Hitomi Matsuzaki

Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation

Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.

A Randomly Integrated Transgenic H19 Imprinting Control Region Acquires Methylation Imprinting Independently of Its Establishment in Germ Cells

ABSTRACT The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human β-globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the β-globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto, M. Shimotsuma, H. Matsuzaki, A. Omori, J. Bungert, J. D. Engel, and A. Fukamizu, Proc. Natl. Acad. Sci. USA 102:10250-10255, 2005). Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.

CTCF binding is not the epigenetic mark that establishes post-fertilization methylation imprinting in the transgenic H19 ICR

Imprinted expression of the mouse Igf2/H19 locus is controlled by parent-of-origin-specific methylation of the imprinting control region (ICR). We previously demonstrated that when placed in a heterologous genomic context, the H19 ICR fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells. In the present study, we investigated the requirements for the CTCF-binding sites of the ICR in the acquisition of post-fertilization methylation. To this end, two mutant ICR fragments were introduced into the human beta-globin locus in a yeast artificial chromosome transgenic mouse (TgM) model: 4xMut had mutations in all four ICR CTCF-binding sites that prevented CTCF binding but retained the methylation target CpG motifs, and -9CG harbored mutations in the CpG motifs within the CTCF-binding sites but each site retained constitutive CTCF-binding activity. In TgM germ cells and pre-implantation blastocysts, the absence of CTCF-binding sites (4xMut) did not lead to hypermethylation of the transgenic H19 ICR. However, after implantation, the mutations of CTCF sites (4xMut and -9CG) affected the maintenance of methylation. These results demonstrated that although the CTCF-binding sites are indispensable for maintenance of the unmethylated state of the maternal ICR in post-implantation embryos, they are not required to establish paternal-allele-specific methylation of the transgenic H19 ICR in pre-implantation embryos.

Sox-Oct motifs contribute to maintenance of the unmethylated H19 ICR in YAC transgenic mice

Abnormal methylation at the maternally inherited H19 imprinted control region (H19 ICR) is one of the causative alterations leading to pathogenesis of Beckwith-Wiedemann syndrome (BWS). Recently, it was shown in human BWS patients, as well as mouse cell culture experiments, that Sox-Oct motifs (SOM) in the H19 ICR might play a role in protecting the maternal ICR from de novo DNA methylation. By grafting a mouse H19 ICR fragment into a human β-globin yeast artificial chromosome (YAC) followed by analysis in transgenic mice (TgM), we showed previously that the fragment carried sufficient information to establish and maintain differential methylation after fertilization. To examine possible functions of the SOM in the establishment and/or maintenance of differential methylation, two kinds of YAC-TgM were generated in this study. In the ΔSOM TgM, carrying the mouse H19 ICR bearing an SOM deletion, a maternally inherited transgenic ICR exhibited increased levels of methylation around the deletion site, in comparison to the wild-type control, after implantation. In the λ + CTCF + b (LCb) TgM, carrying a 2.3 kb λ DNA fragment supplemented with the fragment b including the SOM and four CTCF binding sites, maternally and some of the paternally inherited LCb fragments were significantly less methylated when compared with a control λ + CTCF fragment that was supplemented only with additional CTCF sites; the λ + CTCF was substantially methylated regardless of the parent of origin after implantation. These results demonstrated that the SOM in the maternal H19 ICR was required for maintaining surrounding sequences in the unmethylated state in vivo.

De novo DNA methylation through the 5'-segment of the H19 ICR maintains its imprint during early embryogenesis.

Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here, we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal DNMT3A- and DNMT3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice and found that deletion of the 5′ segment of the endogenous paternal H19 ICR decreased its methylation after fertilization and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis. Highlighted article: In the mouse early embryo, H19 ICR imprinting is achieved through maternally inherited DNMT3A- and DNMT3L-mediated de novo methylation and requires a specific 5′ region of the locus.

Linear Distance from the Locus Control Region Determines ε-Globin Transcriptional Activity

ABSTRACT Enhancer elements modulate promoter activity over vast chromosomal distances, and mechanisms that ensure restrictive interactions between promoters and enhancers are critical for proper control of gene expression. The human β-globin locus control region (LCR) activates expression of five genes in erythroid cells, including the proximal embryonic ε- and the distal adult β-globin genes. To test for possible distance sensitivity of the genes to the LCR, we extended the distance between the LCR and genes by 2.3 kbp within the context of a yeast artificial chromosome, followed by the generation of transgenic mice (TgM). In these TgM lines, ε-globin gene expression decreased by 90%, while the more distantly located γ- or β-globin genes were not affected. Remarkably, introduction of a consensus EKLF binding site into the ε-globin promoter rendered its expression distance insensitive; when tested in an EKLF-null genetic background, expression of the mutant ε-globin gene was severely compromised. Thus, the ε-globin gene differs in its distance sensitivity to the LCR from the other β-like globin genes, which is, at least in part, determined by the transcription factor EKLF.

The H19 imprinting control region mediates preimplantation imprinted methylation of nearby sequences in yeast artificial chromosome transgenic mice.

ABSTRACT In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a λ DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited λ sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.

DNAse I hypersensitivity and ϵ-globin transcriptional enhancement are separable in LCR HS1 mutant human β-globin YAC transgenic mice

Expression of the five β-like globin genes (ϵ, Gγ, Aγ, δ, β) in the human β-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5′HS1 through 5′HS5). We report here a novel enhancer activity in 5′HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5′HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human β-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, ϵ-, but not γ-globin, gene expression in primitive erythroid cells was severely attenuated, while adult β-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5′HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5′HS1 is critical for efficient ϵ-globin gene expression, hypersensitive site formation per se is independent of 5′HS1 function, if any, in definitive erythroid cells.

DNase I Hypersensitivity and ϵ-Globin Transcriptional Enhancement Are Separable in Locus Control Region (LCR) HS1 Mutant Human β-Globin YAC Transgenic Mice

Expression of the five β-like globin genes (ϵ, Gγ, Aγ, δ, β) in the human β-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5′HS1 through 5′HS5). We report here a novel enhancer activity in 5′HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5′HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human β-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, ϵ-, but not γ-globin, gene expression in primitive erythroid cells was severely attenuated, while adult β-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5′HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5′HS1 is critical for efficient ϵ-globin gene expression, hypersensitive site formation per se is independent of 5′HS1 function, if any, in definitive erythroid cells.

All of the human β-type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

ABSTRACT In primitive erythroid cells of human β‐globin locus transgenic mice (TgM), the locus control region (LCR)‐proximal ε‐ and γ‐globin genes are transcribed, whereas the distal δ‐ and β‐globin genes are silent. It is generally accepted that the β‐globin gene is competitively suppressed by γ‐globin gene expression at this developmental stage. Previously, however, we observed that ε‐globin gene expression was severely attenuated when its distance from the LCR was extended, implying that β‐globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the β‐globin gene silencing mechanism, we established TgM lines carrying either γ‐ or ε‐ plus γ‐globin promoter deletions, without significantly altering the distance between the β‐globin gene and the LCR. Precocious expression of δ‐ and β‐globin genes was observed in primitive erythroid cells of mutant, but not wild‐type TgM, which was most evident when both the ε and γ promoters were deleted. Thus, we clearly demonstrated that the repression of the δ‐ and β‐globin genes in primitive erythroid cells is dominated by competitive silencing by the ε‐ and γ‐globin gene promoters, and that ε‐ and the other β‐like globin genes might be activated by two distinct mechanisms by the LCR.—Okamura, E., Matsuzaki, H., Campbell, A. D., Engel, J. D., Fukamizu, A., Tanimoto, K. All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice. FASEB J. 23, 4335‐4343 (2009). www.fasebj.org