Unknown,Transcriptomics,Genomics,Proteomics

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Quantitative analysis of chromatin interactions upon copy number variation at mouse 4E2 [4C-seq]

ABSTRACT: The three-dimensional organization of chromatin has a critical impact on the regulation of gene expression. However, little is known about the effect of DNA copy number variants (CNVs) on chromatin organization. We performed an allele-specific chromatin conformation and gene expression analysis of wild-type (WT) mouse chromosome 4 and an engineered 4.3Mb chromosome 4 deletion in mouse embryonic fibroblasts. A newly developed quantitative framework for the analysis of paired-end 4C-seq data revealed a number of significant differentially interacting regions (DIRs) and higher-order chromatin compaction changes present in the deletion chromosome. Selected DIRs were validated by 3D DNA FISH experiments. We discovered a significant enrichment of CTCF and Smc1 binding sites within DIRs, as well as differentially expressed genes determined by RNA-Seq. Together, our findings highlight the complex effects of a 4E2 CNV on chromatin structure and function, both locally in the deletion chromosome and globally in the WT copy. Examination of chromatin contacts and gene expression in wild type chromosome 4 and a 4.3Mb deletion in chromosome 4 in mouse

ORGANISM(S): Mus musculus

SUBMITTER: Cinthya Zepeda-Mendoza

PROVIDER: E-GEOD-64358 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Many diseases have been associated with gut microbiome abnormalities. The root cause of such diseases is not only due to bacterial dysbiosis, but also to change in bacterial functions, which are best studied by proteomic approaches. Although bacterial proteomics is well established, metaproteomics is hindered by challenges associated with the sample physical structure, contaminating proteins, the simultaneous analysis of hundreds of species and the subsequent data analysis. Here, we present a systematic assessment of sample preparation and data analysis methodologies applied to LC-MS/MS metaproteomics experiment. We could show that low speed centrifugation (LSC) has a significant impact on both peptide identifications and reproducibility. LSC led to increase in peptide and proteins identifications compare to no LSC. Notably, the dominant bacterial phyla, i.e. Firmicutes and Bacteroidetes, showed divergent representation between LSC and no-LSC. In terms of data processing, protein sequence databases derived from mouse faeces metagenome provided at least four times more MS/MS identification compared to databases of concatenated single organisms. We also demonstrated that two-steps database search strategy comes at the expense of a dramatic rise in number of false positives compared to single-step strategy. Overall, we found a positive correlation between matching metaproteome and metagenome abundance, which could be linked to core microbial functions, such as glycolysis-gluconeogenesis, citrate cycle and carbon metabolism. We observed significant overlap and correlation at the phylum, class, order and family taxonomic levels between taxonomy-derived from metagenome and metaproteome. Notably, nearly all functional categories (e.g., membrane transport, translation, transcription) were differentially abundant in the metaproteome (activity) compared to what would be expected from the metagenome (potential). In conclusion, these results highlight the need to perform metaproteomics when studying complex microbiome samples.

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Quantitative analysis of chromatin interactions upon copy number variation at mouse 4E2 [4C-seq]

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