both DNA methylation and silencing at a small but distinct subset of RNA-directed DNA methylation target loci. These regions are characterized by poised transcriptional potential and a low density of sites for symmetric cytosine methylation. These results provide insight into the biological function of MORC proteins in higher eukaryotes.

Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae.

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).