Domain Requirements and Sequence Specificity of DNA Binding for the Forkhead Transcription Factor FOXP3

The forkhead, winged-helix transcription factor FOXP3 is preferentially expressed in T regulatory (Treg) cells and is critical for their immunosuppressive function. Mutations that abolish FOXP3 function lead to systemic autoimmunity in mice and humans. However, the manner by which FOXP3 recognizes cognate DNA elements is unclear. Here we identify an in vitro optimized DNA sequence to assess FOXP3 DNA binding by electrophoretic mobility shift assay (EMSA). The optimized sequence contains two tandem copies of a core DNA element resembling, but not identical to, the canonical forkhead (FKH) binding element. The tandem nature of this optimized FOXP3-binding oligonucleotide suggests a requirement for multimerization, and EMSA experiments confirm that both the DNA-binding FKH domain and an intact leucine-zipper domain, which mediates homo-multimerization of FOXP3, are required for DNA binding. These results establish a practical framework for understanding the molecular basis by which FOXP3 regulates gene transcription and programs Treg suppressive function

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

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.Chemistry and Chemical Biolog

Tet1 and Tet2 Regulate 5-Hydroxymethylcytosine Production and Cell Lineage Specification in Mouse Embryonic Stem Cells

SummaryTET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs

Tumour hypoxia causes DNA hypermethylation by reducing TET activity

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylatio

DNA methylation and methylcytosine oxidation in cell fate decisions

Changes in cellular phenotypes and identities are fundamentally regulated by epigenetic mechanisms including DNA methylation, post-translational histone modifications and chromatin remodeling. Recent genome-wide profiles of the mammalian DNA 'methylome' suggest that hotspots of dynamic DNA methylation changes during cell fate transitions occur at distal regulatory regions with low or intermediate CpG densities. These changes are most prevalent early during the course of cellular differentiation and can be locally influenced by binding of cell-type specific transcription factors. With the advent of next-generation quantitative base-resolution maps of 5-methylcytosine and its oxidized derivatives and better coverage of the genome, we expect to learn more about the true significance of these DNA modifications in the regulation of cell fate choices.status: publishe

FOXP3:DNA binding is regulated by spacing between tandem elements.

<p>(A) Sequences of the oligonucleotides used as probes in EMSA assays. Each FOXP3 binding site is underlined with the first two nucleotides of each site highlighted in red text. The space (in number of nucleotides) separating the 5′ ends of the two binding sites is indicated below each sequence. The total number of base pairs in each sequence is listed next to the probe name in parentheses. (B) <i>In vitro</i>-translated firefly luciferase (Luc), or FOXP3-ΔN were incubated with each labeled probe as indicated. Protein:DNA complexes and free probe are indicated. Quantification values of bound probe were indicated below the lanes. All data shown are representative of at least 2 separate experiments.</p

Definition of the FOXP3 consensus binding site.

<p>(A) <i>Left</i> – Using the A′ oligonucleotide sequence as a reference, the two 5′ nucleotides were randomized within each of the two putative FOXP3 binding sites. Each FOXP3 binding site is underlined and the randomized 5′ dinucleotide motifs are indicated by red text. <i>Right</i> – Computationally-determined putative FOXP3 binding site as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone-0008109-g002" target="_blank">figure 2</a> legend. (B) <i>In vitro</i>-translated firefly luciferase (Luc), full-length FOXP3 or FOXP3-ΔN were incubated with each labeled probe as indicated. Protein:DNA complexes and free probe are indicated by arrowhead and square brackets as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone-0008109-g002" target="_blank">Figure 2</a>. (C) The same experiment as described above in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone-0008109-g003" target="_blank">Figure 3B</a> was performed with <i>in vitro</i>-translated full-length Foxp1 or Foxp1-ΔN. Quantification values of bound probe were indicated below the lanes. These data represent at least 2 independent experiments.</p

Deletion of the FOXP3 N-terminal region allows binding to a Foxp1-like DNA element.

<p>(A) Fragments of human FOXP3 translated <i>in vitro</i> (see materials) for use in EMSA assays. Residue numbering is listed above full-length FOXP3. N-term – the N-terminal region (1–181) of FOXP3, ZF – zinc finger, Zip – leucine zipper, FKH – forkhead domain. (B) <i>Top</i> – Full-length Foxp1 or FOXP3 fragments were expressed and incubated with radiolabelled A probe. Arrowheads indicate Protein:DNA complexes and free probe. <i>Bottom</i> – Anti-HA monoclonal antibodies or FOXP3 antiserum were used to detect expression of Foxp1 or FOXP3 fragments, respectively, by western blotting. Arrowheads indicate the position of each construct. Data are representative of at least 2 independent experiments.</p

DNA binding specificities of Foxp1 and FOXP3.

<p>(A) Sequences of the A probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Wang1" target="_blank">[34]</a>, A′ and A″ oligonucleotides used for EMSA experiments. The 5′ putative FOXP3-binding site (A1) is similar to a predicted Foxp3 binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Zheng2" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Marson1" target="_blank">[30]</a> (see insert adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Marson1" target="_blank">[30]</a> below sequence text). The 3′ binding site (A2) represents the Foxp1 consensus site (blue text) as determined previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008109#pone.0008109-Wang1" target="_blank">[34]</a>. A′ has two putative FOXP3-binding sites (A1-A1). A″ has two putative Foxp1 sites (A2-A2). (B) <i>In vitro</i>-translated firefly luciferase (Luc), Foxp1 (full-length or ΔN) or FOXP3 (full-length or ΔN) were incubated with each labeled probe as indicated. Protein:DNA complexes and free probe are indicated at left (by arrowhead in one case and a square bracket in the other). NS – non-specific. Quantification values of bound probe (shown as % bound of total detected probe in each lane) were indicated below the lanes. (D) Expression of Foxp1 or FOXP3 constructs was evaluated by western blotting using anti-Foxp1 monoclonal or anti-FOXP3 polyclonal antisera respectively. Loading amounts of <i>in vitro</i>-translated lysates were 4 µl for Luc and 1 µl, 2 µl and 4 µl for the Foxp1 and FOXP3 constructs as in (C). Data are representative of at least two independent experiments.</p

Coordination of germ-layer lineage choice by TET1 during primed pluripotency

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.status: accepte