C/EBP-Family Redundancy Determines Patient Survival and Lymph Node Involvement in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPß or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan-Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPß and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPß as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPß and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBß and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

[Homeostatic and Pathophysiological Regulation of Toll-like Receptor 4 Signaling by GM3 Ganglioside Molecular Species].

Chronic inflammation plays an important role in the pathogenesis of obesity and metabolic disorders. In obesity, pattern-recognition receptors in innate immune system, such as Toll-like receptor 4 (TLR4), cause chronic inflammation through prolonged activation by various endogenous ligands, including fatty acids and its metabolites. Gangliosides and other glycosphingolipids are important metabolites of fatty acids and saccharides. GM3, the simplest ganglioside comprising α2,3-sialyllactose, is expressed in insulin-sensitive peripheral tissues such as liver and adipose tissue, and furthermore secreted abundantly into serum. It has been shown that GM3 regulates the signal transduction of insulin receptor in adipose tissue as a component of membrane microdomains, and elevation in GM3 level causes insulin resistance. However, the homeostatic and pathophysiological functions of extracellularly secreted GM3 are poorly understood. We recently reported that GM3 species with differing fatty acid structures act as pro- and anti-inflammatory endogenous TLR4 ligands. GM3 with very long-chain fatty acid (VLCFA) and α-hydroxyl VLCFA strongly enhanced TLR4 activation. Conversely, GM3 with long-chain fatty acid (LCFA) and ω-9 unsaturated VLCFA inhibited TLR4 activation, counteracting the VLCFA species. GM3 interacted with the extracellular complex of TLR4 and promoted dimerization/oligomerization. In obesity and metabolic disorders, VLCFA species were increased in serum and adipose tissue, whereas LCFA species was relatively decreased; their imbalances were correlated to disease progression. Our findings suggest that GM3 species are disease-related endogenous TLR4 ligands, and "glycosphingolipid sensing" by TLR4 controls the homeostatic and pathological roles of innate immune signaling.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes.

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.

Thyroid hormone receptor α controls larval intestinal epithelial cell death by regulating the CDK1 pathway.

Thyroid hormone (T3) regulates adult intestine development through T3 receptors (TRs). It is difficult to study TR function during postembryonic intestinal maturation in mammals due to maternal influence. We chose intestinal remodeling during Xenopus tropicalis metamorphosis as a model to study TR function in adult organ development. By using ChIP (chromatin immunoprecipitation)-Seq, we identified over 3000 TR-bound genes in the intestine of premetamorphic wild type or TRα (the major TR expressed during premetamorphosis)-knockout tadpoles. Surprisingly, cell cycle-related GO (gene ontology) terms and biological pathways were highly enriched among TR target genes even though the first major event during intestinal metamorphosis is larval epithelial cell death, and TRα knockout drastically reduced this enrichment. More importantly, treatment of tadpoles with cell cycle inhibitors blocked T3-induced intestinal remodeling, especially larval epithelial cell death, suggesting that TRα-dependent activation of cell cycle is important for T3-induced apoptosis during intestinal remodeling.

The Antarctic Weddell seal genome reveals evidence of selection on cardiovascular phenotype and lipid handling.

The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes were SUMO2 and EP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.

The effect of the brood and the queen on early gene expression in bumble bee workers' brains.

Worker reproduction in social insects is often regulated by the queen, but can be regulated by the brood and nestmates, who may use different mechanisms to induce the same outcomes in subordinates. Analysis of brain gene expression patterns in bumble bee workers (Bombus impatiens) in response to the presence of the queen, the brood, both or neither, identified 18 differentially expressed genes, 17 of them are regulated by the queen and none are regulated by the brood. Overall, brain gene expression differences in workers were driven by the queen's presence, despite recent studies showing that brood reduces worker egg laying and provides context to the queen pheromones. The queen affected important regulators of reproduction and brood care across insects, such as neuroparsin and vitellogenin, and a comparison with similar datasets in the honey bee and the clonal raider ant revealed that neuroparsin is differentially expressed in all species. These data emphasize the prominent role of the queen in regulating worker physiology and behavior. Genes that serve as key regulators of workers' reproduction are likely to play an important role in the evolution of sociality.

Deficiency in RCAT-1 Function Causes Dopamine Metabolism Related Behavioral Disorders in Caenorhabditis elegans.

When animals are faced with food depletion, food search-associated locomotion is crucial for their survival. Although food search-associated locomotion is known to be regulated by dopamine, it has yet to investigate the potential molecular mechanisms governing the regulation of genes involved in dopamine metabolism (e.g., cat-1, cat-2) and related behavioral disorders. During the studies of the pheromone ascaroside, a signal of starvation stress in C. elegans, we identified R02D3.7, renamed rcat-1 (regulator of cat genes-1), which had previously been shown to bind to regulatory sequences of both cat-1 and cat-2 genes. It was found that RCAT-1 (R02D3.7) is expressed in dopaminergic neurons and functions as a novel negative transcriptional regulator for cat-1 and cat-2 genes. When a food source becomes depleted, the null mutant, rcat-1(ok1745), exhibited an increased frequency of high-angled turns and intensified area restricted search behavior compared to the wild-type animals. Moreover, rcat-1(ok1745) also showed defects in state-dependent olfactory adaptation and basal slowing response, suggesting that the mutants are deficient in either sensing food or locomotion toward food. However, rcat-1(ok1745) has normal cuticular structures and locomotion genes. The discovery of rcat-1 not only identifies a new subtype of dopamine-related behaviors but also provides a potential therapeutic target in Parkinson's disease.

Functional Domain Mapping of Werner Interacting Protein 1 (WRNIP1).

Werner helicase-interacting protein 1 (WRNIP1) belongs to the AAA+ ATPase family and is conserved from Escherichia coli to human. In addition to an ATPase domain in the middle region of WRNIP1, WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain and two leucine zipper motifs in the N-terminal and C-terminal regions, respectively. Here, we report that the UBZ domain of WRNIP1 is responsible for the reduced levels of UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitylation in POLH-disrupted (polymerase η (Polη)-deficient) cells, and that the ATPase domain of WRNIP1 is involved in regulating the level of the PrimPol protein. The suppression of UV sensitivity of Polη-deficient cells by deletion of WRNIP1 was abolished by expression of the mutant WRNIP1 lacking the UBZ domain or ATPase domain, but not by the mutant lacking the leucine zipper domain in WRNIP1/POLH double-disrupted cells. The leucine zipper domain of WRNIP1 was required for its interaction with RAD18, a key factor in TLS (DNA translesion synthesis), and DNA polymerase δ catalytic subunit, POLD1. On the basis of these findings, we discuss the possible role of WRNIP1 in TLS.

Integrative analysis reveals multiple modes of LXR transcriptional regulation in liver.

The nuclear receptors liver X receptor (LXR) α and ß play crucial roles in hepatic metabolism. Many genes induced in response to pharmacologic LXR agonism have been defined; however, the transcriptional consequences of loss of LXR binding to its genomic targets are less well characterized. Here, we addressed how deletion of both LXRα and LXRß from mouse liver (LXR double knockout [DKO]) affects the transcriptional regulatory landscape by integrating changes in LXR binding, chromatin accessibility, and gene expression. Many genes involved in fatty acid metabolism showed reduced expression and chromatin accessibility at their intergenic and intronic regions in LXRDKO livers. Genes that were up-regulated with LXR deletion had increased chromatin accessibility at their promoter regions and were enriched for functions not linked to lipid metabolism. Loss of LXR binding in liver reduced the activity of a broad set of hepatic transcription factors, inferred through changes in motif accessibility. By contrast, accessibility at promoter nuclear factor Y (NF-Y) motifs was increased in the absence of LXR. Unexpectedly, we also defined a small set of LXR targets for direct ligand-dependent repression. These genes have LXR-binding sites but showed increased expression in LXRDKO liver and reduced expression in response to the LXR agonist. In summary, the binding of LXRs to the hepatic genome has broad effects on the transcriptional landscape that extend beyond its canonical function as an activator of lipid metabolic genes.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

The Therapeutic Roles of Recombinant Hsp90α on Cornea Epithelial Injury.

Purpose: The purpose of this study was to explore the therapeutic role of heat shock protein 90 (Hsp90) in wound healing of injury cornea epithelium. Methods: The right eye of C57BL/6N male mice were performed the debridement wounds in the center of the cornea using an algerbrush II blade. The injured area was determined by staining the cornea with fluorescein sodium and measured with image-J. Immunoblotting, ELISA and immunochemistry were used for determining protein expression. The quantitation PCR was performed to measure mRNA expression. Results: Hsp90α is upregulated at both the mRNA and protein levels, and is secreted extracellularly into the corneal stroma and tear film during the healing process after corneal injury in mice. This upregulation is associated with activation of HSF1. Administration of recombinant exogenous Hsp90α (eHsp90α) speeds up wound healing of injured corneal epithelium. The eHsp90α binds to low-density lipoprotein (LDL)-related protein-1 (LRP-1) on the corneal epithelial cells and increases phosphorylation of AKT at S473, which is associated with proliferation and migration corneal epithelial cells in vitro or vivo. Inhibition of AKT by its inhibitor LY294002 abolishes eHsp90α-induced migration and proliferation of corneal epithelial cells. Conclusion: Hsp90α is upregulated and secreted after corneal injury and acts to promote the healing process. Recombinant Hsp90α may be a promising therapeutic drug candidate for corneal injury.

MircoRNA-126-5p inhibits apoptosis of endothelial cell in vascular arterial walls via NF-κB/PI3K/AKT/mTOR signaling pathway in atherosclerosis.

Atherosclerosis is considered as a chronic inflammatory disease. MircoRNA-126-5p (miR-126-5p) may be pathophysiological relevant with the apoptotic processes in the endothelial cells in the arterial wall. Here, this study determined the role of circulating atherosclerosis-regulatory miR-126-5p in atherosclerotic mice and explored the possible mechanism in human aortic endothelial cells (HAECs). Atherosclerotic mice model was established, oxidative stress-induced apoptosis of HAECs was analyzed, and nuclear factor kappa B (NF-κB)/PI3K/AKT/mTOR signaling pathway was investigated both in vitro and in vivo. This study showed that miR-126-5p mice had less coronary atherosclerotic plaque and lower blood lipid than control mice after being induced by high cholesterol diet. Apoptosis of endothelial cells was inhibited and NF-κB/PI3K/AKT/mTOR signal pathway was downregulated in miR-126-5p mice compared to control. MiR-126-5p increased proliferation and inhibited apoptosis of HAECs induced by oxidative stress. In vitro assay showed that miR-126-5p regulated apoptosis of HAECs via downregulation of NF-κB-mediated PI3K/AKT/mTOR signaling pathway. In conclusion, these data indicated that transfection of miR-126-5p rescued apoptosis of HAECs and limited atherosclerosis, introducing a potential therapeutic approach for atherosclerosis.

Increased expression of osteopontin in subchondral bone promotes bone turnover and remodeling, and accelerates the progression of OA in a mouse model.

Osteopontin (OPN) has been proved to be closely related to the pathogenesis of osteoarthritis (OA), but the role of OPN in the pathogenesis of OA has not been fully clarified. Current studies on OPN in OA mostly focus on articular cartilage, synovial membrane and articular fluid, while ignoring its role in OA subchondral bone turnover and remodeling. In this study, we used a destabilization OA mouse model to investigate the role of OPN in OA subchondral bone changes. Our results indicate that increased expression of OPN accelerates the turnover and remodeling of OA subchondral bone, promotes the formation of h-type vessels in subchondral bone, and mediates articular cartilage degeneration induced by subchondral bone metabolism. In addition, our results confirmed that inhibition of PI3K/AKT signaling pathway inhibits OPN-mediated OA subchondral bone remodeling and cartilage degeneration. This study revealed the role and mechanism of OPN in OA subchondral bone, which is of great significance for exploring specific biological indicators for early diagnosis of OA and monitoring disease progression, as well as for developing drugs to regulate the metabolism and turnover of subchondral bone and alleviate the subchondral bone sclerosis of OA.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

Different phases of aging in mouse old skeletal muscle.

With a graying population and increasing longevity, it is essential to identify life transition in later years and discern heterogeneity among older people. Subclassifying the elderly population to inspect the subdivisions for pathophysiological differences is particularly important for the investigation of age-related illnesses. For this purpose, using 24- and 28-month-old mice to represent the "young-old" and "old-old", respectively, we compared their skeletal muscle transcriptomes and found each in a distinct stage: early/gradual (E-aging) and late/accelerated aging phase (L-aging). Principal component analysis showed that the old-old transcriptomes were largely disengaged from the forward transcriptomic trajectory generated in the younger-aged group, indicating a substantial change in gene expression profiles during L-aging. By calculating the transcriptomic distance, it was found that the 28-month group was closer to the two-month group than to the 24-month group. The divergence rate per month for the transcriptomes was the highest in L-aging, twice as fast as the rate in E-aging. Indeed, many of the L-aging genes were significantly altered in transcription, although the changes did not seem random but rather coordinated in a variety of functional gene sets. Of 2,707 genes transcriptionally altered during E-aging, two-thirds were also significantly changed during L-aging, to either downturning or upturning way. The downturn genes were related to mitochondrial function and translational gene sets, while the upturn genes were linked to inflammation-associated gene sets. Our results provide a transcriptomic muscle signature that distinguishes old-old mice from young-old mice. This can help to methodically examine muscle disorders in the elderly.

Endothelial and systemic upregulation of miR-34a-5p fine-tunes senescence in progeria.

Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless.

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.

An autonomous TCR signal-sensing switch influences CD4/CD8 lineage choice in mice.

How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases.

A genetic compensatory mechanism regulated by Jun and Mef2d modulates the expression of distinct class IIa Hdacs to ensure peripheral nerve myelination and repair.

The class IIa histone deacetylases (HDACs) have pivotal roles in the development of different tissues. Of this family, Schwann cells express Hdac4, 5, and 7 but not Hdac9. Here, we show that a transcription factor regulated genetic compensatory mechanism within this family of proteins, blocks negative regulators of myelination ensuring peripheral nerve developmental myelination and remyelination after injury. Thus, when Hdac4 and 5 are knocked-out from Schwann cells in mice, a JUN-dependent mechanism induces the compensatory overexpression of Hdac7 permitting, although with a delay, the formation of the myelin sheath. When Hdac4, 5, and 7 are simultaneously removed, the myocyte-specific enhancer-factor d (MEF2D) binds to the promoter and induces the de novo expression of Hdac9, and although several melanocytic lineage genes are misexpressed and Remak bundle structure is disrupted, myelination proceeds after a long delay. Thus, our data unveil a finely tuned compensatory mechanism within the class IIa Hdac family, coordinated by distinct transcription factors, that guarantees the ability of Schwann cells to myelinate during development and remyelinate after nerve injury.

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns.

TET dioxygenases convert 5-methylcytosine (5mC) preferentially in a CpG context into 5-hydroxymethylcytosine (5hmC) and higher oxidized forms, thereby initiating DNA demethylation, but details regarding the effects of the DNA sequences flanking the target 5mC site on TET activity are unknown. We investigated oxidation of libraries of DNA substrates containing one 5mC or 5hmC residue in randomized sequence context using single molecule readout of oxidation activity and sequence and show pronounced 20 and 70-fold flanking sequence effects on the catalytic activities of TET1 and TET2, respectively. Flanking sequence preferences were similar for TET1 and TET2 and also for 5mC and 5hmC substrates. Enhanced flanking sequence preferences were observed at non-CpG sites together with profound effects of flanking sequences on the specificity of TET2. TET flanking sequence preferences are reflected in genome-wide and local patterns of 5hmC and DNA demethylation in human and mouse cells indicating that they influence genomic DNA modification patterns in combination with locus specific targeting of TET enzymes.