Unknown,Transcriptomics,Genomics,Proteomics

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The developmental patterning of 5-hydroxymethylcytosine during neuronal differentiation

ABSTRACT: Recent analyses of 5-hydroxymethylcytosine (5hmC) suggest that the modified base influences transcription by acting as an intermediate to demethylation. However, elevated levels of 5hmC within neural tissues suggest that the mark is stable and may directly contribute to gene regulation. Here, we characterize the in vivo genomic patterning and stability of 5hmC in three defined developmental states of the mouse main olfactory epithelium – multipotent stem cells, neuronal progenitors, and mature olfactory sensory neurons. 5hmC is globally enriched in neuronal progenitors and neurons relative to the stem population with significant enrichment on the bodies of transcriptionally active genes and intergenic enhancers. Although gene body 5hmC levels are positively correlated with cell type-specific gene expression in all developmental stages, genes that are commonly expressed within both neuronal progenitors and mature neurons experience the greatest increases in 5hmC levels. Moreover, enriched levels of the modified base on intergenic conserved regions correlate with cell type-specific expression of nearby genes and coincide with developmentally regulated enhancer-promoter interaction at one locus examined. Finally, an analysis of two-month old neurons indicates that elevated 5hmC levels persist in fully differentiated neurons. Together, these data suggest that 5hmC stabilizes the expression of developmentally active genes through effects at both gene bodies and intergenic regulatory elements. 9 Samples

ORGANISM(S): Mus musculus

SUBMITTER: Bradley Colquitt

PROVIDER: E-GEOD-38604 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Alteration of genic 5-hydroxymethylcytosine patterning in olfactory neurons correlates with changes in gene expression and cell identity.

Colquitt Bradley M BM Allen William E WE Barnea Gilad G Lomvardas Stavros S

Proceedings of the National Academy of Sciences of the United States of America 20130822 36

The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene regulation and cellular identity. To determine how 5hmC influences gene expression in an in vivo neuronal population, we assessed the patterning and function of the base along the developmental lineage of the main olfactory epithelium-from multipotent stem cells through neuronal progenitors to mature olfactory sensory neurons (mOSNs). We find that 5hmC increases over gene bodies during mOS ...[more]

PMID: 23969834

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Project description:During differentiation, neurons experience a reorganization of DNA modification patterns within their genomes. However, the mechanisms underlying this developmental patterning and its role in defining the neuronaÂÂl state are currently unclear. Here, we find that the dÂÂe novo DNA methyltransferase Dnmt3a is necessary for elevated levels of 5-hydroxymethylcytosine (5hmC), a derivative of 5-methylcytosine (5mC), in olfactory sensory neurons (OSNs). Through an analysis of genome-wide 5mC and 5hmC distributions in isolated OSNs, we find that Dnmt3a-dependent 5mC and 5hmC occurs within regions of high accessibility, neural enhancers, and the transcription start sites of transcribed genes. Its loss results in the global disruption of gene expression patterns, including the upregulation of silent genes, the downregulation of mOSN-expressed genes, and the alteration of odorant-induced transcriptional responses of immediate early genes. Together, these results demonstrate that Dnmt3a is necessary to define the neuronal transcriptional state and may be broadly involved in refining expression profiles within differentiated cells. To determine the contributions of Dnmt3a to the DNA modification and transcriptional landscapes of a post-mitotic neuronal population, we performed DNA immunoprecipitation (DIP-seq) using antibodies specific for 5mC and 5hmC and rRNA-depleted transcriptional profiling (RNA-seq) coupled to high-throughput sequencing using genomic DNA or RNA from FACS-isolated mature olfactory sensory neurons (mOSNs) from main olfactory epithelium (MOE) of Dnmt3a wildtype (WT), heterozygous-null (Het), or homozygous-null (KO) 3-week old mice. Similarly, to compare this information with other epigenetic features of the MOE, we performed H3K4me1 (WT), H3K27ac (WT), and H3K27me3 (WT and KO) chromatin immunoprecipitation (ChIP)-seq and DNase I hypersensitivity assays (DNase-seq) using MOE nuclei from 3-week old mice. In addition, we assayed the influence of Dnmt3a-deficiency on the induction of odorant-responsive genes by exposing 3-week old Dnmt3a WT, Het, and KO mice to either water or a 1:1:1 mixture of amyl acetate:acetophenone:octanal for 1 hour and performed rRNA-depleted RNA-seq using RNA isolated from their MOEs.

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Dataset Information

The developmental patterning of 5-hydroxymethylcytosine during neuronal differentiation

Publications

Alteration of genic 5-hydroxymethylcytosine patterning in olfactory neurons correlates with changes in gene expression and cell identity.

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