Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules.

Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC)) and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC) bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC) isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins.

LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation.

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers.

Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2'-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes.

Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.

Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways.

Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1(-/-) embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ((hm)C), is generated by enzymatic oxidation of 5-methylcytosine ((m)C). This discovery has raised fundamental questions about the functional relevance of (hm)C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare (hm)C against the far more abundant (m)C is technically challenging and to date no methods for direct sequencing of (hm)C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves (hm)C-containing sequences. We determined the consensus cleavage site of PvuRts1I as (hm)CN(11-12)/N(9-10)G and show first data on its potential to interrogate (hm)C patterns in mammalian genomes.

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.