Biomedical subjects

Qianjin Lu

Publications and source records attributed to Qianjin Lu.

13 recordsLinked to original sources

PubMed2026

Multimodal computational framework resolves B cell maturation in autoimmunity and ageing.

Identification of the origin of pathogenic immune cells is crucial for therapeutic interventions and diagnosis but pseudotime methods struggle to trace immune cells accurately. Current trajectory inference methods for B cell development and response in health and disease either ignore or underutilize antigen receptor sequence information, limiting their ability to resolve developmental pathways, particularly for pathogenic populations. Widely used methods such as Monocle 3 reconstruct developmental paths from transcriptomic similarity alone, discarding the features from immune receptors. Dandelion has combined the immune receptor features with transcriptomics but it struggles to simulate the trajectory path of B cells. Here we present ClonoTrace, a computational framework that integrates BCR sequence features with transcriptomic trajectory inference through gated fusion of multimodal embeddings. In fetal B cell development and germinal centre development, ClonoTrace demonstrates closer concordance with the canonical reference ordering than Monocle 3 and Dandelion. Applied to systemic lupus erythematosus, ClonoTrace indicates a memory B cell extrafollicular maturation route alongside the naïve B cell route, accompanied by induction of ZEB2 with a concomitant decline of BACH2 along the trajectory, as a candidate alternative route to pathogenic double negative 2 B cells (DN2) in systemic lupus erythematosus (SLE) patients. In healthy ageing, ClonoTrace resolved three candidate age-related B cell maturation routes, from naïve, IgM+ memory and switched-memory B cells, each passing through a DN2-associated transcriptional state that is ordered before age-associated B cells along the inferred trajectory. ClonoTrace's fate probability algorithm indicated that IgM+ memory B cell to ABC transition as the leading candidate age-associated transition, which may be distinct from SLE DN2 maturation. ClonoTrace provides a generalizable framework for receptor-informed trajectory inference, describing candidate developmental routes of pathogenic B cell populations in autoimmunity and ageing.

Humans↗

PubMed2006

Aging in heterozygous Dnmt1-deficient mice: effects on survival, the DNA methylation genes, and the development of amyloidosis.

We previously reported that heterozygous DNA methyltransferase 1-deficient (Dnmt1(+/-)) mice maintain T-cell immune function and DNA methylation levels with aging, whereas controls develop autoimmunity, immune senescence, and DNA hypomethylation. We therefore compared survival, cause of death, and T-cell DNA methylation gene expression during aging in Dnmt1(+/-) mice and controls. No difference in longevity was observed, but greater numbers of Dnmt1(+/-) mice developed jejunal apolipoprotein AII amyloidosis. Both groups showed decreased Dnmt1 expression with aging. However, expression of the de novo methyltransferases Dnmt3a and Dnmt3b increased with aging in stimulated T cells from control mice. MeCP2, a methylcytosine binding protein that participates in maintenance DNA methylation, increased with age in Dnmt1(+/-) mice, suggesting a mechanism for the sustained DNA methylation levels. This model thus provides potential mechanisms for DNA methylation changes of aging, and suggests that changes in DNA methylation may contribute to some forms of amyloidosis that develop with aging.

Aging↗

PubMed2005

Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs.

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4(+) lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.

5' Flanking Region↗

PubMed2004

Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression.

Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoid-specific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.

Journal Article↗

PubMed2004

Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4+ lupus T cells.

Inhibiting DNA methylation in CD4+ T cells causes aberrant gene expression and autoreactive monocyte/macrophage killing in vitro, and the hypomethylated cells cause a lupus-like disease in animal models. Similar decreases in T cell DNA methylation occur in idiopathic lupus, potentially contributing to disease pathogenesis. The genes affected by DNA hypomethylation are largely unknown. Using DNA methylation inhibitors and oligonucleotide arrays we have identified perforin as a methylation-sensitive gene. Our group has also reported that DNA methylation inhibitors increase CD4+ T cell perforin by demethylating a conserved methylation-sensitive region that is hypomethylated in primary CD8+ cells, which express perforin, but is largely methylated in primary CD4+ cells, which do not. As lupus T cells also have hypomethylated DNA and promiscuously kill autologous monocytes/macrophages, we hypothesized that perforin may be similarly overexpressed in lupus T cells and contribute to the monocyte killing. We report that CD4+ T cells from patients with active, but not inactive, lupus overexpress perforin, and that overexpression is related to demethylation of the same sequences suppressing perforin transcription in primary CD4+ T cells and demethylated by DNA methylation inhibitors. Further, the perforin inhibitor concanamycin A blocks autologous monocyte killing by CD4+ lupus T cells, suggesting that the perforin is functional. We conclude that demethylation of specific regulatory elements contributes to perforin overexpression in CD4+ lupus T cells. Our results also suggest that aberrant perforin expression in CD4+ lupus T cells may contribute to monocyte killing.

Adult↗

PubMed2004

Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors.

OBJECTIVE: Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance. METHODS: Oligonucleotide arrays, real-time reverse transcription-polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzyme-linked immunosorbent assay. The results were compared with those of T cells from lupus patients. RESULTS: SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70. CONCLUSION: SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.

Adult↗

PubMed2004

DNaseI hypersensitivity analysis of chromatin structure.

Transcriptionally inactive DNA is packaged into condensed chromatin such that it is unavailable to the transcription initiation complex. Activation of the silenced genes during processes such as differentiation first requires that the chromatin structure be remodeled into a transcriptionally permissive configuration, with the DNA "exposed" and accessible to transcription factors. The change in chromatin structure associated with transcriptional competence can be detected as increased sensitivity of the exposed DNA to digestion with DNaseI. This increased susceptibility is referred to as DnaseI-hypersensitivity. DNaseI hypersensitive sites are often located in the recognition sites for transcription factors, including promoters and enhancers. This chapter describes the protocols necessary to perform and analyze DNaseI hypersensitivity assays, a technique becoming increasingly important given the rapid advances in our understanding of the chromatin remodeling processes.

Blotting, Southern↗

PubMed2003

DNA methylation and chromatin structure regulate T cell perforin gene expression.

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4(+) and CD8(+) T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4(+) and CD8(+) cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at -1 kb and the distal promoter at -0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5' flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.

5' Untranslated Regions↗

PubMed2003

Hydralazine may induce autoimmunity by inhibiting extracellular signal-regulated kinase pathway signaling.

OBJECTIVE: To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal-regulated kinase (ERK) pathway signaling. METHODS: The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c-Jun N-terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti-DNA antibodies were measured by enzyme-linked immunosorbent assay. RESULTS: In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen-activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti-double-stranded DNA antibodies in a murine model of drug-induced lupus, similar to the effect of hydralazine. CONCLUSION: Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine-induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic lupus.

Animals↗

PubMed2002

Effect of DNA methylation and chromatin structure on ITGAL expression.

LFA-1 (CD11a/CD18, alpha L beta 2) is an integrin expressed in a tissue-specific fashion and is important in inflammatory and immune responses. Promoter analysis has identified transcription factors that may be involved in CD11a expression, but the mechanisms contributing to its tissue-specific expression are incompletely characterized. In this report we have asked if DNA methylation and/or chromatin structure could contribute to tissue-specific CD11a expression. Bisulfite sequencing was used to compare methylation patterns in the promoter and 5' flanking regions of the ITGAL gene, encoding CD11a, in normal human T cells, which express LFA-1, and fibroblasts, which do not. The region was found to be heavily methylated in fibroblasts but not T cells, and methylation correlated with an inactive chromatin configuration as analyzed by deoxyribonuclease 1 sensitivity. Patch methylation of the promoter region revealed that promoter activity was methylation-sensitive but that methylation of the 5' flanking regions more than 500 base pairs 5' to the transcription start site could also suppress promoter function. Treating fibroblasts with a DNA methylation inhibitor decreased ITGAL promoter methylation and increased CD11a messenger RNA. The results thus indicate that methylation and chromatin structure may contribute to the tissue-specific expression of CD11a.

5' Flanking Region↗

PubMed2002

Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus.

OBJECTIVE: Inhibition of T cell DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo, suggesting that T cell DNA hypomethylation may contribute to autoimmunity. The hypomethylation effects are due, in part, to overexpression of lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18). Importantly, T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, suggesting that the same mechanism could contribute to human lupus. The present study investigated the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus. METHODS: Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter and flanking regions in T cells from lupus patients and healthy subjects, and in T cells treated with DNA methylation inhibitors. "Patch" methylation of promoter sequences in reporter constructs was used to determine the functional significance of the methylation changes. RESULTS: Hypomethylation of specific sequences flanking the ITGAL promoter was seen in T cells from patients with active lupus and in T cells treated with 5-azacytidine and procainamide. Patch methylation of this region suppressed ITGAL promoter function. CONCLUSION: DNA methylation changes occur in specific sequences that regulate LFA-1 expression in lupus T cells and in the hypomethylation model, indicating that altered methylation of specific genes may play a role in the pathogenesis of lupus.

Adult↗

PubMed2002

Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter.

DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (d(m)C) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.

Adult↗

PubMed2002

Infrequent occurrence of age-dependent changes in CpG island methylation as detected by restriction landmark genome scanning.

Hypermethylation of CpG islands, resulting in the inactivation of tumor suppressor genes, is an early event in the development of some malignancies. Recent studies suggest that this abnormal methylation may be a function of aging. The number of CpG islands that methylate with age is unknown. We used restriction landmark genome scanning (RLGS) to approximate the extent to which CpG islands change methylation status during aging. Comparison of more than 2000 loci in T lymphocytes isolated from newborn, middle age, and elderly people revealed that 29 loci ( approximately 1%) changed methylation status during aging, with 23 increasing methylation, and six decreasing. The same subset also changed methylation status with age in the esophagus, lung, and pancreas, but in variable directions. Virtual genome scanning identified one of these loci as a member of the forkhead family, recently implicated in aging, and another as an EST fragment. The methylation status of both correlated with level of expression. Confirming studies in multiple tissues from normal and DNMT1(+/-) mice demonstrated only one age dependent change in the methylation of more than 2000 loci, occurring in liver and kidney. These results indicate that the methylation status of the majority of CpG islands in both mice and humans is tightly controlled during aging, and that changes are infrequent and in humans confined to a specific subset of genes.

Adult↗