Javier Rodríguez-Ubreva

Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus.

Monozygotic (MZ) twins are partially concordant for most complex diseases, including autoimmune disorders. Whereas phenotypic concordance can be used to study heritability, discordance suggests the role of non-genetic factors. In autoimmune diseases, environmentally driven epigenetic changes are thought to contribute to their etiology. Here we report the first high-throughput and candidate sequence analyses of DNA methylation to investigate discordance for autoimmune disease in twins. We used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis, and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Gene ontology analysis revealed enrichment in categories associated with immune function. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. These changes occurred in parallel with a global decrease in the 5-methylcytosine content that was concomitantly accompanied with changes in DNA methylation and expression levels of ribosomal RNA genes, although no changes in repetitive sequences were found. Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease.

Geriatric muscle stem cells switch reversible quiescence into senescence

Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16INK4a is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.

Autophagy maintains stemness by preventing senescence

During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia.During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia.

A Robust and Highly Efficient Immune Cell Reprogramming System

Here we describe a lineage reprogramming system consisting of a B cell line with an estradiol-inducible form of C/EBPalpha where cells can be converted into macrophage-like cells at 100% efficiency within 2 to 3 days. The reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. Time-lapse experiments showed that the cells acquire a macrophage morphology and increased migratory activity as early as 10 hr. During induction, thousands of genes become up- or downregulated, including several dozen transcription and chromatin-remodeling factors. Time-limited exposure of cells to the inducer showed that the reprogrammed cells become transgene independent within 1 to 2 days. The reprogramming can be inhibited, at least partially, by perturbation experiments with B cell and macrophage transcription factors. The tightness, robustness, and speed of the system described make it a versatile tool to study biochemical and biological aspects of lineage reprogramming.

PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation

BackgroundDNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved.ResultsHere we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation.ConclusionsThe work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.

Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells

The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.

Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes

Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

Long-Range Epigenetic Silencing Associates with Deregulation of Ikaros Targets in Colorectal Cancer Cells

Transcription factors are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of transcription factors can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of Ikaros family genes, which code for essential transcription factors in cell differentiation and exhibit genetic defects in hematologic neoplasias. Unexpectedly, our analysis revealed that Ikaros undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Dukes stages. Ikaros hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis, and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma. Mol Cancer Res; 9(8); 1139–51. ©2011 AACR.

IL-4 orchestrates STAT6-mediated DNA demethylation leading to dendritic cell differentiation

BackgroundThe role of cytokines in establishing specific transcriptional programmes in innate immune cells has long been recognized. However, little is known about how these extracellular factors instruct innate immune cell epigenomes to engage specific differentiation states. Human monocytes differentiate under inflammatory conditions into effector cells with non-redundant functions, such as dendritic cells and macrophages. In this context, interleukin 4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) drive dendritic cell differentiation, whereas GM-CSF alone leads to macrophage differentiation.ResultsHere, we investigate the role of IL-4 in directing functionally relevant dendritic-cell-specific DNA methylation changes. A comparison of DNA methylome dynamics during differentiation from human monocytes to dendritic cells and macrophages identified gene sets undergoing dendritic-cell-specific or macrophage-specific demethylation. Demethylation is TET2-dependent and is essential for acquiring proper dendritic cell and macrophage identity. Most importantly, activation of the JAK3-STAT6 pathway, downstream of IL-4, is required for the acquisition of the dendritic-cell-specific demethylation and expression signature, following STAT6 binding. A constitutively activated form of STAT6 is able to bypass IL-4 upstream signalling and instruct dendritic-cell-specific functional DNA methylation changes.ConclusionsOur study is the first description of a cytokine-mediated sequence of events leading to direct gene-specific demethylation in innate immune cell differentiation.

The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion

BackgroundEpstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth.ResultsWe investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation.ConclusionsOur data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.