Regenerative potential of cTECs was not affected in Foxn1-Stat3-CKO mice.

<p>(A) Experimental procedure for (B) and (C). Foxn1-Cre::Stat3-f/f mice were lethally irradiated and then rescued by bone marrow transplantation from wild type mice. After 4 weeks, mice were sacrificed and thymic tissue was examined. (B) Macroscopy of the thymus 4 weeks after hematopoietic stem cell transplantation. (C) Cryostat sections of the thymus were stained with anti-K14 antibody (green) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Experimental design for data presented in panels (E) and (F). Foxn1-Cre::Stat3-f/f fetal thymic lobes (15 dpc) were cultured in vitro for 6 days in the presence of deoxyguanosine and subsequently transplanted under kidney capsule of wild type mice. After 4 weeks, mice were sacrificed and thymic grafts were examined. (E) Gross anatomical analysis of the thymic grafts 4 weeks after grafting into C57BL/6 wild type recipients. (F) Comparison of maximum cross-section area of thymic grafts of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 4). (G) Cryostat sections of thymic grafts were stained with anti-K8 (red) and anti-K14 antibody (green). Sections were counterstained with DAPI (blue). Scale bars: 200 μm.</p

Medullary regions are severely affected in Foxn1-Stat3-CKO mice.

<p>(A) Macroscopy of the thymus derived from Stat3-flox/flox (Stat3f/f) and Foxn1Cre::Stat3f/f mice at 7 weeks of age. (B) Flowcytometric profiles of developing thymocytes derived from Stat3f/f and Foxn1Cre::Stat3f/f mice, at 7 weeks of age. (C) Cryostat sections of thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice (7 weeks of age) were stained with the mTEC specific antibody, ER-TR5 (red) and counterstained with DAPI (blue). Scale bars: 400 μm. (D) Cryostat sections of neonatal thymic tissue from Stat3f/f and Foxn1Cre::Stat3f/f mice were stained with antibody directed to K14 (green) and counterstained with DAPI (blue) Scale bars: 100 μm. (E) Total thymic cellularity of control (containing Cre-f/+ and f/f) and Foxn1Cre::Stat3f/f (Cre-f/f) mice at indicated ages. Bar stands for the average value of each experimental group. ns denotes a non-significant difference (P>0.1) in Student’s t test. (F) Changes in the proportional area of medullary regions in thymus tissue sections of control (containing Cre-f/+ and f/f, n = 8, 6, 7, 3 for neo, 3, 6, 12 week, respectively) and Foxn1Cre::Stat3f/f (Cre-f/f, n = 5, 4, 6, 5 for neo, 3, 6, 12 week, respectively) mice in the first 12 weeks of life. The area occupied by mTECs in thymus was quantitatively measured in sections stained with K14 antibody using Axiovision4 software (Carl Zeiss). Error bar stands for the standard deviation. ns denotes a non-significant difference (P>0.1) in Student’s t test. **;P<0.005, ***;P<0.0005. (G) Representative flow cytometric profiles showing frequencies of major TEC populations from 12 weeks old Stat3f/f and Foxn1Cre::Stat3f/f mice. EpCAM⁺CD45^- fraction represents whole TEC population, and UEA1 vs Ly51 profile was displayed for the cells gated on EpCAM⁺CD45^- fraction, where UEA1^highLy51^low and UEA1^lowLy51^high fraction were defined as mTEC and cTEC population, respectively. (H) Ratio of mTEC vs cTECs in flow cytometric analysis of control (containing Cre-f/+ and f/f, n = 4) and Foxn1Cre::Stat3f/f mice (n = 5) at 12 weeks of age is shown.</p

Maturation of mTECs was not affected in Foxn1-Stat3-CKO.

<p>Cryostat sections of the thymus were stained with antibodies directed to K14 (green) and UEA1 (red) (A), and with antibodies to ERTR5 (red) and AIRE (green) (B). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

Ash1l Methylates Lys36 of Histone H3 Independently of Transcriptional Elongation to Counteract Polycomb Silencing

<div><p>Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, we found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES) cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR) to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. We also found that Brg1 is another key player involved in the response. Our results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing.</p></div

Skeletal phenotypes observed in progenies by intercrossing of heterozygotes.

<p>Skeletal phenotypes observed in progenies by intercrossing of heterozygotes.</p

A proposed role of Ash1l with RAR in the establishment of transcriptional activation.

<p>In the upper panel, Ash1l is preloaded in the promoter-proximal coding region with the condensed bivalent chromatin (thick and short gene body) that mainly generates immature short transcripts (represented in orange on the gene body). Enzymatic activity of Ash1l is inactivated under the condition (light-red). During the establishment of transcriptional activation, retinoic acid and its receptor (RA&RAR) promote activation of Ash1l (dark-red), as well as association of the other Lys36-methylases with the target chromatin. These Lys36-methylases, including Ash1l, orchestrate the downstream mechanisms directly or indirectly, thereby further promoting RA response through alleviating the repressive effect of the PRCs and opening the condensed chromatin (represented by the extended shape of the gene body in the bottom panel) independently of transcriptional elongation. The Brg1 complex may indirectly target Lys36me2/3 through Lys16ac.</p

mTECs are reduced in Foxn1-EGF-R-CKO mice.

<p>(A) Gross anatomical analysis of the thymus derived from Foxn1Cre::EGF-Rf/+ mice (control) and Foxn1Cre::EGF-Rf/f mice at 6 weeks of age. (B) Quantitative analysis for the proportion of medullary regions in thymus of control (containing Cre-f/+ and f/f, n = 4) and mutant (Cre-f/f, n = 5) mice. (C) Cryostat sections of the thymus were stained with ER-TR5 antibody (green) and anti-AIRE antibody (red). Sections were counterstained with DAPI (blue). Scale bars: 400 μm.</p

Lys36me2/3 and exclusion of the PRCs occur independently of productive transcriptional elongation.

<p>(<b>A</b>) Protocol for RA-induced differentiation of ES cells under DRB pretreatment. DRB was added to the culture medium 1 hour before the addition of RA, then the ES cells were cultured for another 16 hours in the presence of 100 nM RA. (<b>B</b>) Nuclear run-on assay in combination with RT-qPCR analyses of <i>Hoxd4</i> mRNA expression either with or without DRB pretreatment. The results are represented as values relative to <i>Gapdh</i> mRNA in each cell type. Error bars represent s.d. (Student's t-test, *P<0.05). ND: not detected. (<b>C</b> and <b>D</b>) ChIP assays of differentiating ES cells either with (+) or without (−) DRB pretreatment. A promoter-proximal coding region of <i>Hoxd4</i> was analyzed. The antibodies used are indicated above each graph. The results are represented as means and s.d. (Student's t-test, *P<0.05). Broken lines indicate approximate levels of ChIP signals in either <i>Il2ra</i> promoter (C) or <i>Gapdh</i> coding region (D) as controls.</p

Functional links of Ash1l to the Tip60, Mof, and Brg1 complexes.

<p>(<b>A</b>–<b>C</b> and <b>F</b>–<b>I</b>) ChIP assays of <i>Hoxd4</i> and <i>Gapdh</i> in differentiating ES cells either with (+) or without (−) DRB treatment. Regions that were analyzed were divided into two parts as indicated in each panel: promoter-proximal (<i>proximal</i>) and distal (<i>distal</i>) coding regions. The antibodies used are indicated above each graph or in panels. The results are represented as means and s.d. (Student's t-test, *P<0.05). Broken lines indicate approximate levels of ChIP signals in <i>Il2ra</i> promoter as a control. (<b>D</b>) A diagram of the <i>Gapdh</i> gene is shown. Black bars under the diagram indicate the regions analyzed by ChIP assays. (<b>E</b>) Whole-cell extracts were analyzed by immunoblot using the antibodies against the indicated histone modifications.</p

Basic characterization of Ash1l mutant ES cells and Hox gene expression in response to RA.

<p>(<b>A</b>) Schematic representation of the strategy used for targeted disruption of the <i>Ash1l</i> gene. Exons 11–12 encoding the AWS-SET domain with their flanking introns were floxed by loxP sequences. Cre-mediated germ-line recombination resulted in the generation of the ΔSET allele. (<b>B</b>) A PCR primer-pair of SET_flank_F/R was used to distinguish between expression from the wild-type allele and that from the ΔSET allele as shown in (A). A PCR primer-pair of Ab3_F/R was used to determine total expression from both alleles. The PCR primer-pairs are listed with their sequences in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003897#pgen.1003897.s015" target="_blank">Table S4</a>. (<b>C</b>) Comparison of <i>Ash1l</i> mRNA expression among E10.5-litter-mate embryos by conventional RT-PCR. Expression levels of <i>Gapdh</i> mRNA are shown as controls. (<b>D</b>) Protocol for RA-induced differentiation of ES cells. (<b>E</b>) Conventional RT-PCR analyses of <i>Hoxb4</i> and <i>Hoxd4</i> mRNA expression levels in response to various concentrations of RA. (<b>F</b> and <b>G</b>) Quantitative RT-PCR analysis of <i>Hoxd4</i> mRNA expression levels. RA-titration analysis after 48 hours of induction (F), and a time-course analysis using 1 nM RA (G). The results are represented as relative expression levels between wild-type and ΔSET ES cells. Wild-type cells (blue bars or line) and homozygous ΔSET ES cells (orange bars or line) are shown. These results represent means and standard deviations (s.d.) of three independent PCR reactions (Student's t-test, *P<0.05).</p