Regulation of CBP and Tip60 coordinates histone acetylation at local and global level during Ras induced transformation

Cell transformation is clearly linked to epigenetic changes. However, the role of the histone modifying enzymes in this process is still poorly understood. In this study we investigated the contribution of the Histone Acetyltransferase (HAT) enzymes to Ras mediated transformation. Our results demonstrated that lysine acetyltransferase 5 (KAT5), also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27) tumour suppressor gene promoters to facilitate Ras mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both, local and global level to facilitate Ras induced transformation. SUMMARY The contribution of the histone modifying enzymes to Ras transformation is still poorly understood. This study demonstrates that two essential HAT enzymes, CBP and Tip60, coordinate histone acetylation at local and global level to facilitate Ras induced transformation. Sánchez-Molina et al. 3 INTRODUCTION The transformation of a normal cell into a cancer cell is associated with a disruption of cellular controls that regulate cell division and/or cell death. At present, many of the genetic changes associated with this process are known. In addition to these genetic changes, increasing evidence suggests that epigenetic alterations are essential in establishing the transformed phenotype [1-3]. The initial indications of the epigenetic link to cancer came from studies of gene expression and DNA methylation (for review see [4]). For example, several tumour suppressors are silenced in human cancers by DNA methylation and aberrant changes in specific histone modifications have been described [2]. These modifications often target regulatory regions of proto oncogenes or tumour suppressor genes [2]. In addition to changes in the specific promoters, changes in global levels of individual histone modifications are also associated with cancer cells [5,6]. Specifically, alteration in H3K9me3, H4K16ac, H4K20me3, H3K56ac and H3K9ac levels are related to tumourigenesis [6-8]. In accordance with that, changes in the expression levels, mutations and translocations of histone acetyltransferases and methyltransferases have been linked to different types of cancer [13,9]. All these data remark the important contribution of epigenetic processes to cancer biology. The RAS oncogene is mutated in an elevated proportion (30%) of human tumours [10]. In particular RAS mutations at codons 12, 13 or 61 significantly downgrade its GTPase ability, which are thus rendered constitutively active and able to transform mammalian cells. These mutations activate the Raf-MEK-ERK, Ral GDS-Ral, and PI3K-AKT pathways, which ultimately modulate different cellular functions [11,12]. In vitro, Ras transformed cells fail to activate many checkpoint controls: they lack contact inhibition and loss the requirement for mitogens and anchorage in order to proliferate [13]. These characteristics are similar to those Sánchez-Molina et al. 4 that contribute to the loss of controls that takes place in Ras induced tumours. Different epigenetic changes have been linked to Ras mediated transformation; in particular alteration of histone acetylation patterns have been reported [8,14]; moreover, activation of oncogenic Ras leads to increased DNA methyltransferase activity [15] and induces degradation of CBP in NIH3T3 cells [8,16]. Although changes on histone modifications have been correlated to Ras mediated oncogenic processes, the particular contribution of the histone modifying enzymes to cell transformation is still poorly understood. In this study we address this issue by studying the contribution of HAT enzymes to Ras mediated transformation. Our results indicate that Tip60 facilitates histone acetylation of bulk chromatin in Ras transformed cells rendering a more accessible chromatin than in parental cells. Meanwhile, low levels of CBP lead to hypoacetylation of pRb and p27 tumour suppressor gene promoter contributing to Ras mediated transformation. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global level to facilitate Ras induced transformation. MATERIALS AND METHODS Cell Culture NIH3T3 fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% calf serum (CS, Invitrogen) or starved with 0.5% calf serum for 18h as indicated. Stable transfections using pCEFL-KZ-AU5, pCEFL-KZ-AU5-H-Ras (V12) and pCEFL-KZ-AU5-N-Ras (V12) [17] vectors were done using the calcium phosphate precipitation technique. Transient transfections were performed using Lipofectamin reagent (Invitrogen). DNA constructs and antibodies The plasmids pCEFL-KZ-AU5, pCEFL-KZ-AU5-H-Ras (V12), pCEFL-KZ-AU5-NRas (V12), pCEFL-KZ-AU5-H-Ras (V12S35), pCEFL-KZ-AU5-H-Ras (V12G37), pCEFLSánchez-Molina et al. 5 KZ-AU5-H-Ras (V12C40), pCEFL-KZ-AU5-N-Ras (V12S35), pCEFL-KZ-AU5-N-Ras (V12G37), pCEFL-KZ-AU5-N-Ras (V12C40) have been previously described [17,18]. pCDNA3-HA-CBP and pCDNA3-HA-CBPΔHAT have been described elsewhere [19]. pCDNA3-p27 was kindly provided by Dr. O. Bachs. pCDNA3-Tip60-HA was a gift of Dr. D. Trouche. Used antibodies are described in Supplementary Table 1. Indirect immunofluorescence Cells on cover slips were fixed in paraformaldehyde, permeabilized with methanol and incubated with antibodies as previously described [20,21]. RT Quantitative Real-Time PCR (qPCR) Total cellular RNA was obtained with Ultraspec RNA Isolation System (Biotecx). cDNA was generated using Omniscript Reverse Transcription kit (Qiagen) and 2 μg of extracted RNA . Differences in the RNA content were determined by real time PCR using the ABI 7700 Sequence Detection System and SYBR Green Master Mix protocol (Applied Biosystems). PCR reactions were carried out triplicate in 20 μl with 2 μl from 1/5 reverse transcription dilution and 0,25 μM of specific primers at 95oC for 10 min, followed by 40 cycles of 15 s at 95oC and 1 min at 60oC. The primer oligonucleotide sequences are described in Supplementary Table 2 and 3. All PCR products were approximately 50 bp to increase the efficiency of real-time reactions. Relative expression was calculated using the standard curve method. A standard curve was made for each gene of interest and the housekeeping by plotting in a base-10 semi-logarithmic graph the number of cycles at which the fluorescence crossed the threshold (Ct) against increasing amounts of DNA template. The relative quantification for the gene of interest is normalized to that of tubulin in the same sample and then the normalized numbers are compared between samples to get a fold change in expression. FACS staining and analysis Sánchez-Molina et al. 6 FACS analysis was performed as described elsewhere [22]. Basically, cells transfected with the plasmid/s of interest and pCDNA3-GFP were harvested following trypsinization, washed twice in PBS and resuspended in 1 ml of DMEM. Non-permeabilized cells were stained during 30 min with 5 μg/ml Hoechst 33342 and DNA content of GFP population was analyzed by flow cytometry with a Becton Dickinson FACscan according to the manufactures’ procedure. Analysis of chromatin condensation with Micrococcal Nuclease (MNase) The protocol is described in Supplementary Materials and Methods. HAT/Histone deacetylase (HDAC) assays In vitro acetylation/deacetylation assay was performed as described elsewhere [23,24]. Foci Formation Assay Foci formation assays were performed as described elsewhere [18]. RNA interference by siRNA Target sequences for small interfering RNAs are as follows. Tip60: 5 ́ACGGAAGGUGGAGGUGGUU-dTdT-3 ́, control: 5 ́-CAUGUCAUGUGUCACAUCUdTdT-3 (BioNova). The siRNAs were transfected using JetPei Kit (Polyplus). Cell extract preparation. Immunoblotting Total cell extracts were prepared in IPH buffer as previously described [25]. Histones were extracted by treatment of the whole cells with 0.25 M HCl at 4o C under rotation. After centrifugation at 12 000xg for 10 min at 4oC, the histones were dialyzed against 0.1 M Acetic acid twice and against H2O three times. The antibodies used are described in Supplementary Table 1. Immunoblotting was performed with standard procedures and visualized by means of an ECL kit (Amersham). Chromatin immunoprecipitation (ChIP) analysis. Quantitative Real-Time PCR and ChIP quantification Sánchez-Molina et al. 7 ChIP assays were performed as described elsewhere [26]. A detailed protocol is described in Supplementary Materials and Methods. RESULTS Global HAT activity increases in Ras transformed cells It has been previously described that chromatin from Ras transformed cell lines is more accessible than chromatin from normal cells [27]. This feature should correlate with epigenetic changes, in particular with new acetylation/deacetylation equilibrium. In order to gain further insight into this possibility we determined the global HAT and HDAC activities in control and Ras transformed NIH3T3 cells by in vitro assays. We used as a transformation model the well established NIH3T3 cell line stably transfected with Hras mutated at position 12 (RasV12), which makes Ras proteins constitutively active [16]. While originally NIH3T3 are immortal, they become transformed after constitutive activation of the Hras oncogene [[16] and Supplementary Figure 1]. Figure 1A shows that global HAT activity in Ras transformed cells was higher than in control cells while HDAC activity did not change. These results suggest that the equilibrium between HAT’s and HDAC’s is globally moved towards