The TET methylcytosine dioxygenase 1 (TET1) enzyme is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. Decreased expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we show that deletion of Tet1 promoted the development of B cell lymphoma in mice. Tet1 was required for maintaining normal content of 5hmC, preventing DNA hypermethylation and in the regulation of B cell lineage, chromosome maintenance and DNA repair genes. Whole-exome sequencing of Tet1-deficient tumors revealed mutations frequently found in Non-Hodgkin B cell lymphoma, where TET1 was hypermethylated and transcriptionally silenced. These findings provide in vivo evidence of TET1 function as a tumor suppressor of hematopoietic malignancy.

The BCL2 family plays important roles in acute myeloid leukemia (AML). Venetoclax, a selective BCL2 inhibitor, has received FDA approval for the treatment of AML. However, drug resistance ensues after prolonged treatment, highlighting the need for a greater understanding of the underlying mechanisms. Using a genome-wide CRISPR/Cas9 screen in human AML, we identifi ed genes whose inactivation sensitizes AML blasts to venetoclax. Genes involved in mitochondrial organization and function were signifi cantly depleted throughout our screen, including the mitochondrial chaperonin CLPB. We demonstrated that CLPB is upregulated in human AML, it is further induced upon acquisition of venetoclax resistance, and its ablation sensitizes AML to venetoclax. Mechanistically, CLPB maintains the mitochondrial cristae structure via its interaction with the cristae-shaping protein OPA1, whereas its loss promotes apoptosis by inducing cristae remodeling and mitochondrial stress responses. Overall, our data suggest that targeting mitochondrial architecture may provide a promising approach to circumvent venetoclax resistance. SIGNIFICANCE: A genome-wide CRISPR/Cas9 screen reveals genes involved in mitochondrial biological processes participate in the acquisition of venetoclax resistance. Loss of the mitochondrial protein CLPB leads to structural and functional defects of mitochondria, hence sensitizing AML cells to apoptosis. Targeting CLPB synergizes with venetoclax and the venetoclax/azacitidine combination in AML in a p53-independent manner.

The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.epigenomics | transcription | mRNA-Seq H untington disease (HD) is a fatal, neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene, which encodes an abnormally long polyglutamine repeat in the HTT protein. In the early stages of the disease, patients are largely asymptomatic, although they may suffer from mild cognitive impairment and behavioral changes. With time, they develop severe motor dysfunction as well as more pronounced cognitive and psychiatric symptoms. Transcriptional dysregulation is a major component of the early stages of HD, before significant neuronal death. Changes in transcription have been detected in human postmortem tissue (1), mouse models (2-4), and cell culture models (5). Genes reproducibly shown to be differentially expressed across HD models are associated with processes including neurotransmission, neurotrophin receptor signaling, signal transduction, calcium ion transport, synaptic organization, chromatin remodeling, G-protein receptor-coupled signaling, and metabolism (6). The polyglutamine expanded form of HTT may have a direct role in causing these expression changes. A number of DNA-binding proteins have been shown to physically interact with either wild-type or mutant HTT. These include NRSF/REST, CBP, PGC1α, Sp1, BCL11b, p53, LXRα, polycombgroup proteins, SIN3A, and NCOR1 (6). Any effect of HTT on the subcellular localization, activity, or concentration of these proteins would be likely to directly change gene expression.Two lines of evidence suggest that alterations in HTT could also influence DNA methylation. First, histone marks are altered in HD (7-11) and SETD2, a SET domain regulator of H3K36me3, has been previously reported to interact with mutant HTT (12). Due to biochemical...

Diet-induced obesity predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but it is unknown what aspects of the in vivo responses are captured by these models. We use global RNA-sequencing (RNA-Seq) to investigate changes induced by TNFα, hypoxia, dexamethasone, high insulin, and a combination of TNFα and hypoxia, comparing the results to the changes in white adipose tissue from diet-induced obese (DIO) mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNFα and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing (DNase-Seq), we further examined the transcriptional regulation of TNFα-induced insulin resistance, and we found that C/EPBβ is a potential key regulator of adipose insulin resistance.

Summary MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.