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LINE-1 Silencing by Retinoblastoma Proteins Is Effected Through the Nucleosomal and Remodeling Deacetylase Multiprotein Complex Diego E

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LINE-1 Silencing by Retinoblastoma Proteins Is Effected Through the Nucleosomal and Remodeling Deacetylase Multiprotein Complex Diego E Montoya-Durango et al. BMC Cancer (2016) 16:38 DOI 10.1186/s12885-016-2068-9 RESEARCHARTICLE Open Access LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex Diego E. Montoya-Durango1, Kenneth A. Ramos1, Pasano Bojang2, Lorell Ruiz1, Irma N. Ramos3 and Kenneth S. Ramos2* Abstract Background: Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Methods: Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. Results: Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5′-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. Conclusions: RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function. Keywords: Chromatin, Gene silencing, Long interspersed nuclear element-1, Retinoblastoma proteins, Nucleosomal and remodeling deacetylase complex Background promoter [2]. While L1s are highly active during early em- Human L1s are non-LTR mammalian retrotransposons bryonic development, they are targeted for epigenetic that consist of an internal bidirectional promoter, two silencing via DNA methylation and histone covalent mod- open reading frames encoding for ORF1p (RNA-binding ifications during the course of differentiation [3]. L1 re- protein) and ORF2p (reverse transcriptase and endonucle- activation is strongly associated with the acquisition of ase activities), a 3′- untranslated region (UTR), and a oncogenic phenotypes resulting from insertion mutagen- polyA tail [1]. Murine L1s share similar structural and esis and/or reprogramming of gene expression [1]. functional features, except that the 5′-UTR is organized We have previously shown that recruitment of E2F/RB into monomeric units that function as an upstream (E2F-retinoblastoma) to the L1 promoter, along with his- tone deacetylases 1 and 2 (HDAC1 and HDAC2, respect- * Correspondence: [email protected] ively) [4, 5] and methyl binding protein 2 (MBD2) [5] are 2Department of Medicine, University of Arizona College of Medicine, Tucson, critical to L1 silencing. Mouse embryo fibroblasts (MEFs) AZ 85721, USA lacking all RB family members (pRb, p107, and p130) and Full list of author information is available at the end of the article © 2016 Montoya-Durango et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Montoya-Durango et al. BMC Cancer (2016) 16:38 Page 2 of 8 referred to as triple knockout (TKO) MEFs, show im- with PBS and incubated in 1 μg/ml Hoescht nuclear dye paired recruitment of HDACs to the L1 promoter and for 20 min and then washed twice. Cells were visualized in markedly enhanced L1 expression compared to wild type a CARL ZEISS AXIOVERT 200 inverted microscope at a MEFs. Methyl CpG binding protein 2 (MeCP2) represses magnification of 63X. the transcriptional activity of L1 in transient reporter as- says [6] and in vivo [7], and this activity is related to Protein immunoprecipitation HDAC recruitment to methylated DNA [8]. The identity Cells were lysed with buffer and centrifuged at 4000 rpm of the repressor complex that silences L1 has not yet been for 2 min. Immunoprecipitation using anti-Mi-2β and elucidated. anti-RbAp46 were completed as described previously [4]. Methods RNA interference assays Cell models Cells were transfected with an EGFP-tagged L1RP vec- MCF7 cells, HeLa cells, primary mouse embryo fibroblasts tor. After the disappearance of EGFP fluorescence by (MEFs) and RB null MEFs lacking all three family members epigenetic silencing, cells were replated and treated at (RB, p105 and p107) (TKO) were cultured in Dulbecco’s 30-50 % confluence with 4 nM siRNA for 48 h followed Modified Eagle’s medium (MultiCel; Cytosystems Pty. Ltd., by direct measurements of fluorescence. Castle Hill, NSW, Australia) supplemented with 10 % fetal calf serum (FCS), 200 mg/ml streptomycin, and 200 U/ml Chemical treatments penicillin G at 5 % CO2 and humidified air at 37 °C. Cells were challenged with 3 μM BaP or 0.06 % dimethyl sulfoxide (DMSO) as vehicle control for 16 h to reacti- Quantitative real time PCR vate L1. Total RNA was extracted and 1 μg used for cDNA synthe- sis (Invitrogen Superscript II). For each reaction, 25 μLof Ethics statement 2X SYBR green (Biorad) was mixed with forward and re- No ethics approval was required for the experimental verse primers to give a final concentration of 10 μm. One work performed in this study. μL of cDNA mixture was brought up to 50 μLusingDEPC water. Cycling conditions included an initial denaturation Results at 95 °C for 3 min followed by 50 cycles at 95 °C for 30 s, The NuRD corepressor complex is recruited to the L1 55 °C for 30 s and 72 °C for 45 s. The homogeneity of PCR promoter products was confirmed using a real time PCR melting The NuRD multiprotein complex includes the curve. dermatomyositis-specific Mi2 autoantigen (Mi2-β), RB-associated proteins 46 and 48 (RbAp46 and RbAp48), Chromatin immunoprecipitation MBD2 and MBD3, and metastasis-associated proteins 2 Cells were grown in 150 mm petri dishes to 90 % conflu- and 3 (MTA2 and MTA3) [9–11]. Validation trials were ence, fixed in 1 % formaldehyde for 7 min and rinsed 3X completed using soluble chromatin of human MCF7 cells with cold PBS. Chromatin was sheared to fragments and antisera against components of the NuRD corepressor below 1 Kb and 20 μg immunoprecipitated using se- complex compared to rabbit IgG as a control for non- lected antibodies. After purification, 0.5 to 2.0 μLof specific interactions. Snail, a master switch for epithelial- DNA were used for PCR. DNA from non-precipitated to-mesenchymal transition in breast cancer that is regu- chromatin samples was extracted and used as input for lated by the Mi2-β/NuRD complex [12], was used as a PCR. Controls included isotype-matched IgG or Mock positive control in these experiments. As expected, spe- reactions which included all reagents except chromatin. cific enrichment for NuRD proteins on the Snail promoter was observed (Fig. 1a). PCR amplification of the human Indirect immunofluorescence L1RP 5′-UTR was compared to Snail and glucose 6- Cells were grown on glass coverslips to 30 % confluence, phosphate dehydrogenase (G6PD) gene promoters as rinsed 2X with PBS (phosphate-buffered saline, pH 7.4), positive and negative control sequences, respectively. All fixed in 3 % paraformaldehyde for 15 min at 4 °C and NuRD subunits specifically enriched the sample for the rinsed twice with PBS. Fixed cells were permeabilized with human L1RP 5′-UTR DNA sequence, but not the G6PD 0.1 % Triton-X100 for 5 min, blocked in 2 % dry milk for sequence (Fig. 1b). The presence of MBD3, MTA2 and 15 min, rinsed with PBS twice and incubated with primary MTA3 readily identified the complex as the MeCP1 multi- antibody (anti-Mi2-β, ABCAM, MA) in PBS containing 1 protein complex. The NuRD complex is involved in re- % dry milk and 0.1 % Triton-X100 overnight. Secondary modeling of nucleosomal particles to mediate formation antibody, mouse mAB-Alexa-488 conjugate, was incu- of heterochromatin and localized domains of repressive bated for 1 h at room temperature. Cells were washed 3X chromatin. Mi2-β functions as an ATP-dependent helicase Montoya-Durango et al. BMC Cancer (2016) 16:38 Page 3 of 8 three member of the RB family were genetically ablated [5]. In these studies, we tested antisera against Mi2-β, MBD3, MTA-2, and RbAp46/48, as well as histone H4 and a control rabbit serum. Specific enrichment for L1 5′-UTR was seen for Mi2-β, MBD2/3, MTA-2, and RbAp46 in wild type MEFs (Fig. 1c). Immunoprecipita- tion with total histone H4 antisera, but not control IgG yielded high levels of L1 DNA enrichment (not shown). In sharp contrast, markedly reduced enrichment was seen when chromatin from TKO MEFs was immunopre- cipitated with serum against Mi2-β, MBD2/3, or MTA-2 (Fig. 1c). This pattern of response was specific to L1 se- quences, as evidenced by markedly different profiles of NuRD protein recruitment to IL-4 intron 2 sequences (Fig. 1d). A comparison with IL-4 intron 2 sequences is pertinent given that genetic ablation of MTA-2 corre- lates with hyperinduction of IL-4 and abnormal T-cell activation in mice [17], and this response is partly medi- ated by a cis-acting element located in the second intron of the murine IL-4 gene [18].
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