Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution.

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall's corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution.

Hypoxia-sensitive cells trigger NK cell activation via the KLF4-ASH1L-ICAM-1 axis, contributing to impairment in the rat epididymis.

Male infertility is a global health problem especially prevalent in high-altitude regions. The epididymis is essential for sperm maturation, but the influence of environmental cues on its reshaping remains poorly understood. Here, we use single-cell transcriptomics to track the cellular profiles of epidydimal cells in rats raised under normoxia or extended hypoxia. The results show that hypoxia impairs epididymal function, evident in reduced epithelial cells, compromised blood-epididymis barrier integrity, and increased natural killer cells. Through combined analysis of gene-regulatory networks and cell-cell interaction maps, we identify epididymal hypoxia-sensitive cells that communicate with natural killer (NK) cells via increased intercellular adhesion molecule 1 (ICAM-1) driven by KLF4 recruitment of the histone methyltransferase ASL1L to the Icam1 promoter. Taken together, our study offers a detailed blueprint of epididymal changes during hypoxia and defines a KLF4-ALSH1L-ICAM-1 axis contributing to NK cell activation, yielding a potential treatment targeting hypoxia-induced infertility.

A positive-feedback loop between HBx and ALKBH5 promotes hepatocellular carcinogenesis.

BACKGROUND: Hepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms. The newly defined epigenetics, epitranscriptomics regulation, has been reported to involve in multiple cancers including Hepatocellular Carcinoma (HCC). Our previous study found that HBx, HBV encodes X protein, mediated H3K4me3 modification in WDR5-dependent manner to involve in HBV infection and contribute to oncogene expression. AlkB Homolog 5 (ALKBH5), one of epitranscriptomics enzymes, has been identified to be associated with various cancers. However, whether and how ALKBH5 is dysregulated in HBV-related HCC remains unclear yet. This study aims to investigate ALKBH5 function, clinical significance and mechanism in HBV related HCC (HBV-HCC) patients derived from Chinese people. METHODS: The expression pattern of ALKBH5 was evaluated by RT-qPCR, Western blot, data mining and immunohistochemistry in total of 373 HBV-HCC tissues and four HCC cell lines. Cell Counting Kit 8 (CCK8) assay, Transwell and nude mouse model were performed to assess ALKBH5 function by both small interference RNAs and lentiviral particles. The regulation mechanism of ALKBH5 was determined in HBx and WDR5 knockdown cells by CHIP-qPCR. The role of ALKBH5 in HBx mRNA N6-methyladenosine (m6A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells. RESULT: ALKBH5 increased in tumor tissues and predicts a poor prognosis of HBV-HCC. Mechanically, the highly expressed ALKBH5 is induced by HBx-mediated H3K4me3 modification of ALKBH5 gene promoter in a WDR5-dependent manner after HBV infection. The increased ALKBH5 protein catalyzes the m6A demethylation of HBx mRNA, thus stabilizing and favoring a higher HBx expression level. Furthermore, there are positive correlations between HBx and ALKBH5 in HBV-HCC tissues, and depletion of ALKBH5 significantly inhibits HBV-driven tumor cells' growth and migration in vitro and in vivo. CONCLUSIONS: HBx-ALKBH5 may form a positive-feedback loop to involve in the HBV-induced liver carcinogenesis, and targeting the loop at ALKBH5 may provide a potential way for HBV-HCC treatment.

Profiling single-cell histone modifications using indexing chromatin immunocleavage sequencing.

Recently, multiple single-cell assays were developed for detecting histone marks at the single-cell level. These techniques are either limited by the low cell throughput or sparse reads which limit their applications. To address these problems, we introduce indexing single-cell immunocleavage sequencing (iscChIC-seq), a multiplex indexing method based on TdT terminal transferase and T4 DNA ligase-mediated barcoding strategy and single-cell ChIC-seq, which is capable of readily analyzing histone modifications across tens of thousands of single cells in one experiment. Application of iscChIC-seq to profiling H3K4me3 and H3K27me3 in human white blood cells (WBCs) enabled successful detection of more than 10,000 single cells for each histone modification with 11 K and 45 K nonredundant reads per cell, respectively. Cluster analysis of these data allowed identification of monocytes, T cells, B cells, and NK cells from WBCs. The cell types annotated from H3K4me3 single-cell data are specifically correlated with the cell types annotated from H3K27me3 single-cell data. Our data indicate that iscChIC-seq is a reliable technique for profiling histone modifications in a large number of single cells, which may find broad applications in studying cellular heterogeneity and differentiation status in complex developmental and disease systems.

Multiplex indexing approach for the detection of DNase I hypersensitive sites in single cells.

Single cell chromatin accessibility assays reveal epigenomic variability at cis-regulatory elements among individual cells. We previously developed a single-cell DNase-seq assay (scDNase-seq) to profile accessible chromatin in a limited number of single cells. Here, we report a novel indexing strategy to resolve single-cell DNase hypersensitivity profiles based on bulk cell analysis. This new technique, termed indexing single-cell DNase sequencing (iscDNase-seq), employs the activities of terminal DNA transferase (TdT) and T4 DNA ligase to add unique cell barcodes to DNase-digested chromatin ends. By a three-layer indexing strategy, it allows profiling genome-wide DHSs for >15 000 single-cells in a single experiment. Application of iscDNase-seq to human white blood cells accurately revealed specific cell types and inferred regulatory transcription factors (TF) specific to each cell type. We found that iscDNase-seq detected DHSs with specific properties related to gene expression and conservation missed by scATAC-seq for the same cell type. Also, we found that the cell-to-cell variation in accessibility computed using iscDNase-seq data is significantly correlated with the cell-to-cell variation in gene expression. Importantly, this correlation is significantly higher than that between scATAC-seq and scRNA-seq, suggesting that iscDNase-seq data can better predict the cellular heterogeneity in gene expression compared to scATAC-seq. Thus, iscDNase-seq is an attractive alternative method for single-cell epigenomics studies.

C5a/C5aR1 mediates IMQ-induced psoriasiform skin inflammation by promoting IL-17A production from γδ-T cells.

Psoriasis is a chronic relapsing inflammatory skin disease, affecting up to 3% of the global population. Accumulating evidence suggests that the complement system is involved in its pathogenesis. Our previous study revealed that the C5a/C5aR1 pathway is crucial for disease development. However, the underlying mechanisms remain largely unknown. To explore potential mechanisms, psoriatic skin lesions and histological changes were assessed following imiquimod (IMQ) cream treatment. Inflammatory cytokine expression was tested by real-time RT-PCR. Immunohistochemistry and flow cytometry were used to identify inflammatory cell infiltration and interleukin (IL-17A) IL-17A expression. A C5aR1 antagonist (C5aR1a) and PI3K inhibitor (wortmannin) were used for blocking experiments (both in vivo and in vitro) to explore the mechanism. C5a/C5aR1-pathway inhibition significantly attenuated psoriasis-like skin lesions with decreased epidermal hyperplasia, downregulated type 17-related inflammatory gene expression, and reduced IL-17A-producing γδ-T cell responses. Mechanistically, C5a/C5aR1 promoted the latter phenotype via PI3K-Akt signaling. Consistently, C5aR1 deficiency clearly ameliorated IMQ-induced chronic psoriasiform dermatitis, with a significant decrease in IL-17A expression. Finally, blocking C5aR1 signaling further decreased psoriasiform skin inflammation in IL-17-deficient mice. Results suggest that C5a/C5aR1 mediates experimental psoriasis and skin inflammation by upregulating IL-17A expression from γδ-T cells. Blocking C5a/C5aR1/IL-17A axis is expected to be a promising strategy for psoriasis treatment.

HBx Protein Contributes to Liver Carcinogenesis by H3K4me3 Modification Through Stabilizing WD Repeat Domain 5 Protein.

BACKGROUND AND AIMS: Cancer is typically considered as a genetic and epigenetic disease. Although numerous studies have indicated that an aberrant structure, function, or expression level of epigenetic enzymes contribute to many tumor types, precisely how the epigenetic mechanisms are involved in the hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remains unknown. APPROACH AND RESULTS: In this study, we found that the WD repeat domain 5 protein (WDR5)-a core subunit of histone H3 lysine 4 methyltransferase complexes, which catalyze the generation of histone H3 lysine 4 trimethylation (H3K4me3) modification-is highly expressed in HBV-related HCC and promotes HCC development. WDR5 plays a critical role in HBV-driven cell proliferation and tumor growth in mice, and the WDR5-0103 small-molecule inhibitor of WDR5 activity compromises HBV- and hepatitis B x protein (HBx)-driven tumor proliferation. The aberrantly high WDR5 protein level was found to involve HBx through its stabilization of the WDR5 protein by inhibiting the interaction between the damage-specific DNA-binding protein 1/cullin-4 and WDR5, causing decreased ubiquitination of the WDR5 protein. HBx was found to colocalize with WDR5 on chromatin genome wide and promotes genome-wide H3K4me3 modification by means of WDR5. Furthermore, the recruitment of HBx to promoters of target genes relied on its interaction with WDR5 through its α-helix domain. WDR5 was also found to promote HBV transcription through H3K4 modification of covalently closed circular DNA minichromosome, and WDR5-0103 was able to inhibit HBV transcription. Finally, the in vitro and in vivo data further proved that HBx exerted its tumor-promoting function in a WDR5-dependent manner. CONCLUSIONS: Our data reveals that WDR5 is a key epigenetic determinant of HBV-induced tumorigenesis and that the HBx-WDR5-H3K4me3 axis may be a potential therapeutic target in HBV-induced liver pathogenesis.

Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.

Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To generate scMNase-seq libraries, single cells are isolated by FACS sorting, lysed and digested by MNase. DNA is purified, end-repaired and ligated to Y-shaped adaptors. Following PCR amplification with indexing primers, the subnucleosome-sized (fragments with a length of ≤80 bp) and mononucleosome-sized (fragments with a length between 140 and 180 bp) DNA fragments are recovered and sequenced on Illumina HiSeq platforms. On average, 0.5-1 million unique mapped reads are obtained for each single cell. The mononucleosome-sized DNA fragments precisely define genome-wide nucleosome positions in single cells, while the subnucleosome-sized DNA fragments provide information on chromatin accessibility. Library preparation of scMNase-seq takes only 2 d, requires only standard molecular biology techniques and does not require sophisticated laboratory equipment. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification.

Our method for analyzing histone modifications, scChIC-seq (single-cell chromatin immunocleavage sequencing), involves targeting of the micrococcal nuclease (MNase) to a histone mark of choice by tethering to a specific antibody. Cleaved target sites are then selectively PCR amplified. We show that scChIC-seq reliably detects H3K4me3 and H3K27me3 target sites in single human white blood cells. The resulting data are used for clustering of blood cell types.

Circular RNA expression profiles of peripheral blood mononuclear cells in hepatocellular carcinoma patients by sequence analysis.

Circular RNAs (circRNAs) are a large class of noncoding RNAs that have potential regulatory roles in disease pathogenesis and progression. Recently, circRNAs have been found to be expressed in hepatocellular carcinoma (HCC) tissues and involved in the development and metastasis of HCC. However, the significance of circRNAs in peripheral blood mononuclear cells (PBMCs) of HCC patients remains unclear. In this study, RNA sequencing analysis was performed to identify circRNAs from four HCC patients and three healthy controls to determine the expression pattern of circRNAs in the PBMCs and the circRNAs' molecular regulatory networks in HCC pathogenesis. A total of 58 circRNAs were found to be significantly changed (≥2 or ≤0.5-fold) in the PBMCs of HCC patients compared with those of the healthy cases. Six random representative circRNAs (three up- and three down-regulated) were further validated by real-time RT-PCR in 72 samples of PBMCs from HCC patients and 30 control subjects. Chi-square test indicated that one of the up-regulated circRNA candidates-circ_0000798-was correlated with clinical variables. Highly expressed circ_0000798 was associated with poor overall survival of HCC patients. Receiver operating characteristic curve analysis further revealed that circ_0000798 was discriminating HCC patients from healthy controls. Finally, the predicted competing endogenous RNA network of circ_0000798 showed that it might act as a "sponge" of target microRNAs, that would subsequently regulate the expression of target genes in PBMCs. In summary, this is the first study to comprehensively identify dysregulated circRNAs in PBMCs of HCC patients, and its findings suggest that dysregulated circ_0000798 in PBMCs has potential as a convenient biomarker for diagnosing or prognosticating HCC.

Prognostic Values of CD38+CD101+PD1+CD8+ T Cells in Pancreatic Cancer.

Programmed death-1 (PD-1), a key immune checkpoint molecule, has been developed as an oncotherapy target for various carcinomas. However, treatment with anti-PD-1 elicited only a minimal effect in pancreatic ductal adenocarcinoma (PDAC). Subsequent studies revealed the existence of a subset of PD-1+ T cells coexpressing CD38 and CD101, representing a fixed dysfunctional subpopulation that are not able to be rescued by anti-PD-1 immunotherapy. However, whether this subpopulation of PD-1 expressing CD8+ T cells could be useful in predicting PDAC stage or prognosing survival is unknown. In this study, we used flow cytometry and immunofluorescence assay to analyze the expression of CD38 and CD101 in 183 clinical PDAC samples, including 84 of peripheral blood and 99 of surgical tissues. High coexpression of CD38/CD101 on peripheral PD-1+CD8+ T cells or tumor-infiltrating lymphocytes (TILs) was found to be most significantly correlated with Tumor/Node/Metastasis (T/N/M) classification and clinical stage, in contrast PD-1+CD8+ T cells could not correlate with T classification. CD38/CD101 co-repression on TILs also correlated with the poor survival in these PDAC patient samples. Our data suggest that CD38/CD101 might represent a more helpful biomarker than PD-1 alone for diagnosis and prognosis of PDAC.

Publisher Correction: Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing.

Change history: In Fig. 1c of this Letter, the two graphs were duplicates. The right panel of Fig. 1c has been corrected online.

Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing.

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3-6 and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9 that may be related to heterogeneity in chromatin accessibility10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.

Diverse effects of interleukin-22 on pancreatic diseases.

Interleukin-22 (IL-22) is involved in the development of lymphocytes and serves as a rapid and early source of the effector cytokines that are released in response to pathogen-induced changes in the microenvironment. Recent research has implicated IL-22 as a potential contributing factor to the spectrum of inflammation-related pancreatic diseases, particularly pancreatitis, fibrosis, carcinoma and diabetes. In this review, we summarize the current knowledge on the roles of IL-22 in the various pancreatic pathogenesis, providing insights into the underlying cellular and signaling mechanisms that will help guide future research into promising interventional targets with therapeutic potential.

Critical effects of long non-coding RNA on fibrosis diseases.

The expression or dysfunction of long non-coding RNAs (lncRNAs) is closely related to various hereditary diseases, autoimmune diseases, metabolic diseases and tumors. LncRNAs were also recently recognized as functional regulators of fibrosis, which is a secondary process in many of these diseases and a primary pathology in fibrosis diseases. We review the latest findings on lncRNAs in fibrosis diseases of the liver, myocardium, kidney, lung and peritoneum. We also discuss the potential of disease-related lncRNAs as therapeutic targets for the clinical treatment of human fibrosis diseases.

Multifunctional YY1 in Liver Diseases.

The transcription factor Yin Yang 1 (YY1) is a multifunctional protein that can activate or repress gene expression, depending on the cellular context. While YY1 is ubiquitously expressed and highly conserved between species, its role varies among the diverse cell types and includes proliferation, differentiation, and apoptosis. Upregulated YY1 expression is found in pathogenic conditions, such as human hepatocellular carcinoma and hepatitis B virus infection, and its roles in the molecular pathogenic mechanisms in liver (i.e., fibrosis, carcinogenesis, viral-induced injury) are currently being elucidated. The most recent studies have revealed that YY1 is deeply involved in such dysregulated cellular metabolisms as glycometabolism, lipid metabolism, and bile acid metabolism, which are all involved in various diseases. In this review, we will summarize the current knowledge on YY1 in liver diseases, providing a focused discussion on the characterized and probable underlying mechanisms, as well as a reasoned evaluation of the potential for YY1-mediated pathology as drug targets in liver disease therapies.

Dual roles of IL-22 at ischemia-reperfusion injury and acute rejection stages of rat allograft liver transplantation.

Interleukin-22 (IL-22) is a recently identified regulator of inflammation, but little is known about its role in liver transplantation. Therefore, in this study, we explored the roles and the underlying mechanisms of IL-22 in acute allograft rejection by using a rat allogeneic liver transplantation model. Results showed that allograft liver transplantation led to damage of the parent liver and to significantly increased IL-22 expression in the allograft liver and plasma of the recipient rats compared with the rats who received isografts. Moreover, the significantly increased IL-22 expression was accompanied by markedly increased level of phospho-STAT3 in the allogeneic liver tissues after transplantation. Of note, neutralization of the IL-22 protein in recipient rats significantly worsened the function of the allograft liver at 1 day post-transplantation (ischemia-reperfusion injury, IRI) but improved the function at 7 days post-transplantation (acute rejection, AR). At IRI stage, IL-22 protected liver function through the increase of anti-apoptosis and pro-regeneration cytokines. However, IL-22 led to the increase of pro-inflammation factors at AR stage, accompanied by the marked increase of the Th17 and the marked decrease of Treg cells in allograft recipient rats through modulating the expression of chemokines for different cell types, which however were reversed by in vivo IL-22 neutralization. Results indicate the dual roles of IL-22 and suggest the differential potential clinical application of IL-22 at different stage of allograft liver transplantation.

Histone Deacetylase 1 Regulates the Expression of Progesterone Receptor A During Human Parturition by Occupying the Progesterone Receptor A Promoter.

The functional withdrawal of progesterone is mediated by the enhanced expression ratio of the 2 progesterone receptor (PR) isoforms, PRA and PRB, and causes the pregnant human myometrium to switch from a quiet state to a state of excitation-contraction and subsequent laboring. However, the precise mechanisms responsible for alterations in PRA and PRB expression during human parturition have yet to be resolved. In the present study, we report that PRA expression was increased in myometrium samples during labor (P < .001), concomitant with reduced expression of histone deacetylase 1 (HDAC1; P < .01). These results were further confirmed in the laboratory using cultured primary myometrial cells to investigate the effects of HDAC1 knockdown or overexpression. Finally, we verified that HDAC1 downregulated PRA expression by binding to the promoter region of PRA as confirmed by chromatin immunoprecipitation assays (P < .01) and real-time polymerase chain reaction (P < .001). Therefore, the present study not only demonstrates the epigenetic mechanisms underlying human labor but also provides a potential clinical strategy with which to intervene and prevent labor disorders.

Role of Ets Proteins in Development, Differentiation, and Function of T-Cell Subsets.

Through positive selection, double-positive cells in the thymus differentiate into CD4(+) or CD8(+) T single-positive cells that subsequently develop into different types of effective T cells, such as T-helper and cytotoxic T lymphocyte cells, that play distinctive roles in the immune system. Development, differentiation, and function of thymocytes and CD4(+) and CD8(+) T cells are controlled by a multitude of secreted and intracellular factors, ranging from cytokine signaling modules to transcription factors and epigenetic modifiers. Members of the E26 transformation specific (Ets) family of transcription factors, in particular, are potent regulators of these CD4(+) or CD8(+) T-cell processes. In this review, we summarize and discuss the functions and underlying mechanisms of the Ets family members that have been characterized as involved in these processes. Ongoing research of these factors is expected to identify practical applications for the Ets family members as novel therapeutic targets for inflammation-related diseases.

Mutual interaction between BCL6 and microRNAs in T cell differentiation.

The transcription factor B-cell CLL/lymphoma 6 (BCL6) and the regulatory factor microRNAs (miRNAs) are of great importance in the differentiation of T cell subsets. An increasing body of evidence has demonstrated that BCL6 and miRNAs can target one another and mutually adjust their expression in T cell subsets, such as T helper (Th)-2, Th17, CD8+ regulatory T (CD8+Treg) and T follicular helper (Tfh) cells. Here, we discuss the most recent advances and emerging concepts in how BCL6 and miRNAs regulate one another, and the effects of such mutual regulations on T cell subset differentiation.