Ivo Teneng

Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins.

Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

Reactivation of L1 retrotransposon by benzo(a)pyrene involves complex genetic and epigenetic regulation

Benzo(a)pyrene (BaP), is an environmental pollutant present in tobacco smoke and a byproduct of fossil fuel combustion which likely contributes to the tumorigenic processes in human cancers including lung and esophageal. Long Interspersed Nuclear Element-1 (LINE-1) or L1 is a mobile element within the mammalian genome that propagates via a “copy-and-paste” mechanism using reverse transcriptase and RNA intermediates. L1 is strongly expressed during early embryogenesis and then silenced as cells initiate differentiation programming. Although the complex transcriptional control mechanisms of L1 are not well understood, L1 reactivation has been described in several human cancers and following exposure of mouse or human cells to BaP. In this study we investigated the molecular mechanisms and epigenetic events that regulate L1 reactivation following BaP exposure. We show that challenge of HeLa cells with BaP induces early enrichment of the transcriptionally-active chromatin markers histone H3 trimethylated at lysine 4 (H3K4Me3) and histone H3 acetylated at lysine 9 (H3K9Ac), and reduces association of DNA methyltransferase-1 (DNMT1) with the L1 promoter. These changes are followed by proteasome-dependent decreases in cellular DNMT1 expression and sustained reduction of cytosine methylation within the L1 promoter CpG island. Pharmacological inhibition of the proteasome signaling pathway with the inhibitor MG132 blocks degradation of DNMT1 and alters BaP-mediated histone epigenetic modifications. We conclude that genetic reactivation of L1 by BaP involves an ordered cascade of epigenetic events that begin with nucleosomal histone modifications and is completed with alterations in DNMT1 recruitment to the L1 promoter and reduced DNA methylation of CpG islands.

Context-specific regulation of LINE-1.

The present study was conducted to evaluate the contextual specificity of long interspersed nuclear element‐1 (LINE‐1 or L1) activation by cellular stress and the role of the aryl hydrocarbon receptor (AHR) transcription factor and oxidative stress in the gene activation response. Activation of the AHR by the genotoxic carcinogen benzo(a)pyrene (BaP) increased L1 expression in human cervical carcinoma (HeLa) cells, human microvascular endothelial cells (HMEC), mouse vascular smooth muscle cells (mVSMC) and mouse embryonic kidney cells (mK4). In contrast, challenge with a different AHR ligand 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), or UV irradiation (10–20 J/m2), induced L1 only in HeLa cells. Transactivation of the mouse L1Md‐A5 promoter was observed in all cell types challenged with BaP, while TCDD was without effect, and UV only activated L1 in HeLa cells. Genetic and pharmacological experiments implicated the AHR and oxidative stress as contextual determinants of L1 inducibility by cellular stress.

Validation of a Mathematical Model of Gene Transcription in Aggregated Cellular Systems: Application to L1 Retrotransposition

We present a methodology aimed at partial validation and accuracy-precision assessment of a mathematical model of gene transcription at the cellular level. The method is based on the analysis of time-series measurements aggregated over a large number of cells. Such measurements are typically obtained via reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. The validation procedure presented herein uses as an example data on L1 retrotransposon gene in HeLa cells. The procedure compares model predicted values with the RT-PCR data for L1 by means of the standard Bayesian statistical techniques with the help of modern Markov-Chain Monte-Carlo methodology.

The Intersection of Genetics and Epigenetics: Reactivation of Mammalian LINE-1 Retrotransposons by Environmental Injury

Transposable elements such as LINE-1 (long interspersed nuclear element-1 or L1) are mobile genetic moieties within the genome. L1 retrotransposons comprise 21 % of the human genome by mass, and up to 100 are believed to remain retrotransposition competent within the human genome. During embryonic development, the genome undergoes reprogramming events defined by specific patterns of DNA methylation established de novo after implantation and preferentially targeted to repetitive sequences. Recent studies in the Ramos laboratory have shown that the ability of polycyclic aromatic hydrocarbon carcinogens, such as benzo(a)pyrene, to reactivate L1 transcription and retrotransposition in mammalian cells involves dysregulation of epigenetic programming mediated in part via mechanisms involving the aryl hydrocarbon receptor, a ligand-activated transcription factor and regulator of several other biological processes. The most detrimental effect of L1 on the genome is believed to be insertion into functional sequences that severely compromise gene function. Other studies have shown that L1 reactivation mediates changes in genetic programming of differentiation networks. Because L1 insertions can have a profound impact on primary genetic structure as well as epigenetic status of the host, they represent ideal molecular targets for development of novel epigenetic therapies targeting medical conditions that involve derangements of L1 activity.

Abstract 619: CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells.

Reduced DNA repair capacity (DRC) and polymorphisms in genes involved in the repair pathways are associated with increased risk for lung cancer. Studies from our lab have demonstrated an association between increased double strand breaks (DSB) and increased risk for gene promoter hypermethylation. The goal of this study was to identify genes in the DSB repair pathway that may be rate limiting for DNA repair and to determine the effect of gene knockdown on genomic stability, DRC, gene promoter methylation, and cellular transformation. CHK2 is an important DNA damage sensor and cell cycle regulator gene that is important in maintaining genomic stability. DNAPKc is a kinase within the non-homologous end joining (NHEJ) pathway that participates in DNA repair by inducing conformational changes that allows access to DSB sites, and reduced activity is associated with lung cancer. Expression of CHK2 was reduced in 4 of 10 tumor-derived lung cancer cell lines, while 3 cell lines had undetectable levels of DNAPKc. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to carcinogens. CHK2 and DNAPKc were stably knocked down in HBEC2 and HBEC3 to determine their role in DRC, genomic instability and transformation. Knockdown of neither CHK2 nor DNAPKc affected microsatellite instability, nor was deficiency of either gene sufficient to induce spontaneous transformation in HBEC2 and HBEC3. Knockdown of CHK2 or DNAPKc decreased DRC in HBEC2 and HBEC3 in response to bleomycin, (a DSB-inducing carcinogen) indicating an important role for both genes in DSB repair. Knockdown cell lines were treated with bleomycin once a week for 12 weeks to determine the effect of gene deficiency on transformation. DNAPKc deficiency increased transformation efficiency in HBEC2 and HBEC3 by 14- and 4.5- fold, respectively, while CHK2 deficiency increased transformation efficiency in HBEC3 by 6- fold compared to bleomycin treated controls. Studies are ongoing to determine changes induced in the transcriptome and methylome in DNA repair-deficient cells during transformation. These studies will highlight the roles of CHK2 and DNAPKc in DSB repair, malignant transformation, and will shed more light on the role of DNAPKc and CHK2 in reprograming the genome. Supported by R01 ES15262 to SAB Citation Format: Ivo Teneng, Randy Willink, Kieu C. Do, Christopher Dagucon, Steven A. Belinsky. CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2013-619

Abstract 1049: DNMT3b increases transformation efficiency and accelerates carcinogen induced transformation of human bronchial epithelial cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant tumors and cell lines are characterized by increased expression of DNMT1, DNMT3a and DNMT3b. While evidence suggests that DNMT1, DNMT3a and DNMT3b co-operate in methylation and gene silencing during carcinogenesis, very little is known regarding gene targets for DNMT3a and DNMT3b and their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5mM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and DNMT3b were observed in transformed cells. Increased DNMT3a and DNMT3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and DNMT3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and DNMT3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. Over expression of DNMT3a or DNMT3b did not affect DNA repair capacity and was not sufficient to drive spontaneous transformation as evidenced by inability to form colonies on soft agar. Transformation efficiency was increased 2-2.5 fold in the two HBEC2 cell lines over expressing DNMT3b after 12 weeks of carcinogen treatment relative to treated parental HBEC2. In addition, after 8 weeks, colony formation was evident in the DNMT3b over expressing, but not in parental HBECs indicating that increased expression of this de novo DNMT accelerates transformation. Over expressing DNMT3a had no effect on carcinogen-induced transformation. Current studies are evaluating global re-programming of the epigenome in transformed clones using the Illumina 450k promoter array and the effect of over expression of DNMT3b on stem cell markers. This study was supported by R01 ES008801 to S.A.B. and 1F32 CA157082 to I.T. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1049. doi:1538-7445.AM2012-1049

Abstract 2003: Role of de novo DNMTs in carcinogen induced transformation of human bronchial epithelial cells

Malignant tumors and cell lines are characterized by increased expression of DNMT3a and 3b. While evidence suggests that DNMT1, 3a and 3b co-operate in methylation and gene silencing during carcinogenesis, very little is known about targets for DNMT3a and 3b or their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase /cyclin dependent kinase 4 -immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5µM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC1 and HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and 3b were observed in transformed cells. Increased DNMT3a and 3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and 3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and 3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. HBEC1 and HBEC2 clones that expressed DNMT3a or 3b at levels comparable to those observed in tumor derived cell lines were isolated, expanded and characterized. Over expression of DNMT3a or 3b had no effect on DNA repair capacity, did not affect DNMT1 expression and was not sufficient to drive transformation as evidenced by inability to form colonies on soft agar. Two clones from each cell line over expressing DNMT3a or 3b were treated with carcinogen for one hour, once a week for 12 weeks and analyzed for transformation on soft agar. Studies completed for HBEC1 indicate that expression of DNMT3b increases transformation efficiency. Soft agar analyses for HBEC2 clones are on going. Further studies will characterize the mechanism for increased transformation by examining reprogramming of the epigenome and tumorigenicity of transformed cells in xenografts This study was supported by R01 ES008801. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2011-2003