Differential recognition of response elements determines target gene specificity for p53 and p63

p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63+/+ mouse, it is undetectable in these tissues in the p63-/- mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.     

Comparative genomic analysis of allatostatin-encoding (Ast) genes in Drosophila species and prediction of regulatory elements by phylogenetic footprinting

The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3'-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.     

Ubiquitous nuclear factors bind specifically to a 5'-region conserved in carcinoembryonic antigen-related genes.

We recently cloned members of the murine carcinoembryonic antigen (CEA) gene family, some of which are differentially expressed during placental development. By intra- and interspecies sequence comparisons, we identified an element in the putative promoter and/or 5'-nontranslated region which is conserved within all human and rodent CEA-related genes analyzed so far. Using gel retardation analysis and DNaseI hypersensitive site mapping, we now show that ubiquitously expressed nuclear factors specifically bind to the conserved region derived from the mouse gene Cea-2 in vitro and probably also in vivo. Another DNaseI hypersensitive site lies within or close to a simple sequence motif [(GGA)n] located in the first intron of Cea-2. Such sequences have been reported to play a role in the regulation of certain genes. Therefore, this analysis has identified putative regulatory regions for Cea-2 and possibly CEA-related genes in general.     

Isolation, characterization, and mapping of the mouse and human WDR8 genes, members of a novel WD-repeat gene family

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family.     

斑马鱼肌肉高表达基因近端非编码元件分析及功能检测

为鉴定鱼类肌肉组织特异性顺式调控元件,通过分析斑马鱼多个组织的转录组数据,筛选出肌肉高表达基因及低表达基因.通过MEME对肌肉高表达基因和低表达基因非编码区序列特征进行分析,在5个肌肉高表达基因的转录起始位点上游发现了序列保守的DNA区域,包含6个排列顺序一致的DNA基序.将其中一段目标片段插入具有Tol2转座子元件的...     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum,a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

Riboswitches that sense S-adenosylhomocysteine and activate genes involved in coenzyme recycling

We have identified a highly conserved RNA motif that occurs upstream of genes involved in S-adenosyl-L-methionine (SAM) recycling in many Gram-positive and Gram-negative species of bacteria. The phylogenetic distribution and the conserved structural features of representatives of this motif are indicative of riboswitch function. Riboswitches are widespread metabolite-sensing gene control elements that are typically found in the 5' untranslated regions (UTRs) of bacterial mRNAs. We experimentally verified that examples of this RNA motif specifically recognize S-adenosylhomocysteine (SAH) in protein-free in vitro assays, and confirmed that these RNAs strongly discriminate against SAM and other closely related analogs. A representative SAH motif was found to activate expression of a downstream gene in vivo when the metabolite is bound. These observations confirm that SAH motif RNAs are distinct ligand-binding aptamers for a riboswitch class that selectively binds SAH and controls genes essential for recycling expended SAM coenzymes.     

Evolution of Conserved Non-Coding Sequences Within the Vertebrate Hox Clusters Through the Two-Round Whole Genome Duplications Revealed by Phylogenetic Footprinting Analysis

As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications.     

Characterization of the human patatin-like phospholipase family

Several publications have described biological roles for human patatin-like phospholipases (PNPLAs) in the regulation of adipocyte differentiation. Here, we report on the characterization and expression profiling of 10 human PNPLAs. A variety of bioinformatics approaches were used to identify and characterize all PNPLAs encoded by the human genome. The genes described represent a divergent family, most with a highly conserved ortholog in several mammalian species. In silico characterization predicts that two of the genes function as integral membrane proteins and are regulated by cAMP/cGMP. A structurally guided protein alignment of the patatin-like domain identifies a number of conserved residues in all family members. Quantitative PCR was used to determine the expression profile of each family member. Affymetrix-based profiling of a human preadipocyte cell line identified several members that are differentially regulated during cell differentiation. Cumulative data suggest that patatin-like genes normally expressed at very low levels are induced in response to environmental signals. Given the observed conservation of the patatin fold and lipase motif in all human PNPLAs, a single nomenclature to describe the PNPLA family is proposed.