Suneet Agarwal

Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1

Methylation Mediation Methylation of cytosine bases, 5-methylcytosine (5mC), in DNA plays an important regulatory role in mammalian genomes. Methylation patterns are often inherited across generations, but they can also be dynamic, suggesting that active DNA demethylation pathways exist. One such pathway, best characterized in plants, involves the removal of the 5mC base, and its replacement by C, via a DNA repair mechanism. Kriaucionis and Heintz (p. 929, published online 16 April) now show that, as well as 5mC in mammalian genomes, there are also significant amounts of 5-hydroxymethylcytosine (5hmC) in DNA of Purkinje neurons, which have large nuclei with apparently very little heterochromatin. Tahiliani et al. (p. 930, published online 16 April) find that the protein TET1 is capable of converting 5mC into 5hmC both in vitro and in vivo. 5-Hydroxymethylcytosine is also present in embryonic stem cells, and levels of 5hmC and TET1 show correlated variation during cell differentiation. Methylated C bases, an important epigenetic mark in genomic DNA, can be enzymically converted to 5-hydroxymethylcytosine. DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

SOMATIC CODING MUTATIONS IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.

Modulation of Chromatin Structure Regulates Cytokine Gene Expression during T Cell Differentiation

Differentiating cells undergo programmed alterations in their patterns of gene expression, which are often regulated by structural changes in chromatin. Here we demonstrate that T cell differentiation results in long-range changes in the chromatin structure of effector cytokine genes, which persist in resting Th1 and Th2 cells in the absence of further stimulation. Differentiation of naive T helper cells into mature Th2 cells is associated with chromatin remodeling of the IL-4 and IL-13 genes, whereas differentiation into Th1 cells evokes remodeling of the IFNgamma but not IL-4 or IL-13 genes. IL-4 locus remodeling is accompanied by demethylation and requires both antigen stimulation and STAT6 activation. We propose that chromatin remodeling of cytokine gene loci is functionally associated with productive T cell differentiation and may explain the coordinate regulation of Th2 cytokine genes.

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells

The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome, resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells, suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches, we found that one such lincRNA (lincRNA-RoR) modulates reprogramming, thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells.

Generation of induced pluripotent stem cells from human blood.

Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.

Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3′ deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Reprogramming of T cells from human peripheral blood

A manuscript has appeared online demonstrating isolation of iPSCs from peripheral blood, including a single line that showed evidence for both TCR-β and TCR-γ rearrangement by PCR (Kunisato, A., Wakatsuki, M., Shinba, H., Ota, T., Ishida, I., and Nagao, K. [2010]. Direct generation of induced pluripotent stem cells from human non-mobilized blood. Stem Cells Dev., in press. Published online May 24, 2010. 10.1089/scd.2010.0063).

A 3′ Enhancer in the IL-4 Gene Regulates Cytokine Production by Th2 Cells and Mast Cells

Differentiation of naive T cells into mature Th2 cells is associated with the appearance of a complex pattern of DNase I hypersensitive (DH) sites within the IL-4/IL-13 cytokine gene cluster. We show here that targeted deletion of an inducible DH site, V(A), and the adjacent conserved DH site V (CNS-2) selectively compromises IL-4 gene transcription by differentiated Th2 cells and mast cells. In mast cells, the deletion abrogates IL-4 mRNA induction, an effect mimicked by deletion of the transcription factor NFAT1 (NFATc2), which binds DH site V(A). In T cells, the deletion impairs a process of response maturation, defined by progressive increases in IL-4 levels as Th2 differentiation proceeds. These results identify an essential enhancer which regulates IL-4 gene expression in two important cell lineages in vivo.