Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method

The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications

Porting LHAASO WFCTA simulation job to ARM computing cluster

With the advancement of many large-scale high-energy physics experiments, the amount of data to be processed and analyzed has significantly increased. For example, since the start of the Large High Altitude Air Shower Observatory (LHAASO) experiment in 2020, their simulation jobs have been running on an Intel X86 cluster, producing only a fraction of the planned data for the first phase due to limited CPU resources. Therefore, it is necessary to explore and expand other computing service devices. We built an application ecosystem based on the ARM architecture to support offline data processing for high-energy physics. The main work includes porting the offline software based on LHAASO experiments to run on ARM machines, formulating data transfer and job scheduling strategies in the ARM cluster, and evaluating performance and power consumption in both Intel X86 and ARM clusters. The results show that the LHAASO simulation jobs can run correctly on the ARM computing cluster. The singlecore performance of Intel X86 CPUs is better than ARM CPUs, but for the entire server with a multicore architecture, ARM servers perform better

METTL14 regulates chromatin bivalent domains in mouse embryonic stem cells

METTL14 (methyltransferase-like 14) is an RNA-binding protein that partners with METTL3 to mediate N6-methyladenosine (m6A) methylation. Recent studies identified a function for METTL3 in heterochromatin in mouse embryonic stem cells (mESCs), but the molecular function of METTL14 on chromatin in mESCs remains unclear. Here, we show that METTL14 specifically binds and regulates bivalent domains, which are marked by trimethylation of histone H3 lysine 27 (H3K27me3) and lysine 4 (H3K4me3). Knockout of Mettl14 results in decreased H3K27me3 but increased H3K4me3 levels, leading to increased transcription. We find that bivalent domain regulation by METTL14 is independent of METTL3 or m6A modification. METTL14 enhances H3K27me3 and reduces H3K4me3 by interacting with and probably recruiting the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) and H3K4 demethylase KDM5B to chromatin. Our findings identify an METTL3-independent role of METTL14 in maintaining the integrity of bivalent domains in mESCs, thus indicating a mechanism of bivalent domain regulation in mammals

Loss of 5-hydroxymethylcytosine correlates with increasing morphologic dysplasia in melanocytic tumors

DNA methylation is the most well studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family. We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared to benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis. In this study we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. 175 cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas. Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed. In addition, an analysis of the relationship of nuclear diameter to 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine. Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case. This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions

Tet and TDG Mediate DNA Demethylation Essential for Mesenchymal-to-Epithelial Transition in Somatic Cell Reprogramming

SummaryTet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion

Monitoring the Process and Characterizing Symptoms of Suckling Mouse Inoculation Promote Isolating Viruses from Ticks

Suckling mouse inoculation is an important method that has been used for years to isolate viruses from ticks; however, this method has usually been briefly described in the literature on a case-by-case basis upon successful isolation rather than providing extensive details. This study describes the procedure from preparation of tick homogenates to identification of virus isolation using the suckling mouse inoculation method. The transient and persistent features were characterized and the incidence of manifestations that developed in the suckling mice, especially in mice from which viruses were isolated, is reported. We identified 22 symptoms that developed in mice, including 13 transient symptoms that recovered by the end of the observation period and 7 persistent symptoms that the mice suffered from throughout the observation period. Persistent symptoms (lateral positioning and dead) and transient symptoms (malaise, emaciation, and difficulty turning over) were the main symptoms based on the high overall incidence. Moreover, we showed that mice from which viruses were isolated had a concentrated period and advanced days of disease onset. This study provides detailed information necessary for better use of suckling mouse inoculation to isolate viruses from ticks, which may benefit optimization of this method to identify, discover, and acquire tick-borne viruses

Innate immunity of vascular smooth muscle cells contributes to two-wave inflammation in atherosclerosis, twin-peak inflammation in aortic aneurysms and trans-differentiation potential into 25 cell types

IntroductionVascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in aortic diseases. Innate immunity is the main driving force for cardiovascular diseases. MethodsTo determine the roles of innate immunity in VSMC and aortic pathologies, we performed transcriptome analyses on aortas from ApoE–/– angiotensin II (Ang II)-induced aortic aneurysm (AAA) time course, and ApoE–/– atherosclerosis time course, as well as VSMCs stimulated with danger-associated molecular patterns (DAMPs).ResultsWe made significant findings: 1) 95% and 45% of the upregulated innate immune pathways (UIIPs, based on data of 1226 innate immune genes) in ApoE–/– Ang II-induced AAA at 7 days were different from that of 14 and 28 days, respectively; and AAA showed twin peaks of UIIPs with a major peak at 7 days and a minor peak at 28 days; 2) all the UIIPs in ApoE–/– atherosclerosis at 6 weeks were different from that of 32 and 78 weeks (two waves); 3) analyses of additional 12 lists of innate immune-related genes with 1325 cytokine and chemokine genes, 2022 plasma membrane protein genes, 373 clusters of differentiation (CD) marker genes, 280 nuclear membrane protein genes, 1425 nucleoli protein genes, 6750 nucleoplasm protein genes, 1496 transcription factors (TFs) including 15 pioneer TFs, 164 histone modification enzymes, 102 oxidative cell death genes, 68 necrotic cell death genes, and 47 efferocytosis genes confirmed two-wave inflammation in atherosclerosis and twin-peak inflammation in AAA; 4) DAMPs-stimulated VSMCs were innate immune cells as judged by the upregulation of innate immune genes and genes from 12 additional lists; 5) DAMPs-stimulated VSMCs increased trans-differentiation potential by upregulating not only some of 82 markers of 7 VSMC-plastic cell types, including fibroblast, osteogenic, myofibroblast, macrophage, adipocyte, foam cell, and mesenchymal cell, but also 18 new cell types (out of 79 human cell types with 8065 cell markers); 6) analysis of gene deficient transcriptomes indicated that the antioxidant transcription factor NRF2 suppresses, however, the other five inflammatory transcription factors and master regulators, including AHR, NF-KB, NOX (ROS enzyme), PERK, and SET7 promote the upregulation of twelve lists of innate immune genes in atherosclerosis, AAA, and DAMP-stimulated VSMCs; and 7) both SET7 and trained tolerance-promoting metabolite itaconate contributed to twin-peak upregulation of cytokines in AAA. DiscussionOur findings have provided novel insights on the roles of innate immune responses and nuclear stresses in the development of AAA, atherosclerosis, and VSMC immunology and provided novel therapeutic targets for treating those significant cardiovascular and cerebrovascular diseases

The protective role of DOT1L in UV-induced melanomagenesis

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis

Multifractal Characteristics and Classification of Tight Sandstone Reservoirs: A Case Study from the Triassic Yanchang Formation, Ordos Basin, China

Pore structure determines the ability of fluid storage and migration in rocks, expressed as porosity and permeability in the macroscopic aspects, and the pore throat radius in the microcosmic aspects. However, complex pore structure and strong heterogeneity make the accurate description of the tight sandstone reservoir of the Triassic Yanchang Formation, Ordos Basin, China still a problem. In this paper, mercury injection capillary pressure (MICP) parameters were applied to characterize the heterogeneity of pore structure, and three types of pore structure were divided, from high to low quality and defined as Type I, Type II and Type III, separately. Then, the multifractal analysis based on the MICP data was conducted to investigate the heterogeneity of the tight sandstone reservoir. The relationships among physical properties, MICP parameters and a series of multifractal parameters have been detailed analyzed. The results showed that four multifractal parameters, singularity exponent parameter (αmin), generalized dimension parameter (Dmax), information dimension (D1), and correlation dimension (D2) were in good correlations with the porosity and permeability, which can well characterize the pore structure and reservoir heterogeneity of the study area, while the others didn’t respond well. Meanwhile, there also were good relationships between these multifractal and MICP parameters