Cell-type-specific expression of tRNAs in the brain regulates cellular homeostasis.

Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.

RREB1 regulates neuronal proteostasis and the microtubule network.

Transcription factors play vital roles in neuron development; however, little is known about the role of these proteins in maintaining neuronal homeostasis. Here, we show that the transcription factor RREB1 (Ras-responsive element-binding protein 1) is essential for neuron survival in the mammalian brain. A spontaneous mouse mutation causing loss of a nervous system-enriched Rreb1 transcript is associated with progressive loss of cerebellar Purkinje cells and ataxia. Analysis of chromatin immunoprecipitation and sequencing, along with RNA sequencing data revealed dysregulation of RREB1 targets associated with the microtubule cytoskeleton. In agreement with the known role of microtubules in dendritic development, dendritic complexity was disrupted in Rreb1-deficient neurons. Analysis of sequencing data also suggested that RREB1 plays a role in the endomembrane system. Mutant Purkinje cells had fewer numbers of autophagosomes and lysosomes and contained P62- and ubiquitin-positive inclusions. Together, these studies demonstrate that RREB1 functions to maintain the microtubule network and proteostasis in mammalian neurons.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins.

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.

DOT1L regulates chamber-specific transcriptional networks during cardiogenesis and mediates postnatal cell cycle withdrawal.

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

MEPP: more transparent motif enrichment by profiling positional correlations.

Score-based motif enrichment analysis (MEA) is typically applied to regulatory DNA to infer transcription factors (TFs) that may modulate transcription and chromatin state in different conditions. Most MEA methods determine motif enrichment independent of motif position within a sequence, even when those sequences harbor anchor points that motifs and their bound TFs may functionally interact with in a distance-dependent fashion, such as other TF binding motifs, transcription start sites (TSS), sequencing assay cleavage sites, or other biologically meaningful features. We developed motif enrichment positional profiling (MEPP), a novel MEA method that outputs a positional enrichment profile of a given TF's binding motif relative to key anchor points (e.g. transcription start sites, or other motifs) within the analyzed sequences while accounting for lower-order nucleotide bias. Using transcription initiation and TF binding as test cases, we demonstrate MEPP's utility in determining the sequence positions where motif presence correlates with measures of biological activity, inferring positional dependencies of binding site function. We demonstrate how MEPP can be applied to interpretation and hypothesis generation from experiments that quantify transcription initiation, chromatin structure, or TF binding measurements. MEPP is available for download from https://github.com/npdeloss/mepp.

Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium.

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.

An optimized protocol for rapid, sensitive and robust on-bead ChIP-seq from primary cells.

Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells. For complete details on the use and execution of this protocol, please refer to Brigidi et al. (2019), Carlin et al. (2018), Heinz et al. (2018), Nott et al. (2019), Sakai et al. (2019), and Seidman et al. (2020).

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches.

Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules. For complete details on the use and execution of these protocols, please refer to Muse et al. (2018), Sakai et al. (2019), and Seidman et al. (2020).

Ncor2/PPARα-Dependent Upregulation of MCUb in the Type 2 Diabetic Heart Impacts Cardiac Metabolic Flexibility and Function.

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. In this study, we show that the T2D heart is metabolically inflexible and almost exclusively dependent on mitochondrial fatty acid oxidation as a consequence of mitochondrial calcium uniporter complex (MCUC) inhibitory subunit MCUb overexpression. Using a recombinant endonuclease-deficient Cas9-based gene promoter pulldown approach coupled with mass spectrometry, we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor corepressor 2 repression, and chromatin immunoprecipitation assays identified peroxisome proliferator-activated receptor α as a mediator of MCUb gene expression in T2D cardiomyocytes. Upregulation of MCUb limits mitochondrial matrix Ca2+ uptake and impairs mitochondrial energy production via glucose oxidation by depressing pyruvate dehydrogenase complex activity. Gene therapy displacement of endogenous MCUb with a dominant-negative MCUb transgene (MCUbW246R/V251E) in vivo rescued T2D cardiomyocytes from metabolic inflexibility and stimulated cardiac contractile function and adrenergic responsiveness by enhancing phospholamban phosphorylation via protein kinase A. We conclude that MCUb represents one newly discovered molecular effector at the interface of metabolism and cardiac function, and its repression improves the outcome of the chronically stressed diabetic heart.

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

Phonon Bridge Effect in Superlattices of Thermoelectric TiNiSn/HfNiSn With Controlled Interface Intermixing.

The implementation of thermal barriers in thermoelectric materials improves their power conversion rates effectively. For this purpose, material boundaries are utilized and manipulated to affect phonon transmissivity. Specifically, interface intermixing and topography represents a useful but complex parameter for thermal transport modification. This study investigates epitaxial thin film multilayers, so called superlattices (SL), of TiNiSn/HfNiSn, both with pristine and purposefully deteriorated interfaces. High-resolution transmission electron microscopy and X-ray diffractometry are used to characterize their structural properties in detail. A differential 3 ω -method probes their thermal resistivity. The thermal resistivity reaches a maximum for an intermediate interface quality and decreases again for higher boundary layer intermixing. For boundaries with the lowest interface quality, the interface thermal resistance is reduced by 23% compared to a pristine SL. While an uptake of diffuse scattering likely explains the initial deterioration of thermal transport, we propose a phonon bridge interpretation for the lowered thermal resistivity of the interfaces beyond a critical intermixing. In this picture, the locally reduced acoustic contrast of the less defined boundary acts as a mediator that promotes phonon transition.

ARTDeco: automatic readthrough transcription detection.

BACKGROUND: Mounting evidence suggests several diseases and biological processes target transcription termination to misregulate gene expression. Disruption of transcription termination leads to readthrough transcription past the 3' end of genes, which can result in novel transcripts, changes in epigenetic states and altered 3D genome structure. RESULTS: We developed Automatic Readthrough Transcription Detection (ARTDeco), a tool to detect and analyze multiple features of readthrough transcription from RNA-seq and other next-generation sequencing (NGS) assays that profile transcriptional activity. ARTDeco robustly quantifies the global severity of readthrough phenotypes, and reliably identifies individual genes that fail to terminate (readthrough genes), are aberrantly transcribed due to upstream termination failure (read-in genes), and novel transcripts created as a result of readthrough (downstream of gene or DoG transcripts). We used ARTDeco to characterize readthrough transcription observed during influenza A virus (IAV) infection, validating its specificity and sensitivity by comparing its performance in samples infected with a mutant virus that fails to block transcription termination. We verify ARTDeco's ability to detect readthrough as well as identify read-in genes from different experimental assays across multiple experimental systems with known defects in transcriptional termination, and show how these results can be leveraged to improve the interpretation of gene expression and downstream analysis. Applying ARTDeco to a gene expression data set from IAV-infected monocytes from different donors, we find strong evidence that read-in gene-associated expression quantitative trait loci (eQTLs) likely regulate genes upstream of read-in genes. This indicates that taking readthrough transcription into account is important for the interpretation of eQTLs in systems where transcription termination is blocked. CONCLUSIONS: ARTDeco aids researchers investigating readthrough transcription in a variety of systems and contexts.

Identification of Lineage-Specific Transcription Factors That Prevent Activation of Hepatic Stellate Cells and Promote Fibrosis Resolution.

BACKGROUND & AIMS: Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing myofibroblasts (activated HSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of activated HSCs/myofibroblasts into a quiescent-like state (inactivated HSCs). We aimed to identify molecular features of phenotypes of HSCs from mice and humans. METHODS: We performed studies with LratCre, Ets1-floxed, Nf1-floxed, Pparγ-floxed, Gata6-floxed, Rag2-/-γc-/-, and C57/Bl6 (control) mice. Some mice were given carbon tetrachloride (CCl4) to induce liver fibrosis, with or without a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. Livers from mice were analyzed by immunohistochemistry. Quiescent, activated, and inactivated HSCs were isolated from livers of Col1α1YFP mice and analyzed by chromatin immunoprecipitation and sequencing. Human HSCs were isolated from livers denied for transplantation. We compared changes in gene expression patterns and epigenetic modifications (histone H3 lysine 4 dimethylation and histone H3 lysine 27 acetylation) in primary mouse and human HSCs. Transcription factors were knocked down with small hairpin RNAs in mouse HSCs. RESULTS: Motif enrichment identified E26 transcription-specific transcription factors (ETS) 1, ETS2, GATA4, GATA6, interferon regulatory factor (IRF) 1, and IRF2 transcription factors as regulators of the mouse and human HSC lineage. Small hairpin RNA-knockdown of these transcription factors resulted in increased expression of genes that promote fibrogenesis and inflammation, and loss of HSC phenotype. Disruption of Gata6 or Ets1, or Nf1 or Pparγ (which are regulated by ETS1), increased the severity of CCl4-induced liver fibrosis in mice compared to control mice. Only mice with disruption of Gata6 or Pparγ had defects in fibrosis resolution after CCl4 administration was stopped, associated with persistent activation of HSCs. Administration of a PPARγ agonist accelerated regression of liver fibrosis after CCl4 administration in control mice but not in mice with disruption of Pparγ. CONCLUSIONS: Phenotypes of HSCs from humans and mice are regulated by transcription factors, including ETS1, ETS2, GATA4, GATA6, IRF1, and IRF2. Activated mouse and human HSCs can revert to a quiescent-like, inactivated phenotype. We found GATA6 and PPARγ to be required for inactivation of human HSCs and regression of liver fibrosis in mice.

IL-17 signaling in steatotic hepatocytes and macrophages promotes hepatocellular carcinoma in alcohol-related liver disease.

BACKGROUND & AIMS: Chronic alcohol consumption is a leading risk factor for the development of hepatocellular carcinoma (HCC), which is associated with a marked increase in hepatic expression of pro-inflammatory IL-17A and its receptor IL-17RA. METHODS: Genetic deletion and pharmacological blocking were used to characterize the role of IL-17A/IL-17RA signaling in the pathogenesis of HCC in mouse models and human specimens. RESULTS: We demonstrate that the global deletion of the Il-17ra gene suppressed HCC in alcohol-fed diethylnitrosamine-challenged Il-17ra-/- and major urinary protein-urokinase-type plasminogen activator/Il-17ra-/- mice compared with wild-type mice. When the cell-specific role of IL-17RA signaling was examined, the development of HCC was decreased in both alcohol-fed Il-17raΔMΦ and Il-17raΔHep mice devoid of IL-17RA in myeloid cells and hepatocytes, but not in Il-17raΔHSC mice (deficient in IL-17RA in hepatic stellate cells). Deletion of Il-17ra in myeloid cells ameliorated tumorigenesis via suppression of pro-tumorigenic/inflammatory and pro-fibrogenic responses in alcohol-fed Il-17raΔMΦ mice. Remarkably, despite a normal inflammatory response, alcohol-fed Il-17raΔHep mice developed the fewest tumors (compared with Il-17raΔMΦ mice), with reduced steatosis and fibrosis. Steatotic IL-17RA-deficient hepatocytes downregulated the expression of Cxcl1 and other chemokines, exhibited a striking defect in tumor necrosis factor (TNF)/TNF receptor 1-dependent caspase-2-SREBP1/2-DHCR7-mediated cholesterol synthesis, and upregulated the production of antioxidant vitamin D3. The pharmacological blocking of IL-17A/Th-17 cells using anti-IL-12/IL-23 antibodies suppressed the progression of HCC (by 70%) in alcohol-fed mice, indicating that targeting IL-17 signaling might provide novel strategies for the treatment of alcohol-induced HCC. CONCLUSIONS: Overall, IL-17A is a tumor-promoting cytokine, which critically regulates alcohol-induced hepatic steatosis, inflammation, fibrosis, and HCC. LAY SUMMARY: IL-17A is a tumor-promoting cytokine, which critically regulates inflammatory responses in macrophages (Kupffer cells and bone-marrow-derived monocytes) and cholesterol synthesis in steatotic hepatocytes in an experimental model of alcohol-induced HCC. Therefore, IL-17A may be a potential therapeutic target for patients with alcohol-induced HCC.

Identification and dynamic quantification of regulatory elements using total RNA.

The spatial and temporal regulation of transcription initiation is pivotal for controlling gene expression. Here, we introduce capped-small RNA-seq (csRNA-seq), which uses total RNA as starting material to detect transcription start sites (TSSs) of both stable and unstable RNAs at single-nucleotide resolution. csRNA-seq is highly sensitive to acute changes in transcription and identifies an order of magnitude more regulated transcripts than does RNA-seq. Interrogating tissues from species across the eukaryotic kingdoms identified unstable transcripts resembling enhancer RNAs, pri-miRNAs, antisense transcripts, and promoter upstream transcripts in multicellular animals, plants, and fungi spanning 1.6 billion years of evolution. Integration of epigenomic data from these organisms revealed that histone H3 trimethylation (H3K4me3) was largely confined to TSSs of stable transcripts, whereas H3K27ac marked nucleosomes downstream from all active TSSs, suggesting an ancient role for posttranslational histone modifications in transcription. Our findings show that total RNA is sufficient to identify transcribed regulatory elements and capture the dynamics of initiated stable and unstable transcripts at single-nucleotide resolution in eukaryotes.

Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity.

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-ß) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Genomic Decoding of Neuronal Depolarization by Stimulus-Specific NPAS4 Heterodimers.

Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both a neuron's action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if distinct types of electrical activity can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in mouse hippocampal neurons trigger two spatially segregated and molecularly distinct induction mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate in the formation of stimulus-specific NPAS4 heterodimers that exhibit distinct DNA binding patterns. Thus, NPAS4 differentially communicates increases in a neuron's spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity along the somato-dendritic axis of a neuron.

Paradoxical association of TET loss of function with genome-wide DNA hypomethylation.

Cancer genomes are characterized by focal increases in DNA methylation, co-occurring with widespread hypomethylation. Here, we show that TET loss of function results in a similar genomic footprint. Both 5hmC in wild-type (WT) genomes and DNA hypermethylation in TET-deficient genomes are largely confined to the active euchromatic compartment, consistent with the known functions of TET proteins in DNA demethylation and the known distribution of 5hmC at transcribed genes and active enhancers. In contrast, an unexpected DNA hypomethylation noted in multiple TET-deficient genomes is primarily observed in the heterochromatin compartment. In a mouse model of T cell lymphoma driven by TET deficiency (Tet2/3 DKO T cells), genomic analysis of malignant T cells revealed DNA hypomethylation in the heterochromatic genomic compartment, as well as reactivation of repeat elements and enrichment for single-nucleotide alterations, primarily in heterochromatic regions of the genome. Moreover, hematopoietic stem/precursor cells (HSPCs) doubly deficient for Tet2 and Dnmt3a displayed greater losses of DNA methylation than HSPCs singly deficient for Tet2 or Dnmt3a alone, potentially explaining the unexpected synergy between DNMT3A and TET2 mutations in myeloid and lymphoid malignancies. Tet1-deficient cells showed decreased localization of DNMT3A in the heterochromatin compartment compared with WT cells, pointing to a functional interaction between TET and DNMT proteins and providing a potential explanation for the hypomethylation observed in TET-deficient genomes. Our data suggest that TET loss of function may at least partially underlie the characteristic pattern of global hypomethylation coupled to regional hypermethylation observed in diverse cancer genomes, and highlight the potential contribution of heterochromatin hypomethylation to oncogenesis.