Keisuke Nimura

A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf–Hirschhorn syndrome

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals. In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription; however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf–Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf–Hirschhorn syndrome (WHS). We found that mouse Whsc1, one of five putative Set2 homologues, governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies.

PDGFRα-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia

The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin−/PDGFRα+) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin−/PDGFRα+ cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.

Essential Role for miR-196a in Brown Adipogenesis of White Fat Progenitor Cells

Brown adipocytes can differentiate from white fat progenitor cells in mice exposed to cold or β3-adrenergic stimulation, and this process is regulated by a microRNA that regulates the expression of Hoxc8, a master regulator of brown adipogenesis.

Rad51 siRNA delivered by HVJ envelope vector enhances the anti-cancer effect of cisplatin

Every cancer therapy appears to be transiently effective for cancer regression, but cancers gradually transform to be resistant to the therapy. Cancers also develop machineries to resist chemotherapy. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. Suppression of the machineries using siRNA may enhance the sensitivity to chemotherapy in cancers when combined with an effective delivery system.

Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling

Basic helix-loop-helix (bHLH) transcription factors are known as key regulators for mesenchymal differentiation. The present study showed that overexpression of Twist-1, a bHLH transcription factor, suppresses bone morphogenetic protein (BMP)-induced osteoblast differentiation, and downregulation of endogenous Twist-1 enhances BMP signaling. Maximal inhibition of BMP signaling was observed when Twist-1 was bound to E47, which markedly enhanced the stability of Twist-1. Co-immunoprecipitation assays revealed that Twist-1 formed a complex with Smad4 and histone deacetylase (HDAC) 1 in MC3T3-E1 cells stably expressing Twist-1. With trichostatin, an HDAC inhibitor, osteogenic factors such as alkaline phosphatase, Runx2 and osteopontin increased. Those results suggested that Twist-1 inhibited BMP signaling by recruiting HDAC1 to Smad4. Furthermore, the inhibitory effects of Twist-1 on BMP signaling were overcome by Id1 through induction of Twist-1 degradation. These findings suggest that Twist-1 can act as an inhibitor of BMP signaling, and Id1 can regulate BMP signaling through a positive feedback loop repressing Twist-1 function. These two molecules may therefore regulate differentiation of mesenchymal cells into progeny such as osteoblasts by controlling BMP signaling.

Bone Marrow Cell Transfer into Fetal Circulation Can Ameliorate Genetic Skin Diseases by Providing Fibroblasts to the Skin and Inducing Immune Tolerance

Recent studies have shown that skin injury recruits bone marrow-derived fibroblasts (BMDFs) to the site of injury to accelerate tissue repair. However, whether uninjured skin can recruit BMDFs to maintain skin homeostasis remains uncertain. Here, we investigated the appearance of BMDFs in normal mouse skin after embryonic bone marrow cell transplantation (E-BMT) with green fluorescent protein-transgenic bone marrow cells (GFP-BMCs) via the vitelline vein, which traverses the uterine wall and is connected to the fetal circulation. At 12 weeks of age, mice treated with E-BMT were observed to have successful engraftment of GFP-BMCs in hematopoietic tissues accompanied by induction of immune tolerance against GFP. We then investigated BMDFs in the skin of the same mice without prior injury and found that a significant number of BMDFs, which generate matrix proteins both in vitro and in vivo, were recruited and maintained after birth. Next, we performed E-BMT in a dystrophic epidermolysis bullosa mouse model (col7a1(-/-)) lacking type VII collagen in the cutaneous basement membrane zone. E-BMT significantly ameliorated the severity of the dystrophic epidermolysis bullosa phenotype in neonatal mice. Type VII collagen was deposited primarily in the follicular basement membrane zone in the vicinity of the BMDFs. Thus, gene therapy using E-BMT into the fetal circulation may offer a potential treatment option to ameliorate genetic skin diseases that are characterized by fibroblast dysfunction through the introduction of immune-tolerated BMDFs.

Sall4 Is Essential for Stabilization, But Not for Pluripotency, of Embryonic Stem Cells by Repressing Aberrant Trophectoderm Gene Expression

Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri‐implantation and that Sall4‐null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4‐null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder‐free Sall4‐null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1‐null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4‐null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression. STEM CELLS 2009;27:796–805

Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation

DNA methylation is involved in fundamental cellular processes such as silencing of genes and transposable elements, but the underlying mechanism of regulation of DNA methylation is largely unknown. DNA methyltransferase 3‐like protein (Dnmt3L), a member of the Dnmt3 family of proteins, is required during the establishment of DNA methylation patterns in germ cells. Dnmt3L does not possess enzymatic activity. Rather, in vitro analysis indicates that Dnmt3L stimulates DNA methylation by both Dnmt3a and Dnmt3b through direct binding to these proteins. In the current study, we demonstrated that in vivo, Dnmt3L physically and functionally interacted with the Dnmt3 isoform Dnmt3a2. In wild‐type embryonic stem (ES) cells, but not in cells lacking Dnmt3a, endogenous Dnmt3L was concentrated in chromatin foci. In ES cells deficient in both Dnmt3a and Dnmt3b, Dnmt3L was distributed diffusely throughout the nucleus and cytoplasm, and ectopic expression of Dnmt3a2, but not Dnmt3a or Dnmt3b, restored wild‐type Dnmt3L localization. We showed that endogenous Dnmt3L physically interacted with Dnmt3a2, but not Dnmt3a or Dnmt3b, in ES cells and embryonic testes. We also found that specific CpG sites were demethylated upon depletion of either Dnmt3a or Dnmt3L, but not Dnmt3b, in ES cells. These results provide evidence for a physical and functional interaction between Dnmt3L and Dnmt3a2 in the nucleus. We propose that Dnmt3a2 recruits Dnmt3L to chromatin, and induces regional DNA methylation in germ cells.

Histone methyltransferases: regulation of transcription and contribution to human disease

Histone modifications contribute to the precise regulation of transcription by recruiting non-histone proteins and controlling chromatin conformation. These covalent modifications are dynamically regulated by many enzymes that modify histones at specific residues in different ways. Histone modifiers contribute to development as well as cellular responses to extracellular stimuli. Mutations in the genes encoding them cause various diseases, including developmental disorders and certain malignancies. Haploinsufficiency for some histone methyltransferases, one of the principal modifiers of the histone modification network, are associated with particular congenital diseases, including Sotos syndrome, Wolf–Hirschhorn syndrome, and 9q syndrome. In this review, we discuss the molecular function of the histone methyltransferases and the human diseases associated with their dysfunction.

The Transcription Factors Tbx18 and Wt1 Control the Epicardial Epithelial-Mesenchymal Transition through Bi-Directional Regulation of Slug in Murine Primary Epicardial Cells

During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (Tbx18) and Wilms’ tumor 1 homolog (Wt1), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the Slug promoter region and regulated Slug expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.