Dataset Information

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

ABSTRACT: Label-free quantification is a powerful method for studying cellular protein phosphorylation dynamics. However, whether current data normalization methods achieve sufficient accuracy has not been examined systematically. Here, we demonstrate that a large uni-directional shift in the phosphopeptide abundance distribution is problematic for global median centering and quantile-based normalizations and may mislead the biological conclusion from unlabeled phosphoproteome data. Instead, we present a novel normalization strategy, named pairwise normalization, which is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of pairwise normalization was validated by statistical methods, western blotting analysis, and by bioinformatics analysis. In addition, we demonstrate that the choice of normalization method influences the downstream analyses of the data and perceived pathway activities. Furthermore, we demonstrate that the developed normalization method, combined with pathway analysis algorithms, revealed a novel biological synergism between Ras signalling and PP2A inhibition by CIP2A.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Susumu Imanishi

LAB HEAD: Susumu Y. Imanishi

PROVIDER: PXD001374 | Pride | 2015-08-24

REPOSITORIES: Pride

ACCESS DATA

Publications

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

Kauko Otto O Laajala Teemu Daniel TD Jumppanen Mikael M Hintsanen Petteri P Suni Veronika V Haapaniemi Pekka P Corthals Garry G Aittokallio Tero T Westermarck Jukka J Imanishi Susumu Y SY

Scientific reports 20150817

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification ...[more]

PMID: 26278961

Similar Datasets

Project description:Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the DNA methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET family of enzymes and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide distribution map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. Our method exploits the unique reactivity of 5fC to link a biotin tag for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, a histone mark associated with active transcription, and were frequently bound by RNA Polymerase II. Downregulation of TDG led to accumulation of 5fC in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation and in mouse embryonic fibroblasts derived from TDG knockout embryos. Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming. The formation and removal of this cytosine modification are confined to specific genomic regions, which are in part controlled by TDG. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation, and hence, for appropriate patterns of gene expression during development. We devised a method to map 5-formylcytosine (5fC) by linking a biotin tag to 5fC for pulldown and high-throughput sequencing. We mapped 5fC in the following samples of mouse embryonic stem cells (J1): Wild-type ES cells (two replicates); ES cells transfected with siRNA targeting TDG (two replicates); ES cells transfected with non-targeting siRNA (two replicates). One genomic input library was also sequenced to detect and correct biases in fragment enrichment.

Project description:Purpose: Development of resistance to tamoxifen is an important clinical issue in the treatment of patients with breast cancer. Tamoxifen resistance may be the result of the acquisition of epigenetic regulation such as DNA methylation within breast cancer cells resulting in changed mRNA expression of genes being pivotal for estrogen dependent growth. Alternatively, tamoxifen resistance may be due to selection of preexisting resistant cells, which may exhibit cancer stem-like characteristics or a combination of the two mechanisms. Methods: To evaluate the contribution of these possible mechanisms to tamoxifen resistance, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massively parallel sequencing to analyze a well-established tamoxifen resistant cell line model: MCF-7/S0.5 (tamoxifen sensitive parental cell line) and 4 high-dosage tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8). MMSDK uses BssHII as mapping enzyme (DNA methylation sensitive enzyme). Both MMSDK and DGE use NlaIII and MmeI to produce 20-21 bp tag. The indexed single-end sequencing was performed by Illumina HiSeq 2000 in BGI-Shenzhen. A dynamic programming algorithm-FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the BurrowsM-bM-^@M-^SWheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied. Results: MMSDK libraries using BssHII/NlaIII were generated from the parental tamoxifen sensitive subline MCF-7/S0.5 and the 4 TAMR cell lines: TAMR-1, TAMR-4, TAMR-7 and TAMR-8. The 5 indexed MMSDK libraries were sequenced in one lane and 1.38 Gb clean tag data for all 5 cell lines were obtained, with an average sequencing amount of ~270 Mb per library. On average, 59.5 % of the tags with mapping quality M-bM-^IM-% 20 were mapped back to the simulated BssHII/NlaIII reference library. DGE libraries were also generated from MCF-7/S0.5 and the 4 TAMR cell lines. The 5 indexed DGE libraries were sequenced in one lane and obtained 1.71 Gb clean tag data for all 5 cell lines with an average sequencing amount of ~340 Mb per library. On average, 40.8 % with mapping quality M-bM-^IM-% 20 were mapped back to the simulated NlaIII human transcriptome (refMrna reference library). Our present study demonstrates large differences in global gene expression and DNA methylation profiles between parental tamoxifen-sensitive cell line and 4 high-dosage tamoxifen treatment selected resistant sublines. The tamoxifen resistant cell lines exhibited globally higher methylation level than the parental cell line and an inverse relationship between gene expression and DNA methylation in the promoter regions were noticed. High expression of SOX2 and alterations of other SOX gene family members, E2F gene family members and RB-related pocket protein genes as well as highlighted stem cell pathways imply that cancer initiating cells/stem cells are involved in the resistance to tamoxifen. DNA methylation and mRNA expression profiles from tamoxifen sensitive parental cell line MCF-7/S0.5 and 4 high dosage of tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8) were analyzed by MMSDK and DGE methods, respectively, in combination of massively parallel sequencing, using Illumina HiSeq 2000

Project description:Recurrent mutations in histone modifying enzymes in multiple cancer types imply key roles in tumorigenesis. However, the functional relevance of these mutations remains unknown. Here we show that the JARID1B histone H3 lysine 4 demethylase is frequently amplified and overexpressed in luminal breast tumors and a somatic point mutation of JARID1B leads to the gain of luminal-specific gene expression programs. Downregulation of JARID1B in luminal breast cancer cells induces the expression of basal cell-specific genes and growth arrest, which is partially rescued by the inhibition of TGFBR thereby indicating a key role for TGFb signaling. Integrated genome-wide analysis of JARID1B chromatin binding, histone H3 lysine trimethyl (H3K4me3) and dimethyl (H3K4me2) patterns, and gene expression profiles in luminal and basal-like breast cancer cells suggest a key role for JARID1B in luminal cell-specific gene expression programs. A significant fraction of JARID1B binding-sites overlaps with CTCF in both luminal and basal-like breast cancer cells. CTCF also co-immunoprecipitates with JARID1B and it may influence its histone demethylase (HDM) activity as the H3K4me3/me2 ratio is lower at the CTCF-overlapping compared to JARID1B-unique sites. Additionally, a heterozygous JARID1B missense mutation (K1435R) in the HCC2157 basal-like breast cancer cell line is associated with unique JARID1B chromatin-binding and gene expression patterns implying gain of luminal features. In line with this, exogenous expression of this mutant in basal-like breast cancer cells leads to a gain of JARID1B binding at many luminal-specific genes. A PARADIGM score reflecting JARID1B activity in luminal breast cancer cells is associated with poor clinical outcome in patients with luminal breast tumors. Together, our data imply that JARID1B is a luminal lineage-driving oncogene and that its therapeutic targeting may represent a novel therapeutic strategy in treatment-resistant luminal breast tumors. ChIP-Seq on JARID1B, H3K4me2, H3K4me3, CTFC, and input on breast cancer cell-lines. 50 cycles of sequencing on Illumina platform in 6 cell-lines.

Dataset Information

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

Publications

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure