Interrogating transcriptional regulatory sequences in Tol2-mediated Xenopus transgenics.

Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate in silico predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative cis-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in Xenopus laevis to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the Xenopus laevis tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ~200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific cis-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in Xenopus.

Genomic organization and imprinting of the Peg3 domain in bovine.

Using multiple mammalian genomic sequences, we have analyzed the evolution and imprinting of several genes located in the Peg3 domain, including Mim1 (approved name, Mimt1), Usp29, Zim3, and Zfp264. A series of comparative analyses shows that the overall genomic structure of this 500-kb imprinted domain has been well maintained throughout mammalian evolution but that several lineage-specific changes have also occurred in each species. In the bovine domain, Usp29 has lost its protein-coding capability, Zim3 has been duplicated, and the expression of Zfp264 has become biallelic in brain and testis, which differs from paternal expression of mouse Zfp264 in brain. In contrast, the two transcript genes of cow, Mim1 and Usp29, both lacking protein-coding capability, are still expressed mainly from the paternal allele, indicating the imprinting of these two genes in cow. The imprinting of Mim1 and Usp29 along with Peg3 is the most evolutionarily selected feature in this imprinted domain, suggesting significant function of these three genes, either as protein-coding or as untranslated transcript genes.

Identification of clustered YY1 binding sites in imprinting control regions.

Mammalian genomic imprinting is regulated by imprinting control regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In this study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.

Lineage-specific imprinting and evolution of the zinc-finger gene ZIM2.

We have carried out an in-depth comparative analysis of a 100-kb genomic interval containing two imprinted genes, PEG3 and ZIM2, using sequences derived from human, mouse, and cow. In all three mammals, ZIM2 is located at a similar genomic distance and in the same orientation relative to PEG3, indicating the basic structural conservation of this imprinted locus. However, several lineage-specific changes have occurred that affect the exon structure and imprinting status of ZIM2. Human ZIM2 and PEG3 share a set of 5' exons and a common promoter, and both genes are paternally expressed. In contrast, mouse and cow Zim2 genes do not share 5' exons with Peg3, and Zim2 employs a separate downstream promoter in both species. The imprinting status of Zim2 is also not conserved among mammals; mouse Zim2 is expressed biallelically in testis but predominantly from the maternal allele in brain, while cow Zim2 is expressed biallelically in testis. The separate transcription of Zim2 and Peg3 and the change in promoter usage and imprinting status appear to have resulted from independent insertional events that have placed unrelated genes, Zim1 and Ast1, respectively, between Zim2 and Peg3 in mouse and cow. Our results suggest that PEG3 and ZIM2 represent the two original genes at this locus and that rearrangements have occurred independently in different mammalian lineages in recent evolutionary times. Our data also suggest that exon-sharing of human PEG3 and ZIM2 was not ancestral, but may represent a fusion event joining the two neighboring genes and bringing ZIM2 under paternal expression control. These observations are striking in light of the structural and functional conservation that typifies other imprinted domains and suggest that the PEG3/ZIM2 imprinted domain may have evolved in an unusual lineage-specific pattern.

Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3.

The 5'-ends of two paternally expressed mouse genes, Peg3 and Usp29, are jointly associated with a CpG island that exhibits allele-specific methylation. Sequence comparison of the regions derived from human, mouse and cow revealed the presence of two evolutionarily conserved sequence motifs including one that is repeated multiple times within the first intron of Peg3 in all three mammals. DNA mobility shift and chromatin immunoprecipitation (ChIP) assays clearly demonstrated that this motif is an in vivo binding site for the Gli-type transcription factor YY1. The YY1-binding site contains one CpG dinucleotide, and methylation of this CpG site abolishes the binding activity of YY1 in vitro. The Peg3 YY1-binding sites are methylated only on the maternal chromosome in vivo, and ChIP assays confirmed that YY1 binds specifically to the paternal allele of the gene. Promoter, enhancer and insulator assays with deletion constructs of sequence surrounding the YY1-binding sites indicate that the region functions as a methylation-sensitive insulator that may influence the imprinted expression of Peg3 and neighboring genes. The current study is the first report demonstrating the involvement of YY1 in methylation-sensitive insulator activity and suggests a potential role of this highly conserved protein in mammalian genomic imprinting.