5-hydroxymethylcytosine Promotes Productive Transcription and Inhibits Spurious Promoters
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA529472 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8795010.fastq.gz | Fastqsanger.gz | |||
| SRR8795020.fastq.gz | Fastqsanger.gz | |||
| SRR8795030.fastq.gz | Fastqsanger.gz | |||
| SRR8795040.fastq.gz | Fastqsanger.gz | |||
| SRR8795050.fastq.gz | Fastqsanger.gz |
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Project description:Covalent DNA modifications play vital roles during animal development. While 5-methylcytosine (5mC) is important for imprinting, X chromosome inactivation and transcriptional repression, the function of 5-hydroxymethylcytosine (5hmC) is less well defined. Recent studies indicate that 5hmC can be stable and long-lived in some cell types, suggesting roles beyond a DNA demethylation intermediate. Here we show that oxidation of 5mC to 5hmC enhances transcription of a specific repertoire of genes. 5hmC upregulates transcription by counteracting the repressive effects of promoter-proximal 5mC, while supressing the ability of gene body resident spurious transcription initiation sites to produce mature transcripts. By contrast, the equivalent hypomethylation results in chaotic gene expression potentiating a broad collection of genes and cryptic promoters. Our observations provide an explanation why some post-mitotic cell types favour high 5hmC in the genomes, while equivalent global hypomethylation is rarely observed.
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Project description:The discovery of cytosine hydroxymethylation (5-hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behavior in colon cancer. 5-hmC is globally reduced in proliferating cells such as colon tumors and the gut crypt progenitors, from which tumors can arise. Here, we show that colorectal tumors and cancer cells express Ten-Eleven Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5-hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5-hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. Together our results indicate that promoters that acquire 5-hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5-hmC in tumors. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation. 5 normal colon samples and 4 matching tumor samples were profiled for 5-hydroxymethylcytosine content genomewide using hmeDIP-seq. The colorectal cancer cell line HCT116 was profiled for binding of TET2 genomewide by chromatin immunoprecipitation sequencing (ChIP-seq).
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Project description:The fidelity of signal transmission requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. Understanding the constraints this imposes on cell systems evolution requires the fitness cost of spurious interactions to be quantified. Towards this end, we express human tyrosine kinases in the budding yeast S. cerevisiae. Yeast lacks bona fide tyrosine kinases and so the majority of resulting pY sites are functionless and artificial. We express 24 unique tyrosine kinases in total and perform phosphoproteomics in each case, resulting in ~30,000 phosphosites sites mapping to 3500 phosphoproteins. Examination of the fitness costs in each strain reveals a strong correlation between the number of spurious pY sites generated and negative effects on growth. Moreover, the prediction of pY effects on protein structure and on protein function (conservation-based) reveals potential for the widespread perturbation of the yeast proteome. Comparing the spurious pY sites (pre-selection) with native pY sites in human (post-selection) also demonstrates the recurrent modification of proteins and sites with no homology to native substrates. However, examination of these data together (fitness and phosphoproteomics) strongly suggests that a large number of the pY sites generated have a negligible effect on fitness. Finally, we test the hypothesis of pY counter-selection following the emergence of tyrosine kinases in metazoan species, but find no strong evidence for proteome-wide selection against spurious Y phosphorylation.
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