Significance: Methylation of cytosine bases in DNA is an epigenetic modification that influences gene expression. TET (Ten-Eleven Translocation) enzymes regulate DNA methylation status and facilitate DNA demethylation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in mammalian genomes. Of the three mammalian TET proteins, Tet1 and Tet2 are the major regulators of 5hmC levels in mouse embryonic stem (ES) cells. We show that Tet1 and Tet2 have distinct roles in mouse ES cells: Tet1 primarily regulates 5hmC levels at gene promoters and transcription start sites, whereas Tet2 mainly regulates 5hmC levels in gene bodies and exon boundaries of highly-expressed genes and exons respectively. Our results suggest a complex interplay between the functions of Tet1 and Tet2 proteins in mESC.

ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring–Opitz syndrome. Here we demonstrate that ASXL1 truncations confer enhanced activity on the ASXL1–BAP1 complex. Stable expression of truncated, hyperactive ASXL1–BAP1 complexes in a haematopoietic precursor cell line results in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters are marked by both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes require the catalytic activity of BAP1, indicating that they are downstream consequences of H2AK119Ub erasure. In bone marrow precursors, expression of truncated ASXL1–BAP1 complex cooperates with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. Our data raise the possibility that ASXL1 truncation mutations confer gain-of-function on the ASXL–BAP1 complex.

The majority of cancer antigens identified thus far have limited expression in normal tissues. It has been suggested that autoimmune disease is a necessary price for cancer immunity. This notion is supported by a recent clinical trial involving an anti–CTL antigen-4 (CTLA-4) antibody that showed significant clinical responses but severe autoimmune diseases in melanoma patients. To selectively modulate cancer immunity and autoimmunity, we used anti-CTLA-4 and anti-4-1BB antibodies to treat mice with a preexisting cancer, MC38. The combination of the two antibodies led to CD8 T-cell-mediated rejection of large established MC38 tumors and long-lasting immunity to the same tumor cells, although the same regimen was not effective for B16 melanoma. More importantly, whereas individual antibodies induced inflammation and autoimmune manifestations, combination therapy increased cancer immunity while reducing autoimmunity. The reduction of autoimmune effects correlates with an increased function of regulatory T cells. Our results suggest a novel approach to simultaneously enhance cancer immunity and reduce autoimmunity. (Cancer Res 2006; 66(14): 7276-84)

Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine, facilitating DNA demethylation and generating new epigenetic marks. Here we show that concomitant loss of Tet2 and Tet3 in mice at early B cell stage blocked the pro- to pre-B cell transition in the bone marrow, decreased Irf4 expression and impaired the germline transcription and rearrangement of the Igκ locus. Tet2/3-deficient pro-B cells showed increased CpG methylation at the Igκ 3’ and distal enhancers that was mimicked by depletion of E2A or PU.1, as well as a global decrease in chromatin accessibility at enhancers. Importantly, re-expression of the Tet2 catalytic domain in Tet2/3-deficient B cells resulted in demethylation of the Igκ enhancers and restored their chromatin accessibility. Our data suggest that TET proteins and lineage-specific transcription factors cooperate to influence chromatin accessibility and Igκ enhancer function by modulating the modification status of DNA.