[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) direct CD4(+) T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4(+) T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Two decades of research have fostered the view that MeCP2 is a multifunctional chromatin protein that integrates diverse aspects of neuronal biology. More recently, studies have focused on specific RTT-associated mutations within the protein. This work has yielded molecular insights into the critical functions of MeCP2 that promise to simplify our understanding of RTT pathology.
[Show abstract][Hide abstract] ABSTRACT: The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display 'active' histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a 'bivalent' chromatin state bearing simultaneous 'active' and 'inactive' chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs.
[Show abstract][Hide abstract] ABSTRACT: Background
Trimethylation of histone H3 lysine 4 (H3K4me3) accumulates at promoters in a gene activity dependent manner. The Set1 complex is responsible for most H3K4me3 in somatic cells and contains the conserved subunit Cfp1, which is implicated in targeting the Set1 complex to CpG islands in mammals. In mouse embryonic stem cells, Cfp1 is necessary for H3K4me3 accumulation at constitutively active gene promoters, but is not required to maintain steady-state transcription of the associated gene.ResultsHere we show that Cfp1 is instrumental for targeting H3K4me3 to promoters upon rapid transcriptional induction in response to external stimuli. Surprisingly, H3K4me3 accumulation is not required to ensure appropriate transcriptional output but rather plays gene specific roles. We also show that Cfp1-dependent H3K4me3 deposition contributes to H3K9 acetylation genome wide, suggesting that Cfp1 dependent H3K4me3 regulates overall H3K9 acetylation dynamics and is necessary for histone acetyl transferase recruitment. Finally, we observe increased antisense transcription at the start and end of genes that require Cfp1 for accurate deposition of H3K4me3 and H3K9ac.Conclusions
Our results assign a key role for Cfp1 in establishing a complex active promoter chromatin state and shed light on how chromatin signaling pathways provide context-dependent transcriptional outcomes.
[Show abstract][Hide abstract] ABSTRACT: Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
[Show abstract][Hide abstract] ABSTRACT: BackgroundMethyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.ResultsUsing immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2-/y
) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2-/y
mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2-/y
mice.ConclusionsMECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.
[Show abstract][Hide abstract] ABSTRACT: Th1 and Th2 cell fates are traditionally viewed as mutually exclusive but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4(+) T cells from mice infected with the parasitic helminth Schistosoma mansoni we observed a defined population of IFN-γ/IL-4 double-positive cells. These IFN-γ(+) IL-4(+) cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN-γ(+) IL-4(-) (Th1) and IFN-γ(-) IL-4(+) (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN-γ(+) IL-4(+) cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind co-existence of Th1 and Th2 characteristics. This article is protected by copyright. All rights reserved.
European Journal of Immunology 06/2014; 44(6). DOI:10.1002/eji.201344098 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell-specific gene expression is controlled by epigenetic modifications and transcription factor binding.While genome-wide maps for these protein-DNA interactions have become widely available,quantitative comparison of the resulting ChIP-Seq data sets remains challenging. Current approachesto detect differentially bound or modified regions are mainly borrowed from RNA-Seq data analysis,thus focusing on total counts of fragments mapped to a region, ignoring any information encoded inthe shape of the peaks.
Here, we present MMDiff, a robust, broadly applicable method for detecting differences between sequencecount data sets. Based on quantifying shape changes in signal profiles, it overcomes challengesimposed by the highly structured nature of the data and the paucity of replicates.We first use a simulated data set to compare the performance of MMDiff with results obtained byfour alternative methods. We demonstrate that MMDiff excels when peak profiles change betweensamples. We next use MMDiff to re-analyse a recent data set of the histone modification H3K4me3elucidating the establishment of this prominent epigenomic marker. Our empirical analysis showsthat the method yields reproducible results across experiments, and is able to detect functional importantchanges in histone modifications. To further explore the broader applicability of MMDiff,we apply it to two ENCODE data sets: one investigating the histone modification H3K27ac and onemeasuring the genome-wide binding of the transcription factor CTCF. In both cases, MMDiff provesto be complementary to count-based methods. In addition, we can show that MMDiff is capable ofdirectly detecting changes of homotypic binding events at neighbouring binding sites. MMDiff isreadily available as a Bioconductor package.
Our results demonstrate that higher order features of ChIP-Seq peaks carry relevant and often complementaryinformation to total counts, and hence are important in assessing differential histone modificationsand transcription factor binding. We have developed a new computational method, MMDiff,that is capable of exploring these features and therefore closes an existing gap in the analysis of ChIPSeqdata sets.
[Show abstract][Hide abstract] ABSTRACT: The discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified.
Here we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites.
We conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body.
[Show abstract][Hide abstract] ABSTRACT: De novo mutations in the X-linked gene encoding the transcription factor methyl-CpG binding protein 2 (MECP2) are the most frequent cause of the neurological disorder Rett syndrome (RTT). Hemizygous males usually die of neonatal encephalopathy. Heterozygous females survive into adulthood but exhibit severe symptoms including microcephaly, loss of purposeful hand motions and speech, and motor abnormalities, which appear after a period of apparently normal development. Most studies have focused on male mouse models because of the shorter latency to and severity in symptoms, yet how well these mice mimic the disease in affected females is not clear. Very few therapeutic treatments have been proposed for females, the more gender-appropriate model. Here, we show that self-complementary AAV9, bearing MeCP2 cDNA under control of a fragment of its own promoter (scAAV9/MeCP2), is capable of significantly stabilizing or reversing symptoms when administered systemically into female RTT mice. To our knowledge, this is the first potential gene therapy for females afflicted with RTT.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2013; 33(34):13612-13620. DOI:10.1523/JNEUROSCI.1854-13.2013 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MECP2), a nuclear protein that in neurons regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MECP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3', 5'-cyclic AMP (cAMP), indicating that MECP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MECP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MECP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MECP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MECP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MECP2 at T308 regulates the interaction of MECP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MECP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MECP2 with the NCoR complex.
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is a disorder with a pronounced neurological phenotype and is caused mainly by mutations in the X-linked gene MECP2. A common feature of RTT is an abnormal EEG and a propensity for seizures. In the current study we aimed to assess brain network excitability and seizure propensity in a mouse model of RTT. Mice in which Mecp2 expression was silenced (Mecp2(stop/y)) showed a higher seizure score (mean = 6 ± 0.8 compared to 4 ± 0.2 in wild-type, WT) and more rapid seizure onset (median onset = 10 mins in Mecp2(stop/y) and 32 mins in WT) when challenged with the convulsant drug kainic acid (25mg/Kg). Hippocampal slices from Mecp2(stop/y) brain displayed no spontaneous field potential activities under control conditions but showed higher power gamma frequency field potential oscillations compared to WT in response to kainic acid (400 nM) in vitro. Brain slices challenged with the GABA(A) receptor antagonist bicuculline (0.1-10μM) and the potassium channel blocker 4-aminopyridine (1-50μM) also revealed differences between genotypes with hippocampal circuits from Mecp2(stop/y) mouse slices showing enhanced epileptiform burst duration and frequency. In contrast to these network level findings, single cell analysis of pyramidal cells by whole-cell patch clamp recording revealed no detectable differences in synaptic or biophysical properties between MeCP2-containing and MeCP2-deficient neurons. These data support the proposal that loss of MeCP2 alters network level excitability in the brain to promote epileptogenesis.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2. STEM Cells2012;30:2128-2139.
[Show abstract][Hide abstract] ABSTRACT: Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of "ectopic" H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents "leakage" of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.
Genes & development 08/2012; 26(15):1714-28. DOI:10.1101/gad.194209.112 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rett Syndrome is a neurological disorder caused by mutations in the X-linked MECP2 gene. Mouse models where Mecp2 is inactivated or mutated recapitulate several features of the disorder and have demonstrated a requirement for the protein to ensure brain function in adult mice. We deleted the Mecp2 gene in ~80% of brain cells at three postnatal ages to determine whether the need for MeCP2 varies with age. Inactivation at all three time points induced Rett-like phenotypes and caused premature death of the animals. We find two threshold ages beyond which the requirement for MeCP2 markedly increases in stringency. The earlier threshold (8-14 weeks), when inactivated mice develop symptoms, represents early adulthood in the mouse and coincides with the period when Mecp2-null mice exhibit terminal symptoms. Unexpectedly, we identified a later age threshold (30-45 weeks) beyond which an 80% reduction in MeCP2 is incompatible with life. This finding suggests an enhanced role for MeCP2 in the aging brain.
Human Molecular Genetics 05/2012; 21(17):3806-14. DOI:10.1093/hmg/dds208 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome is a neurological disorder caused by mutation of the X-linked MECP2 gene. Mice lacking functional Mecp2 display a spectrum of Rett syndrome-like signs, including disturbances in motor function and abnormal patterns of breathing, accompanied by structural defects in central motor areas and the brainstem. Although routinely classified as a neurodevelopmental disorder, many aspects of the mouse phenotype can be effectively reversed by activation of a quiescent Mecp2 gene in adults. This suggests that absence of Mecp2 during brain development does not irreversibly compromise brain function. It is conceivable, however, that deep-seated neurological defects persist in mice rescued by late activation of Mecp2. To test this possibility, we have quantitatively analysed structural and functional plasticity of the rescued adult male mouse brain. Activation of Mecp2 in ∼70% of neurons reversed many morphological defects in the motor cortex, including neuronal size and dendritic complexity. Restoration of Mecp2 expression was also accompanied by a significant improvement in respiratory and sensory-motor functions, including breathing pattern, grip strength, balance beam and rotarod performance. Our findings sustain the view that MeCP2 does not play a pivotal role in brain development, but may instead be required to maintain full neurological function once development is complete.