The Mitochondrial Epigenome: An Unexplored Avenue to Explain Unexplained Myopathies?

Mutations in either mitochondrial DNA (mtDNA) or nuclear genes that encode mitochondrial proteins may lead to dysfunctional mitochondria, giving rise to mitochondrial diseases. Some mitochondrial myopathies, however, present without a known underlying cause. Interestingly, methylation of mtDNA has been associated with various clinical pathologies. The present study set out to assess whether mtDNA methylation could explain impaired mitochondrial function in patients diagnosed with myopathy without known underlying genetic mutations. Enhanced mtDNA methylation was indicated by pyrosequencing for muscle biopsies of 14 myopathy patients compared to four healthy controls, at selected cytosines in the Cytochrome B (CYTB) gene, but not within the displacement loop (D-loop) region. The mtDNA methylation patterns of the four healthy muscle biopsies were highly consistent and showed intriguing tissue-specific differences at particular cytosines with control skin fibroblasts cultured in vitro. Within individual myopathy patients, the overall mtDNA methylation pattern correlated well between muscle and skin fibroblasts. Despite this correlation, a pilot analysis of four myopathy and five healthy fibroblast samples did not reveal a disease-associated difference in mtDNA methylation. We did, however, detect increased expression of solute carrier family 25A26 (SLC25A26), encoding the importer of S-adenosylmethionine, together with enhanced mtDNA copy numbers in myopathy fibroblasts compared to healthy controls. To confirm that pyrosequencing indeed reflected DNA methylation and not bisulfite accessibility, mass spectrometry was employed. Although no myopathy-related differences in total amount of methylated cytosines were detected at this stage, a significant contribution of contaminating nuclear DNA (nDNA) was revealed, and steps to improve enrichment for mtDNA are reported. In conclusion, in this explorative study we show that analyzing the mitochondrial genome beyond its sequence opens novel avenues to identify potential molecular biomarkers assisting in the diagnosis of unexplained myopathies.

Adipose Stromal Cell-Secretome Counteracts Profibrotic Signals From IPF Lung Matrices.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by excess deposition and altered structure of extracellular matrix (ECM) in the lungs. The fibrotic ECM is paramount in directing resident cells toward a profibrotic phenotype. Collagens, an important part of the fibrotic ECM, have been shown to be structurally different in IPF. To further understand the disease to develop better treatments, the signals from the ECM that drive fibrosis need to be identified. Adipose tissue-derived stromal cell conditioned medium (ASC-CM) has demonstrated antifibrotic effects in animal studies but has not been tested in human samples yet. In this study, the collagen structural integrity in (fibrotic) lung tissue, its interactions with fibroblasts and effects of ASC-CM treatment hereon were studied. Methods: Native and decellularized lung tissue from patients with IPF and controls were stained for denatured collagen using a collagen hybridizing peptide. Primary lung fibroblasts were seeded into decellularized matrices from IPF and control subjects and cultured for 7 days in the presence or absence of ASC-CM. Reseeded matrices were fixed, stained and analyzed for total tissue deposition and specific protein expression. Results: In both native and decellularized lung tissue, more denatured collagen was observed in IPF tissue compared to control tissue. Upon recellularization with fibroblasts, the presence of denatured collagen was equalized in IPF and control matrices, whereas total ECM was higher in IPF matrices than in the control. Treatment with ASC-CM resulted in less ECM deposition, but did not alter the levels of denatured collagen. Discussion: Our data showed that ASC-CM can inhibit fibrotic ECM-induced profibrotic behavior of fibroblasts. This process was independent of collagen structural integrity. Our findings open up new avenues for ASC-CM to be explored as treatment for IPF.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

Increased miR-142-3p Expression Might Explain Reduced Regulatory T Cell Function in Granulomatosis With Polyangiitis.

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression. Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127-) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry. Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro. Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells.

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28- T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3'UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3'UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3'UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner.

Histone modifications reflect gene activity, but the relationship between cause and consequence of transcriptional control is heavily debated. Recent developments in rewriting local histone codes of endogenous genes elucidated instructiveness of certain marks in regulating gene expression. Maintenance of such repressive epigenome editing is controversial, while stable reactivation is still largely unexplored. Here we demonstrate sustained gene re-expression using two types of engineered DNA-binding domains fused to a H3K4 methyltransferase. Local induction of H3K4me3 is sufficient to allow re-expression of silenced target genes in various cell types. Maintenance of the re-expression is achieved, but strongly depends on the chromatin microenvironment (that is, DNA methylation status). We further identify H3K79me to be essential in allowing stable gene re-expression, confirming its role in epigenetic crosstalk for stable reactivation. Our approach uncovers potent epigenetic modifications to be directly written onto genomic loci to stably activate any given gene.

Age-Associated Differences in MiRNA Signatures Are Restricted to CD45RO Negative T Cells and Are Associated with Changes in the Cellular Composition, Activation and Cellular Ageing.

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.

Dual role of miR-21 in CD4+ T-cells: activation-induced miR-21 supports survival of memory T-cells and regulates CCR7 expression in naive T-cells.

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon αCD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.