Biology subjects

Miller, C.

Publications and source records attributed to Miller, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Elevated peripheral and nervous system inflammation is associated with decreased short-chain fatty acid levels in Zika-virus infected macaques

Zika virus (ZIKV) infection of central nervous system (CNS) tissue is associated with CNS inflammation, which contributes to ZIKV pathology. Similarly, ZIKV infection has been associated with increased vaginal and rectal mucosal inflammation. As mucosal dysfunction may contribute to elevated systemic inflammation, ZIKV-induced mucosal alterations could potentiate CNS disruptions, leading to ZIKV pathogenesis. However, the potential link between mucosal dysfunction, CNS inflammation and the underlying mechanisms causing these disruptions in ZIKV infection has not been well described. Here, we assessed plasma and CSF indicators of inflammation, including neopterin, tryptophan, kynurenine and serotonin by liquid chromatography tandem mass spectrometry. We observed significant increases in neopterin formation, tryptophan catabolism and serotonin levels in the plasma and CSF of ZIKV-infected pigtail macaques (PTM), rhesus macaques (RM) and in the plasma of ZIKV-infected humans. We next examined whether ZIKV infection resulted in microbial translocation across mucosal surfaces by evaluating plasma and cerebrospinal fluid (CSF) levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP) by enzyme-linked immunosorbent assay (ELISA). Increased sCD14 was observed in the CSF of PTM and rhesus macaque (RM), while increased LBP was observed in pigtail macaque (PTM) plasma. Finally, to examine whether ZIKV-induced microbial dysbiosis could underlie increased microbial translocation and inflammation, we characterized intestinal microbial communities by 16s rRNA gene sequencing and microbial functional changes by quantifying short-chain fatty acid (SCFA) concentrations by gas chromatography mass spectrometry. We observed that although ZIKV infection of PTM did not result in significant taxonomic shifts in microbial communities, there were significant reductions in SCFA levels. Loss of microbial function in ZIKV infection could cause decreased intestinal integrity, thereby contributing to elevated microbial translocation and systemic and CNS inflammation, providing a possible mechanism underlying ZIKV pathogenesis. Further, this may represent a mechanism underlying inflammation and pathogenesis in other diseases. Author SummaryZika virus (ZIKV) can be transmitted to humans via the bite of an infected mosquito or between humans during sexual intercourse, typically resulting in mild symptoms, which has been linked to elevated inflammation in the CNS and the development of more serious conditions, including severe neurological syndromes. Previous studies have observed that ZIKV infection is associated with increased mucosal dysfunction, including elevated inflammation in rectal and vaginal mucosal tissue. However, the mechanism of ZIKV-induced mucosal dysfunction may contribute to systemic and CNS inflammation has not been previously investigated. Here, we used the non-human primate (NHP) model and clinical specimens from ZIKV-infected humans to examine markers of systemic and CNS inflammation and microbial translocation. We observed elevated markers indicative of microbial translocation and inflammation in the CNS of ZIKV-infected macaques and humans. A potential association with mucosal dysfunction in ZIKV infection is shifts in microbial dysbiosis. We also observed that there were no significant overall taxonomic shifts in microbial communities, but a reduction of bacterial-derived short-chain fatty acid (SCFA) levels. Finally, we observed that the decrease in SCFA levels significantly negatively correlated with the elevated peripheral and CNS inflammatory markers, suggesting a link between ZIKV-driven disease pathology and microbial function. Taken together, our study provides new insight into a previously unconsidered mechanism underlying ZIKV pathogenesis.

microbiology↗

bioRxiv2023

Reference genome of the bicolored carpenter ant, Camponotus vicinus

Carpenter ants in the genus Camponotus are large, conspicuous ants that are abundant and ecologically influential in many terrestrial ecosystems. The bicolored carpenter ant, C. vicinus Mayr, is distributed across a wide range of elevations and latitudes in western North America, where it is a prominent scavenger and predator. Here, we present a high-quality genome assembly of C. vicinus from a sample collected in Sonoma County, CA, near the type locality of the species. This genome assembly consists of 38 scaffolds spanning 302.74 Mb, with contig N50 of 15.9Mb, scaffold N50 of 19.9 Mb, and BUSCO completeness of 99.2%. This genome sequence will be a valuable resource for exploring the evolutionary ecology of C. vicinus and carpenter ants generally. It also provides an important tool for clarifying cryptic diversity within the C. vicinus species complex, a genetically diverse set of populations, some of which are quite localized and of conservation interest.

genomics↗

bioRxiv2023

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

cell biology↗

bioRxiv2023

Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease

The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimers disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

neuroscience↗

bioRxiv2023

Effects of structural complexity and water depth on the juvenile blue crab Callinectes sapidus in a simulated salt marsh mesocosm

The blue crab (Callinectes sapidus) is ecologically and economically important in Chesapeake Bay. Nursery habitats, which disproportionately contribute individuals to the adult segment of populations, are essential to blue crab population dynamics. Spartina alterniflora salt marshes are productive but fragmented intertidal nursery habitats which may serve as a refuge from predation for juvenile blue crabs. However, the effects of various characteristics of salt marshes on nursery metrics, such as survival, have not been quantified. Using mesocosm experiments, we examined the effects of shoot density and water depth on juvenile blue crab survival using adult blue crabs as predators. Survival increased significantly with shoot density, whereas water depth did not affect survival. Thus, in contrast to several previous studies, water depth did not influence survival of juvenile blue crabs, possibly due to different environmental conditions from prior studies. These findings indicate that salt marsh structural complexity enhances juvenile survival, and that the beneficial effect of shallow water on juvenile survival differs by environmental conditions.

ecology↗

bioRxiv2023

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

bioengineering↗

bioRxiv2022

Syngap1 Regulates Cortical Circuit Assembly by Controlling Membrane Excitability

Gene expression intersects with neural activity to produce cortical circuits during brain development. However, the cell biological mechanisms linking gene expression to activity-dependent cortical circuit assembly remain unclear. Here, we demonstrate in mice that a newly discovered function of the neurodevelopmental disorder gene, Syngap1, is to cell-autonomously control intrinsic membrane excitability (IME) in developing cortical glutamatergic neurons. Syngap1 regulation of IME was mechanistically linked to wiring of a cortical circuit motif required for sensory processing and behavioral action. Restoring depressed IME in Syngap1 deficient neurons through genetic targeting of hyper-functional potassium currents unleashed deficient dendritic morphogenesis in upper lamina sensory cortex pyramidal neurons. Furthermore, enhancing dendritic morphogenesis was sufficient to stimulate assembly of translaminar feed-forward excitatory circuit motifs. Thus, Syngap1 promotes excitatory circuit assembly during cortical development by maintaining IME in a range that enables trophic neuronal activity to maximize pyramidal cell somatodendritic maturation and subsequent synapse formation. HighlightsO_LISyngap1 cell-autonomously tunes cortical pyramidal neuron IME in vivo C_LIO_LISyngap1-IME is regulated in part by control of neuronal potassium currents C_LIO_LISyngap1 enhancement of IME drives dendritic maturation in pyramidal cells C_LIO_LISyngap1 tuning of IME-regulated dendritic maturation promotes circuit assembly C_LI

neuroscience↗

bioRxiv2022

PTEN deficiency exposes a requirement for an ARF GTPase module in integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in ovarian carcinomas. Loss of the tumour suppressor PTEN in high-grade serous ovarian carcinoma (HGSOC) is associated with a patient subgroup with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We utilise long-term time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency does not affect proliferation but rather induces PI(3,4,5)P3-rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor {beta}1-integrin (ITGB1) as key ARF6 interactors regulating the PTEN loss-associated invasion phenotype. ARF6 functions to promote invasion by controlling the recycling of internalised, active {beta}1-integrin complexes to maintain invasive activity into the ECM. The expression of the ARF6-centred complex in HGSOC patients is inversely associated with outcome, allowing identification of patient groups with improved versus poor outcome. ARF6 may represent a new therapeutic vulnerability in PTEN- depleted HGSOC tumours.

cancer biology↗

bioRxiv2022

The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli

The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made towards identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N=N-R), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azo reductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical motifs found in both dietary and pharmaceutical compounds. IMPORTANCEThis work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and non-enzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen and cysteine, control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

microbiology↗

bioRxiv2022

Cross-Modal Representation of Identity in Primate Hippocampus

Faces and voices are the dominant social signals used to recognize individuals amongst human and nonhuman primates (1-4). Yet it is not known how these critical signals are integrated into a cross-modal representation of individual identity in the primate brain. Here we show that while, like humans (5-7), single neurons in the marmoset hippocampus exhibit selective responses when presented with the face or voice of a specific individual conspecific, a parallel mechanism for representing the cross-modal identities for multiple individuals is evident within single neurons and at a population level. Manifold projections likewise showed separability of individuals, as well as clustering for others families, suggesting that multiple learned social categories are encoded as related dimensions of identity in hippocampus. These findings demonstrate that neural representations of identity in hippocampus are both modality-independent and reflect the hierarchical structure of the primate social network. One-Sentence SummaryWe show that cross-modal representations of identity in primate hippocampus can be achieved by at least two distinct neural mechanisms and comprise multiple social categories that reflect different relationships.

neuroscience↗

bioRxiv2022

Genomic patterns of malignant peripheral nerve sheath tumour (MPNST) evolution correlate with clinical outcome and are detectable in cell-free DNA

Malignant peripheral nerve sheath tumour (MPNST) is an aggressive soft-tissue sarcoma that arises in peripheral nerves. MPNST occurs either sporadically or in people with neurofibromatosis type 1 (NF1), a common cancer predisposition syndrome caused by germline pathogenic variants in NF1. Although MPNST is the most common cause of death and morbidity for individuals with NF1, the molecular underpinnings of MPNST pathogenesis remain unclear. Here, we report the analysis of whole-genome sequencing, multi-regional exome sequencing, transcriptomic and methylation profiling data for 95 MPNSTs and precursor lesions (64 NF1-related; 31 sporadic) from 77 individuals. Early events in tumour evolution include biallelic inactivation of NF1 followed by inactivation of CDKN2A and in some cases also TP53 and polycomb repressive complex 2 (PRC2) genes. Subsequently, both sporadic and NF1-related MPNSTs acquire a high burden of somatic copy number alterations (SCNAs). Our analysis revealed distinct pathways of tumour evolution and immune infiltration associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumours with loss of H3K27me3 evolve through extensive chromosomal losses with retention of chromosome 8 heterozygosity followed by whole genome doubling and chromosome 8 amplification. These tumours show lower levels of immune cell infiltration with low cytotoxic activity and low expression of immune checkpoints. In contrast, tumours with retention of H3K27me3 evolve through extensive genomic instability in the absence of recurrent alterations and exhibit an immune cell-rich phenotype. Specific SCNAs detected in both tumour samples and cell-free DNA (cfDNA) act as a surrogate for loss of H3K27me3 and immune infiltration, and predict prognosis. Our results suggest that SCNA profiling of tumour or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.

cancer biology↗

bioRxiv2022

Active vision during prey-capture in wild marmoset monkeys

Here, we examined prey-capture in wild common marmosets (Callithrix jacchus) to explicate the active role of primate vision for high-precision, goal-directed motor actions in challenging natural environments. We observed distinct marmoset hunting strategies that each relied on the close integration of visual processes and dynamic biomechanical motor movements, but differed based on several factors including prey size/speed, substrate, and their relative distance to the prey. Marmoset positional behavior in these complex arboreal settings often involves extending the body, including inversions, while relying on vision to guide prey capture. Applying markerless pose estimation technology, analyses revealed details of how primate vision both guides and corrects rapid motor actions in real-time during prey-capture in the wild. These findings illustrate the significance of active vision during primate ethological behaviors in response to the very challenges the visual system evolved to solve.

animal behavior and cognition↗

bioRxiv2022

Identification of large offspring syndrome during pregnancy through ultrasonography and maternal blood transcriptome analyses

The use of assisted reproductive technologies (ART) in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or ART. Fetal ultrasonographies were taken on gestation day 55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle {approx}280 days). ART fetuses weighing [≥]97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.

developmental biology↗

bioRxiv2021

The C. elegans gonadal sheath Sh1 cells associate selectively with a differentiating germ cell population in the proliferative zone

The C. elegans adult hermaphrodite germ line is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis and ovulation. Recently, we discovered that the distal Sh1 sheath cells associate with mitotic germ cells as they exit the niche (Gordon et al., 2020). Here we report that these sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other.

developmental biology↗

bioRxiv2021

Immunosuppression and Outcomes in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T-cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T-cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T-cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

cancer biology↗

bioRxiv2021

Homeobox transcription factor MNX1 is crucial for restraining the expression of pan-neuronal genes in motor neurons

Motor neurons (MNs) control muscle movement and are essential for breathing, walking and fine motor skills. Motor Neuron and Pancreas Homeobox 1 (MNX1) has long been recognized as a key marker of the MN lineage. Deficiency of the Mnx1 gene in mice results in early postnatal lethality - likely by causing abnormal MN development and respiratory malfunction. However, the genome-wide targets and exact regulatory function of Mnx1 in MNs remains unresolved. Using an in vitro model for efficient MN induction from mouse embryonic stem cells, we identified about six thousand MNX1-bound loci, of which half are conserved enhancers co-bound by the core MN-inducing factors ISL1 and LHX3, while the other half are promoters for housekeeping-like genes. Despite its widespread binding, disruption of Mnx1 affects the activity of only a few dozen MNX1-bound loci, and causes mis-regulation of about one hundred genes, the majority of which are up-regulated pan-neuronal genes with relatively higher expression in the brain compared to MNs. Integration of genome-wide binding, transcriptomic and epigenomic data in the wild-type and Mnx1-disrupted MNs predicts that Pbx3 and Pou6f2 are two putative direct targets of MNX1, and both are homeobox transcription factors highly expressed in the central nervous system. Our results suggest that MNX1 is crucial for restraining the expression of many pan-neuronal genes in MNs, likely in an indirect fashion. Further, the rarity of direct targets in contrast to the widespread binding of MNX1 reflects a distinctive mode of transcriptional regulation by homeobox transcriptional factors.

molecular biology↗

bioRxiv2021

Single-cell analysis uncovers differential regulation of lung γδ T cell subsets by the co-inhibitory molecules, PD-1 and TIM-3

IL-17A-producing {gamma}{delta} T cells within the lung consist of both V{gamma}6+ tissue-resident cells and V{gamma}4+ circulating cells that play important roles in homeostasis, inflammation, infection, tumor progression and metastasis. How these {gamma}{delta} T cell subsets are regulated in the lung environment during homeostasis and cancer remains poorly understood. Using single-cell RNA sequencing and flow cytometry, we show that lung V{gamma}6+ cells express a repertoire of cell surface molecules distinctive from V{gamma}4+ cells, including PD-1 and ICOS. We found that PD-1 functions as a co-inhibitory molecule on V{gamma}6+ cells to reduce IL-17A production, whereas manipulation of ICOS signaling fails to affect IL-17A in V{gamma}6+ cells. In a mammary tumor model, ICOS and PD-1 expression on lung V{gamma}6+ cells remained stable. However, V{gamma}6+ and V{gamma}4+ cells within the lung pre-metastatic niche increased expression of IL-17A, IL-17F, amphiregulin (AREG) and TIM-3 in response to tumor-derived IL-1{beta} and IL-23, where the upregulation of TIM-3 was specific to V{gamma}4+ cells. Inhibition of either PD-1 or TIM-3 in mammary tumor-bearing mice further increased IL-17A by V{gamma}6+ and V{gamma}4+ cells, indicating that both PD-1 and TIM-3 function as negative regulators of IL-17A-producing {gamma}{delta} T cell subsets. Together, these data demonstrate how lung {gamma}{delta} T cell subsets are differentially controlled by co-inhibitory molecules in steady-state and cancer.

immunology↗

bioRxiv2021

Genome-wide analysis of focal DNA hypermethylation in IDH-mutant AML samples

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4,000 focal regions that were uniquely hypermethylated vs. normal CD34+ cells. These regions had modest, but significant, hypermethylation in AMLs with biallelic TET2 mutations, and 5-hydroxymethylation levels that were dependent on functional TET2, indicating that hypermethylation in these regions is caused by inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred in regions with low methylation in normal CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that methylation in these regions is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

cancer biology↗