Thyroid hormone directly activates mitochondrial fission process 1 (Mtfp1) gene transcription during adult intestinal stem cell development and proliferation in Xenopus tropicalis.

Thyroid hormone (T3) regulates vertebrate development via T3 receptors (TRs). T3 level peaks during postembryonic development, a period around birth in mammals or metamorphosis in anurans. Anuran metamorphosis offers many advantages for studying T3 and TR function in vivo largely because of its total dependent on T3 and the dramatic changes affecting essentially all organs/tissues that can be easily manipulated. Earlier studies have shown that TRs are both necessary and sufficient for mediating the metamorphic effects of T3. Many candidate TR target genes have been identified during Xenopus tropicalis intestinal metamorphosis, a process that involves apoptotic degeneration of most of the larval epithelial cells and de novo development of adult epithelial stem cells. Among these putative TR target genes is mitochondrial fission process 1 (Mtfp1), a nuclear-encoded mitochondrial gene. Here, we report that Mtfp1gene expression peaks in the intestine during both natural and T3-induced metamorphosis when adult epithelial stem cell development and proliferation take place. Furthermore, we show that Mtfp1 contains a T3-response element within the first intron that is bound by TR to mediate T3-induced local histone H3K79 methylation and RNA polymerase recruitment in the intestine during metamorphosis. Additionally, we demonstrate that the Mtfp1 promoter can be activated by T3 in a reconstituted frog oocyte system in vivo and that this activation is dependent on the intronic TRE. These findings suggest that T3 activates Mtfp1 gene directly via the intronic TRE and that Mtfp1 in turn facilitate adult intestinal stem cell development/proliferation by affecting mitochondrial fission process.

Cell cycle activation in thyroid hormone-induced apoptosis and stem cell development during Xenopus intestinal metamorphosis.

Amphibian metamorphosis resembles mammalian postembryonic development, a period around birth when many organs mature into their adult forms and when plasma thyroid hormone (T3) concentration peaks. T3 plays a causative role for amphibian metamorphosis. This and its independence from maternal influence make metamorphosis of amphibians, particularly anurans such as pseudo-tetraploid Xenopus laevis and its highly related diploid species Xenopus tropicalis, an excellent model to investigate how T3 regulates adult organ development. Studies on intestinal remodeling, a process that involves degeneration of larval epithelium via apoptosis and de novo formation of adult stem cells followed by their proliferation and differentiation to form the adult epithelium, have revealed important molecular insights on T3 regulation of cell fate during development. Here, we review some evidence suggesting that T3-induced activation of cell cycle program is important for T3-induced larval epithelial cell death and de novo formation of adult intestinal stem cells.

Molecular mechanism of Jmjd3-mediated interleukin-6 gene regulation in endothelial cells underlying spinal cord injury.

The inflammatory response contributes substantially to secondary injury cascades after spinal cord injury, with both neurotoxic and protective effects. However, epigenetic regulations of inflammatory genes following spinal cord injury have yet to be characterized thoroughly. In this study, we found that histone H3K27me3 demethylase Jmjd3 expression is acutely up-regulated in blood vessels of the injured spinal cord. We also observed up-regulation of Jmjd3 gene expression in bEnd.3 endothelial cells that were subjected to oxygen-glucose deprivation/reperfusion injury. When Jmjd3 was depleted by siRNA, oxygen-glucose deprivation/reperfusion injury-induced up-regulation of IL-6 was significantly inhibited. In addition, Jmjd3 associated with NF-κB (p65/p50) and CCAAT-enhancer-binding protein ß at the IL-6 gene promoter. The recruitment of Jmjd3 coincided with decreased levels of tri-methylated H3K27 as well as increased levels of mono-methylated H3K27 at the IL-6 gene promoter. Furthermore, Jmjd3 depletion did not result in significant changes of methylation level of H3K27 at the IL-6 gene promoter. Collectively, our findings imply that Jmjd3-mediated H3K27me3 demethylation is crucial for IL-6 gene activation in endothelial cells, and this molecular event may regulate acute inflammatory response and integrity of the blood-spinal cord barrier following spinal cord injury.

Regulation of XFGF8 gene expression through SRY (sex-determining region Y)-box 2 in developing Xenopus embryos.

Fibroblast growth factors (FGFs) function as mitogens and morphogens during vertebrate development. In the present study, to characterise the regulatory mechanism of FGF8 gene expression in developing Xenopus embryos the upstream region of the Xenopus FGF8 (XFGF8) gene was isolated. The upstream region of the XFGF8 gene contains two putative binding sites for the SRY (sex-determining region Y)-box 2 (SOX2) transcription factor. A reporter assay with serially deleted constructs revealed that the putative SOX2-binding motif may be a critical cis-element for XFGF8 gene activation in developing Xenopus embryos. Furthermore, Xenopus SOX2 (XSOX2) physically interacted with the SOX2-binding motif within the upstream region of the XFGF8 gene in vitro and in vivo. Depletion of endogenous XSOX2 resulted in loss of XFGF8 gene expression in midbrain-hindbrain junction, auditory placode, lens placode and forebrain in developing Xenopus embryos. Collectively, our results suggest that XSOX2 directly upregulates XFGF8 gene expression in the early embryonic development of Xenopus.

Direct activation of tRNA methyltransferase-like 1 (Mettl1) gene by thyroid hormone receptor implicates a role in adult intestinal stem cell development and proliferation during Xenopus tropicalis metamorphosis.

BACKGROUND: Thyroid hormone (T3) plays an important role in vertebrate development. Compared to the postembryonic development of uterus-enclosed mammalian embryos, T3-dependent amphibian metamorphosis is advantageous for studying the function of T3 and T3 receptors (TRs) during vertebrate development. The effects of T3 on the metamorphosis of anurans such as Xenopus tropicalis is known to be mediated by TRs. Many putative TR target genes have been identified previously. Among them is the tRNA methyltransferase Mettl1. RESULTS: We studied the regulation of Mettl1 gene by T3 during intestinal metamorphosis, a process involves near complete degeneration of the larval epithelial cells via apoptosis and de novo formation of adult epithelial stem cells and their subsequent proliferation and differentiation. We observed that Mettl1 was activated by T3 in the intestine during both natural and T3-induced metamorphosis and that its mRNA level peaks at the climax of intestinal remodeling. We further showed that Mettl1 promoter could be activated by liganded TR via a T3 response element upstream of the transcription start site in vivo. More importantly, we found that TR binding to the TRE region correlated with the increase in the level of H3K79 methylation, a transcription activation histone mark, and the recruitment of RNA polymerase II by T3 during metamorphosis. CONCLUSIONS: Our findings suggest that Mettl1 is activated by liganded TR directly at the transcriptional level via the TRE in the promoter region in the intestine during metamorphosis. Mettl1 in turn regulate target tRNAs to affect translation, thus facilitating stem cell formation and/or proliferation during intestinal remodeling.

Thyroid hormone activates Xenopus MBD3 gene via an intronic TRE in vivo.

Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and can be easily manipulated, thus offering a unique opportunity for studying how T3 controls vertebrate development. T3 controls frog metamorphosis through T3 receptor (TR)-mediated regulation of T3 response genes. To identify direct T3 response genes, we previously carried out a ChIP (chromatin immunoprecipitation)-on-chip analysis with a polyclonal anti-TR antibody on the tadpole intestine and identified many putative TR target genes. Among them is the methyl-CpG binding domain protein 3 (MBD3) gene, which has been implicated to play a role in epigenetic regulation of cellular processes as a subunit of the Mi-2/NuRD (Nucleosome Remodeling Deacetylase) complex. We show here that MBD3 is upregulated in the intestine and tail by T3 and its expression peaks at stage 62, the climax of metamorphosis. We further show that a putative TRE within the first intron of the MBD3 gene binds to TR/RXR in vitro and in vivo, and mediates T3 regulation of the MBD3 promoter in vivo.

Dexamethasone suppresses JMJD3 gene activation via a putative negative glucocorticoid response element and maintains integrity of tight junctions in brain microvascular endothelial cells.

The blood-brain barrier (BBB) exhibits a highly selective permeability to support the homeostasis of the central nervous system (CNS). The tight junctions in the BBB microvascular endothelial cells seal the paracellular space to prevent diffusion. Thus, disruption of tight junctions results in harmful effects in CNS diseases and injuries. It has recently been demonstrated that glucocorticoids have beneficial effects on maintaining tight junctions in both in vitro cell and in vivo animal models. In the present study, we found that dexamethasone suppresses the expression of JMJD3, a histone H3K27 demethylase, via the recruitment of glucocorticoid receptor α (GRα) and nuclear receptor co-repressor (N-CoR) to the negative glucocorticoid response element (nGRE) in the upstream region of JMJD3 gene in brain microvascular endothelial cells subjected to TNFα treatment. The decreased JMJD3 gene expression resulted in the suppression of MMP-2, MMP-3, and MMP-9 gene activation. Dexamethasone also activated the expression of the claudin 5 and occludin genes. Collectively, dexamethasone attenuated the disruption of the tight junctions in the brain microvascular endothelial cells subjected to TNFα treatment. Therefore, glucocorticoids may help to preserve the integrity of the tight junctions in the BBB via transcriptional and post-translational regulation following CNS diseases and injuries.

Histone H3K27 Demethylase JMJD3 in Cooperation with NF-κB Regulates Keratinocyte Wound Healing.

Histone H3K27me3 demethylase JMJD3 has been shown to be involved in keratinocyte differentiation and wound healing. However, the exact molecular mechanism underlying JMJD3-mediated keratinocyte wound healing has not been fully elucidated. In this study, we report on the biological function of JMJD3 in keratinocyte wound healing using in vitro cell and in vivo animal models. Our results indicate that JMJD3 up-regulation and NF-κB activation occur in the region of the wound edge during keratinocyte wound healing. We also found that JMJD3 interacts with NF-κB, resulting in increased expression of the inflammatory, matrix metalloproteinase, and growth factor genes via demethylation of H3K27me3 at the gene promoters. Consistently, inactivation of JMJD3 or NF-κB resulted in aberrant keratinocyte wound healing. Our study suggests that regulation of JMJD3 may provide a new therapeutic intervention for treating the chronic skin wound.

17ß-Estradiol Ameliorates Tight Junction Disruption via Repression of MMP Transcription.

The blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) formed by capillary endothelial cells provides a physical wall between the central nervous system (CNS) and circulating blood with highly selective permeability. BBB/BSCB disruption by activation of matrix metalloproteinases (MMPs) has been shown to result in further neurological damage after CNS injury. Recently it has been discovered that estrogen attenuates BBB/BSCB disruption in in vitro and in vivo models. However, the molecular mechanism underlying the estrogen-mediated attenuation of BBB/BSCB disruption has not been elucidated fully. In the present study, we found that 17ß-estradiol (E2) suppresses nuclear factor-κB-dependent MMP-1b, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 gene activation in microvessel endothelial bEnd.3 cells subjected to oxygen and glucose deprivation/reperfusion injury. E2 induced the recruitment of ERα and nuclear receptor corepressor to the nuclear factor-κB binding site on the MMPs' gene promoters. Consistently, ER antagonist ICI 182.780 showed opposite effects of E2. We further found that E2 attenuates tight junction disruption through the decreased degradation of tight junction proteins in bEnd.3 cells subjected to oxygen and glucose deprivation-reperfusion injury. In addition, E2 suppressed the up-regulation of MMP expression, leading to a decreased BSCB disruption in the injured spinal cord. In conclusion, we discovered the molecular mechanism underlying the protective role of estrogenin BBB/BSCB disruption using an in vitro and in vivo model. Our study suggests that estrogens may provide a potential therapeutic intervention for preserving BBB/BSCB integrity after CNS injury.

Regulation of DU145 prostate cancer cell growth by Scm-like with four mbt domains 2.

Mammalian SFMBTs have been considered to be polycomb group repressors. However, molecular mechanisms underlying mammalian SFMBTs-mediated gene regulation and their biological function have not been characterized. In the present study, we identified YY1 and methylated histones as interacting proteins of human SFMBT2. We also found that human SFMBT2 binds preferentially to methylated histone H3 and H4 that are associated with transcriptional repression. Using DU145 prostate cancer cells as a model, we showed that SFMBT2 has a transcriptional repression activity on HOXB13 gene expression. In addition, occupancy of SFMBT2 coincided with enrichment of diand tri-methylated H3K9 and H4K20 as well as tri-methylated H3K27 at the HOXB13 gene promoter. When SFMBT2 was depleted by siRNA in DU145 prostate cancer cells, significant up-regulation of HOXB13 gene expression and decreased cell growth were observed. Collectively, our findings indicate that human SFMBT2 may regulate cell growth via epigenetic regulation of HOXB13 gene expression in DU145 prostate cancer cells.

Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5) as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.