MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-gamma (peroxisome proliferator activated receptor-gamma) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII-TAK1-TAB2-NLK transcriptionally represses PPAR-gamma transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-gamma function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-gamma function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

Nuclear receptors comprise a large and expanding family of transcription factors involved in diverse aspects of animal physiology and development, the functions of which can be modulated in a spatial and temporal manner by access to small lipophilic ligands and/or the specificity of their own localized expression. Here we report the identification of a human nuclear receptor that reveals a unique proximal box (CNGCSG) in the DNA-binding domain. The conservation of this feature in its nematode counterpart suggests the requirement for this type of P box in the genetic cascades mediated by nuclear receptors in a wide variety of animal species. The expression of this receptor, PNR (photoreceptor-specific nuclear receptor), appears strongly restricted in the retina, exclusively in photoreceptor cells. In human cell lines, PNR expression was observed in Y79 retinoblastoma along with other photoreceptor marker genes such as CRX. Among vertebrate receptors, PNR shares structural kinship with an orphan receptor TLX, and despite distinct differences in the DNA binding domain, PNR is able to recognize a subset of TLX target sequences in vitro. Analyses of the human PNR gene revealed its chromosomal position as 15q24, a site that has recently been reported as a susceptible region for retinal degeneration. These data support a role for PNR in the regulation of signalling pathways intrinsic to the photoreceptor cell function.

TLX is an orphan nuclear receptor (also called NR2E1) that regulates the expression of target genes by functioning as a constitutive transrepressor. The physiological significance of TLX in the cytodifferentiation of neural cells in the brain is known. However, the corepressors supporting the transrepressive function of TLX have yet to be identified. In this report, Y79 retinoblastoma cells were subjected to biochemical techniques to purify proteins that interact with TLX, and we identified LSD1 (also called KDM1), which appears to form a complex with CoREST and histone deacetylase 1. LSD1 interacted with TLX directly through its SWIRM and amine oxidase domains. LSD1 potentiated the transrepressive function of TLX through its histone demethylase activity as determined by a luciferase assay using a genomically integrated reporter gene. LSD1 and TLX were recruited to a TLX-binding site in the PTEN gene promoter, accompanied by the demethylation of H3K4me2 and deacetylation of H3. Knockdown of either TLX or LSD1 derepressed expression of the endogenous PTEN gene and inhibited cell proliferation of Y79 cells. Thus, the present study suggests that LSD1 is a prime corepressor for TLX.Nuclear receptors (NRs) are transcriptional regulators that play pivotal roles in a variety of key metabolic and developmental processes (26). NRs constitute a gene superfamily, and NR protein structure is divided into several functional domains. One of the well-characterized domains is a highly conserved DNA-binding domain (DBD), and the other is a moderately conserved ligand-binding domain (LBD). The NR gene superfamily includes both steroid/thyroid hormone receptors and vitamin A/D receptors. The transcriptional function of NRs is regulated by the binding of specific ligands. In addition to the ligand-dependent NRs, there is a subfamily of so-called orphan NRs, the ligands of which have not yet been characterized (10). In the absence of ligand, orphan NRs constitutively activate or suppress transcription (20,49,53).Ligand-dependent transcriptional control by NRs requires a number of positive or negative coregulatory multiprotein complexes, in addition to basic transcription factors (25,35,36,52). These coregulator complexes can be classified into two groups. The first group comprises ATP-dependent chromatin-remodeling complexes (such as the SWI/SNF complex), which reorganize nucleosomal arrays and potentiate the promoter accessibility of NRs (6,18,23,33). The other group consists of histone modifier complexes, which covalently modify histone tails by acetylation, methylation, ubiquitination, or phosphorylation. These modifications lead to transcriptional repression or activation within the chromatin (2, 17, 42). Of these modifications, methylation of histone lysines is generally regarded as the most significant histone modification, as it triggers alterations in chromatin structure (27,37). Methylation of H3K9 leads to chromatin silencing, while H3K4 methylation enhances chromatin activity. A number of histone methyltransferases, e.g.,...

Lipofuscin contains fluorophores, which represent a biomarker for cellular aging. Although it remains unsubstantiated clinically, experimental results support that the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization due to age-related macular degeneration, a leading cause of legal blindness. Here, we report that a major lipofuscin component, A2E, activates the retinoic acid receptor (RAR). In vitro experiments using luciferase reporter assay, competitional binding assay, analysis of target genes, and chromatin immunoprecipitation (ChIP) assay strongly suggest that A2E is a bona fide ligand for RAR and induces sustained activation of RAR target genes. A2E-induced vascular endothelial growth factor (VEGF) expression in a human retinal pigment epithelial cell line (ARPE-19) and RAR antagonist blocked the up-regulation of VEGF. The conditioned medium of A2E-treated ARPE-19 cells induced tube formation in human umbilical vascular endothelial cells, which was blocked by the RAR antagonist and anti-VEGF antibody. These results suggest that A2E accumulation results in the phenotypic alteration of retinal pigment epithelial cells, predisposing the environment to choroidal neovascularization development. This is mediated through the agonistic function of A2E, at least in part. The results of this study provide a novel potential therapeutic target for this incurable condition.Retinal age pigments, or lipofuscin granula, contain the fluorophores that accumulate with age and that are thought to represent a biomarker for cellular aging (1). Lipofuscin results from an incomplete degradation of altered material trapped in lysosomes (2) and the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization (CNV) 2 due to age-related macular degeneration (AMD) (1, 2). AMD is a leading cause of legal blindness in developed countries (3), and even with the recent advent of several treatment options (4), treatment of AMD remains difficult (5). Thus, a better understanding of the pathogenesis is needed to pursue a novel potential pharmaceutical target. Visual loss in AMD is caused by CNV, i.e. the neovascular vessels extending from the choroid below the sensory retina, and the subsequent atrophy of the RPE. The process preceding AMD, early age-related maculopathy (4), is pathologically characterized by age-related changes in the RPE, such as the accumulation of a deposit called drusen in the basement membrane of the RPE, i.e. Bruch's membrane, and fluorescent lipofuscin granules in the RPE cells (6). It is generally considered that the age-related accumulation of these potentially toxic deposits affects normal RPE functions (4). Although it remains unsubstantiated whether the accumulation of lipofuscin is related to the development of exudative AMD, the fact that most abundant accretion is in the RPE cells under the central retina suggests that there may be a causal relationship between lipofuscin accumulation and exudative AMD (2). Laboratory studies also support...

Photoreceptor cell‐specific nuclear receptor (PNR) (NR2E3) acts as a sequence‐specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co‐repressor, Ret‐CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret‐CoR identified a multiprotein complex that included E2F/Myb‐associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex‐related components. Ret‐CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret‐CoR complex exhibited HDAC activity, co‐repressed PNR transrepression function in vitro, and co‐repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret‐CoR protein was cell‐cycle‐stage‐dependent (from G1 to S). Therefore, Ret‐CoR appears to act as a component of an HDAC co‐repressor complex that supports PNR repression function in the developing retina, and may represent a co‐regulator class that supports transcriptional regulator function via cell‐cycle‐dependent expression.

Abstract. Nuclear steroid/thyroid vitamin A/D receptor genes form a gene superfamily and encode DNA-binding transcription factors that control the transcription of target genes in a ligand-dependent manner. It has become clear that chromatin remodeling and the modification of histones, the main components of chromatin, play crucial roles in gene transcription, and many distinct classes of NR-interacting co-regulators have been identified that perform significant roles in gene transcription. Since NR dysfunction can lead to the onset or progression of endocrine disease, elucidation of the mechanisms of gene regulation mediated by NRs, as well as the identification and characterization of co-regulator complexes (especially chromatin remodeling and histone-modifying complexes), is essential not only for better understanding of NR ligand function, but also for pathophysiological studies and the development of therapeutic interventions in humans.

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF −/− mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF −/− neonates and ≈10% of WSTF +/− neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF −/− embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF −/− mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF −/− MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.