David Oxley

Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution

Distinguishing Epigenetic Marks Methylation of the cytosine base in eukaryotic DNA (5mC) is an important epigenetic mark involved in gene silencing and genome stability. Methylated cytosine can be enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), which may function as a distinct epigenetic mark—possibly involved in pluripotency—and it may also be an intermediate in active DNA demethylation. To be able to detect 5hmC genome-wide and at single-base resolution, Booth et al. (p. 934, published online 26 April) developed a 5hmC sequencing chemistry that selectively oxidizes 5hmC to 5-formylcytosine and then to uracil while leaving 5mC unchanged. Using this method, mouse embryonic stem cell genomic DNA was sequenced to reveal that 5hmC is found enriched at intragenic CpG islands and long interspersed nuclear element–1 retrotransposons. A sequencing method can discriminate epigenetically modified cytosine nucleotides within embryonic stem cell DNA. 5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r.

The H19 large intergenic non-coding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extra-embryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded in H19’s first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extra-embryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth-promoting insulin-like growth factor 1 receptor (Igf1r) gene. Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress-response RNA-binding protein HuR. These results suggest that H19’s main physiological role is in limiting growth of the placenta before birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.

Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human

Summary Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

RYBP-PRC1 Complexes Mediate H2A Ubiquitylation at Polycomb Target Sites Independently of PRC2 and H3K27me3

Summary Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Summary Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

BackgroundDNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.ResultsWe compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex).Conclusions0ur screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

BackgroundMethylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation.ResultsOur method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts.ConclusionsCollectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

Disease Biomarkers in Cerebrospinal Fluid of Patients with First-Onset Psychosis

Background Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods. Methods and Findings Surface-enhanced laser desorption ionization mass spectrometry was employed to profile proteins and peptides in a total of 179 cerebrospinal fluid samples (58 schizophrenia patients, 16 patients with depression, five patients with obsessive-compulsive disorder, ten patients with Alzheimer disease, and 90 controls). Our results show a highly significant differential distribution of samples from healthy volunteers away from drug-naïve patients with first-onset paranoid schizophrenia. The key alterations were the up-regulation of a 40-amino acid VGF-derived peptide, the down-regulation of transthyretin at ~4 kDa, and a peptide cluster at ~6,800–7,300 Da (which is likely to be influenced by the doubly charged ions of the transthyretin protein cluster). These schizophrenia-specific protein/peptide changes were replicated in an independent sample set. Both experiments achieved a specificity of 95% and a sensitivity of 80% or 88% in the initial study and in a subsequent validation study, respectively. Conclusions Our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and improved therapeutics. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI–TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (−35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (−30%, P=0.02) and RBCs (−60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (−18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (−35%, P=0.05; −51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

Maintenance of Silent Chromatin through Replication Requires SWI/SNF-like Chromatin Remodeler SMARCAD1

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.