Development of a rod photoreceptor mosaic revealed in transgenic zebrafish

The number and distribution of neurons within the vertebrate retina are tightly regulated. This is particularly apparent in the highly ordered, crystalline-like arrangement of the cone photoreceptors in the teleost. In this report, using a transgenic line of zebrafish, a novel and developmentally regulated mosaic pattern of the rod photoreceptors is described. The spatial and temporal expression of EGFP, under the control of the Xenopus rhodopsin gene promoter, was nearly identical to the endogenous rhodopsin. EGFP was first detected in the ventral nasal retinal in an area of precocious neurogenesis referred to as the "ventral patch". Subsequent expression of EGFP was observed in isolated cells sporadically distributed across the dorsal and central retina. However, confocal microscopy and spatial analysis of larval eyes or retinal explants from adults revealed a precise arrangement to the rod photoreceptors. The rod terminals were arranged in regularly spaced rows with clearly identifiable telodendria linking neighboring cells. The rod inner segments projected through the cone mosaic in a predictable pattern. In the adult, the rod mosaic originated near the retinal margin where clusters of rods differentiated around the immature short single cone. In the embryo, the sporadic differentiation of the rods led to the gradual formation of the mosaic pattern. With the growing interest in neuronal stem cells, revisiting this model of neurogenesis provides an avenue to uncover mechanisms underlying the precise integration of new neuronal elements into a preexisting neural network.     

Developmentally Regulated Linker Histone H1c Promotes Heterochromatin Condensation and Mediates Structural Integrity of Rod Photoreceptors in Mouse Retina

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1⁰ triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.     

组蛋白与hAMFR基因启动子的结合对体外转录活性的影响

采用从鸡血红细胞中分离纯化的组蛋白,从ＨｅＬａ细胞核中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性抽提物,以及从非洲爪蟾卵细胞核中撮以的萃取物热处理物上清用于核小体构建,以含有人自泌移动因子受体基因启动子序列的ＤＮＡ片段为模板进行体外转录实验,结果表明,组蛋白和转录因子在ｈＡＭＦＲ基因启动子序列上的竞争性结合对转录活性具有重要的影响作用,在启动子区域预先构建的核小体能够抑制转录活性;     

Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell-Specific Expression in Transgenic Mice

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.     

An Adenoviral Vector-Based System to Study Neuronal Gene Expression: Analysis of the Rat Tyrosine Hydroxylase Promoter in Cultured Neurons

Abstract: We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.     

高山离子芥CBF/DREB1转录因子基因的分离与表达

以冷胁迫和脱水处理的高山离子芥幼叶为材料,采用RT-PCR技术获得1条新的C重复/脱水应答元件结合因子(CBF/DREB1)基因(CbCBF,登录号AY994127).该基因含651 bp的开放阅读框(ORF),编码216个氨基酸的蛋白质,预测该蛋白具有一个AP2 DAN结合域,一个核定位信号和碳端酸性激活域.多序列比对结果表明,CbCBF蛋白与拟南芥及其他植物CBF具有较高的相似性.Northern杂交结果显示,CbCBF基因不能被冷处理诱导表达,但可被脱水和ABA处理快速诱导;同时发现CbCBF基因也能被紫外辐射和机械刺激诱导表达.表明高山离子芥CbCBF基因可能参与应答多种非生物胁迫过程.     

小麦胁迫相关基因W1的克隆及表达模式分析

应用噬菌体原位杂交技术从干旱胁迫诱导的小麦cDNA文库中克隆到一个胁迫诱导的基因片段别。删全长cDNA为901bp,其中,编码区长498bp,编码166个氨基酸。Southern杂交表明,W1是一个低拷贝基因。RT—PCR结果表明,W1受干旱、低温的诱导,但不受高盐的诱导。氨基酸序列分析发现W1有一个USP保守区（pfam00582）。同源性分析发现W1与一个水稻胁迫诱导蛋白（NM_001061239）的同源性为83％,但该类蛋白的功能尚无报道。肼是小麦第1个被克隆的胁迫相关蛋白基因,该基因的克隆有助于阐明小麦的抗逆机制,并为今后培育抗逆性小麦品种提供候选基因。     

Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells

HO-1 (heme oxygenase-1) is an inducible microsomal enzyme that catalyzes the degradation of pro-oxidant heme. The goal of this study was to characterize a minimal enhancer region within the human HO-1 gene and delineate its role in modulating HO-1 expression by participation with its promoter elements in renal epithelial cells. Deletion analysis and site-directed mutagenesis identified a 220-bp minimal enhancer in intron 1 of the HO-1 gene, which regulates hemin-mediated HO-1 gene expression. Small interfering RNA, decoy oligonucleotides, site-directed mutagenesis, and chromatin immunoprecipitation assays confirmed the functional interaction of Sp1 with a consensus binding sequence within the 220-bp region. Mutations of regulatory elements within the −4.5 kb promoter region (a cyclic AMP response and a downstream NF-E2/AP-1 element, both located at −4.0 kb, and/or an E-box sequence located at −44 bp) resulted in the loss of enhancer activity. A chromosome conformation capture assay performed in human renal epithelial (HK-2) cells demonstrated hemin-inducible chromatin looping between the intronic enhancer and the −4.0 kb promoter region in a time-dependent manner. Restriction digestion with ApaLI (which cleaves the 220-bp enhancer) led to a loss of stimulus-dependent chromatin looping. Sp1 small interfering RNA and mithramycin A, a Sp1 binding site inhibitor, resulted in loss of the loop formation between the intronic enhancer and the distal HO-1 promoter by the chromosome conformation capture assay. These results provide novel insight into the complex molecular interactions that underlie human HO-1 regulation in renal epithelial cells.