Project description:Ten-eleven translocation (TET) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of TET2 are frequently found in human malignancies, highlighting the essential role of TET2 in cellular transformation. However, the factors that control TET enzymatic activity remain largely unknown. Here we found that MBD3 and its analogue MBD3L2 can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5mC into 5hmC. Moreover, MBD3L2 is more effective than MDB3 in promoting Tet2 enzymatic activity via strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2 in modulating its target gene activities in cancer development and have important applications in understanding how dysregulation of TET2 may contribute to human malignancy.
Project description:Ten-eleven translocation (TET) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of TET2 are frequently found in human malignancies, highlighting the essential role of TET2 in cellular transformation. However, the factors that control TET enzymatic activity remain largely unknown. Here we found that MBD3 and its analogue MBD3L2 can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5mC into 5hmC. Moreover, MBD3L2 is more effective than MDB3 in promoting Tet2 enzymatic activity via strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2 in modulating its target gene activities in cancer development and have important applications in understanding how dysregulation of TET2 may contribute to human malignancy.
Project description:The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns. TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function was highly specific to chromatin environment: 5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression. Methylation and hydroxymethylation profiling by affinity-based high throughput sequencing
Project description:The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns. TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function was highly specific to chromatin environment: 5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression. Affymetrix gene expression Human ST1.0 microarray of NCCIT human embryonic carcinoma cells (4 samples in duplicate).
Project description:Examine involvement of MBD3 (methyl-CpG-binding domain protein 3), a protein involved in reading DNA methylation patterns, in epileptogenesis and epilepsy.
Project description:Dynamic DNA methylation and demethylation regulate gene expression during development. While DNA methyltransferases (DNMTs) establish and maintain cytosine methylation patterns, Ten-eleven translocation (Tet) dioxygenases catalyze sequential oxidation of 5-methylcytosine (5mC) to promote demethylation. Target-specific 5mC oxidation requires precise regulation of the Tet enzymatic activity. However, the mechanism underlying their activity control remains largely unexplored. Here we show a large low-complexity domain (LCD), present within the catalytic domain of Tet3, functions to repress the dioxygenase activity. Recombinant LCD-deleted Tet3 exhibits enhanced activity to convert 5mC into oxidized species. Deletion of the Tet3 LCD in mouse oocytes renders 5mC oxidation indiscriminately across the genome, leading to most prominently the derepression of ERVK retrotransposons and upregulation of adjacent genes. The extensive 5mC oxidation is associated with impairments in oocyte development. These findings suggest an intrinsic auto-regulatory mechanism of Tet3 dioxygenase operating to ensure a tight regulation of its enzymatic activity to achieve spatiotemporal specificity of methylome reprogramming during oocyte development.
Project description:Dynamic DNA methylation and demethylation regulate gene expression during development. While DNA methyltransferases (DNMTs) establish and maintain cytosine methylation patterns, Ten-eleven translocation (Tet) dioxygenases catalyze sequential oxidation of 5-methylcytosine (5mC) to promote demethylation. Target-specific 5mC oxidation requires precise regulation of the Tet enzymatic activity. However, the mechanism underlying their activity control remains largely unexplored. Here we show a large low-complexity domain (LCD), present within the catalytic domain of Tet3, functions to repress the dioxygenase activity. Recombinant LCD-deleted Tet3 exhibits enhanced activity to convert 5mC into oxidized species. Deletion of the Tet3 LCD in mouse oocytes renders 5mC oxidation indiscriminately across the genome, leading to most prominently the derepression of ERVK retrotransposons and upregulation of adjacent genes. The extensive 5mC oxidation is associated with impairments in oocyte development. These findings suggest an intrinsic auto-regulatory mechanism of Tet3 dioxygenase operating to ensure a tight regulation of its enzymatic activity to achieve spatiotemporal specificity of methylome reprogramming during oocyte development.
Project description:Dynamic DNA methylation and demethylation regulate gene expression during development. While DNA methyltransferases (DNMTs) establish and maintain cytosine methylation patterns, Ten-eleven translocation (Tet) dioxygenases catalyze sequential oxidation of 5-methylcytosine (5mC) to promote demethylation. Target-specific 5mC oxidation requires precise regulation of the Tet enzymatic activity. However, the mechanism underlying their activity control remains largely unexplored. Here we show a large low-complexity domain (LCD), present within the catalytic domain of Tet3, functions to repress the dioxygenase activity. Recombinant LCD-deleted Tet3 exhibits enhanced activity to convert 5mC into oxidized species. Deletion of the Tet3 LCD in mouse oocytes renders 5mC oxidation indiscriminately across the genome, leading to most prominently the derepression of ERVK retrotransposons and upregulation of adjacent genes. The extensive 5mC oxidation is associated with impairments in oocyte development. These findings suggest an intrinsic auto-regulatory mechanism of Tet3 dioxygenase operating to ensure a tight regulation of its enzymatic activity to achieve spatiotemporal specificity of methylome reprogramming during oocyte development.