Project description:AF9 mutations have been implicated in human neurodevelopmental diseases and murine Af9 mediates histone methylation during cortical neuron generation. However, AF9 function and related mechanisms in human neurodevelopment remain unknown. Here we show that AF9 is necessary and sufficient for human embryonic stem cell (hESC) neural differentiation and neurodevelopmental gene activation. The 5-methylcytosine (5mC) dioxygenase TET2, which was identified in an AF9-associated protein complex, physically interacted with AF9. Both AF9 and TET2 co-localized in 5-hydroxymethylcytosine (5hmC)-positive hESC-derived neurons and were required for appropriate hESC neural differentiation. Upon binding to AAC-containing motifs, AF9 recruited TET2 to occupy the common neurodevelopmental gene loci to direct 5mC-to-5hmC conversion, which was followed by sequential activation of neural target genes and hESC neural commitment. These findings define an AF9-TET2 regulatory complex for modulating human neural development and reveal a novel mechanism by which the AF9 recognition specificity and TET2 hydroxylation activity cooperate to control neurodevelopmental gene activation.
Project description:DNA methylation is tightly regulated throughout mammalian development and altered methylation patterns are a hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in acute myeloid leukemia (AML) and has been suggested to protect CpG islands and promoters from aberrant methylation. By generating a novel mouse model of Tet2-deficient AML we show that loss of Tet2 in hematopoietic cells leads to progressive hypermethylation of active enhancer elements and altered expression of genes implicated in tumorigenesis. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner. Furthermore, we confirm this specific enhancer hypermethylation phenotype in human AML patients. Thus, we propose that TET2 prevents leukemic transformation of hematopoietic cells by protecting enhancers from aberrant DNA methylation. Enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analysis of in vitro-grown hematopoietic cells transduced with AML1-ETO or MLL-AF9
Project description:Ten-eleven translocation-2 (TET2) is a member of the methylcytosine dioxygenase family of enzymes implicated in cancer and in aging due to its role as a global epigenetic modifier. TET2 has a large N-terminal domain followed by a catalytic C-terminal. Previous reports have demonstrated that the catalytic domain remains active independent of the N-terminal domain. As such, the function of the N-terminus of this large protein remains poorly characterized. Here, we identify that several isoforms of the 14-3-3 family of proteins bind TET2. 14-3-3s bind TET2 when phosphorylated at serine 99 (S99). AMPK-mediated phosphorylation at S99 promotes TET2 stability and increases global DNA 5-hydroxymethylcytosine. 14-3-3s’ interaction with TET2 serves to protect S99 phosphorylation. Disruption of this interaction leads to both reduced TET2 phosphorylation and decreased protein stability. Furthermore, we identify that the protein phosphatase 2A (PP2A) can interact with TET2 and dephosphorylates S99. Collectively, our study provides novel insights into the role of the N-terminal domain in TET2 regulation. Moreover, they demonstrate the dynamic nature of TET2 protein regulation that could have therapeutic implications for disease states resulting from reduced TET2 levels and/or activity.
Project description:H3K9 acetylation was enriched in pluripotency genes in hESCs and in neural genes in neural progenitor cells Examination of H3K9 acetylation distributions in hESCs and neural progenitor cells
Project description:To assess whether these proteins directly interact with each other in cells, we performed large-scale co-immunoprecipitation (co-IP) experiments using nuclear extract of HEL cells and the AF9 antibody against the N-terminus of AF9, and analyzed immunoprecipitated proteins by mass spectrometry. We identified the common subunits of the COMPASS-like complexes.
Project description:Studies of AML patient samples have shown that specific combinations of AML disease alleles confer an adverse outcome, however, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Here we show that TET2/FLT3 mutations can cooperate to induce AML in vivo using a genetically engineered mouse model, and that this model has a defined stem-cell population with a characteristic transcriptional and epigenetic profile. TET2 and FLT3 mutations cooperate to induce site-specific changes in DNA methylation and gene expression, including at loci that regulate hematopoietic differentiation. We demonstrate that re-expression of genes that are silenced in TET2/FLT3-mutant AML restores normal differentiation, demonstrating that the epigenetic program of TET2/FLT3-mutant AML cells can be reversed in vitro and in vivo. Using ERRBS, we profiled genome-wide DNA methylation patterns of the hematopoietic stem cells (LSK) population in Wide-type, Flt3-IDT, Tet2-/-, and Tet2-/-Flt3-IDT mice, each in triplicates
Project description:Loss of function TET2 mutations are frequently seen in myelodysplastic syndrome (MDS) patients. Previous studies have demonstrated that TET2 deficiency enhances maintenance of MDS hematopoietic stem and progenitor cells (HSPCs). Nonetheless, the pathogenic role of TET2 in MDS progression remains elusive. Here, we demonstrate Tet2 knockout (KO) markedly accelerated malignant transformation in Nup98-HoxD13 (NHD13) transgenic mice and promotes leukemogenesis of HoxA9 transduction/transplant mice. Consistently, low TET2 level cooperating with high HOXA9 level predicts poor outcome of MDS patients. Notably, Tet2 KO conferred a clonal advantage to the HSPCs of NHD13 mice. Whole-exome sequencing revealed that Tet2 KO facilitates accumulation of mutations at genes associated with leukemogenesis, including Arih2, whose loss of function promotes MDS cells proliferation. Using 5-hydroxymethylcytosine immunoprecipitation coupled with high-throughput sequencing analysis, we found while Tet2 deletion decreased overall 5hmC levels, it also increased 5hmC distribution at certain mutation loci such as Arih2. Vitamin C treatment, which mimics Tet2/Tet3 restoration, blocked disease progression in Tet2-deficient NHD13 mice. Collectively, our findings demonstrate that TET2 activity governs occurrence of secondary mutations in MDS HSPCs, providing a rationale for enhancing TETs function to block MDS-malignant transformation.
Project description:Loss of function TET2 mutations are frequently seen in myelodysplastic syndrome (MDS) patients. Previous studies have demonstrated that TET2 deficiency enhances maintenance of MDS hematopoietic stem and progenitor cells (HSPCs). Nonetheless, the pathogenic role of TET2 in MDS progression remains elusive. Here, we demonstrate Tet2 knockout (KO) markedly accelerated malignant transformation in Nup98-HoxD13 (NHD13) transgenic mice and promotes leukemogenesis of HoxA9 transduction/transplant mice. Consistently, low TET2 level cooperating with high HOXA9 level predicts poor outcome of MDS patients. Notably, Tet2 KO conferred a clonal advantage to the HSPCs of NHD13 mice. Whole-exome sequencing revealed that Tet2 KO facilitates accumulation of mutations at genes associated with leukemogenesis, including Arih2, whose loss of function promotes MDS cells proliferation. Using 5-hydroxymethylcytosine immunoprecipitation coupled with high-throughput sequencing analysis, we found while Tet2 deletion decreased overall 5hmC levels, it also increased 5hmC distribution at certain mutation loci such as Arih2. Vitamin C treatment, which mimics Tet2/Tet3 restoration, blocked disease progression in Tet2-deficient NHD13 mice. Collectively, our findings demonstrate that TET2 activity governs occurrence of secondary mutations in MDS HSPCs, providing a rationale for enhancing TETs function to block MDS-malignant transformation.
Project description:Loss-of-function TET2 mutations (TET2MT) are common in myeloid neoplasia. TET2, a DNA dioxygenase, requires 2-oxoglutarate and Fe(II) to oxidize 5-methylcytosine. TET2MT thus result in hypermethylation and transcriptional repression. Ascorbic acid (AA) increases dioxygenase activity by facilitating Fe(III)/Fe(II) redox reaction and may alleviate some biological consequences of TET2MT by restoring dioxygenase activity. Here, we report the utility of AA in the prevention of TET2MT MN, clarify the mechanistic underpinning of the TET2-AA interactions, and demonstrate that the ability of AA to restore TET2 activity in cells depends on N- and C-terminal lysine acetylation and nature of TET2MT. Consequently, pharmacologic modulation of acetyltransferases and histone deacetylases may regulate TET dioxygenase-dependent AA effects. Thus, our study highlights the contribution of factors that may enhance or attenuate AA effects on TET2 and provides a rationale for novel therapeutic approaches including combinations of AA with class I/II HDAC inhibitor or sirtuin activators in TET2MT leukemia.