A novel Pax-6 binding site in rodent B1 repetitive elements: coevolution between developmental regulation and repeated elements?
Introduction
Pax-6 is a member of the Pax gene family, which was originally identified in Drosophila and has been found in most animals (Noll, 1993, Quiring et al., 1994, Strachan and Read, 1994, Stuart et al., 1994, Tremblay and Gruss, 1994). It encodes a transcription factor with two DNA-binding domains, the paired domain (PD) and the paired-type homeodomain (HD). It has a similarity of 100% between human and mouse, and 97% between human and zebrafish over the entire length of the Pax-6 protein. Pax-6 is initially expressed at the time of neural tube closure and is first detected at 8.5 days p.c. in the mouse. Pax-6 is well known for its role in initiating the cascade for eye development (Callaerts et al., 1997). It presumably initiates and/or amplifies the expression of a specific gene network required for eye development. Mutations in Pax-6 result in brain and eye abnormalities in human and mouse (Grindley et al., 1997, Hanson et al., 1994, Hill et al., 1991, Mastick et al., 1997, Schmahl et al., 1993, Stoykova and Gruss, 1994, Ton et al., 1991), thus demonstrating its importance in the cellular differentiation and organization of the central nervous system (CNS). Identification of genes that depend on Pax-6 for their transcription becomes an important approach to understanding the gene regulatory network involved in the development of the eye and CNS.
Like other transcription factors, Pax-6 regulates the expression of its target genes through direct contact with the target sites in the genes. It contains two DNA-binding domains, the paired domain (PD) and the paired-type homeodomain (HD), and a total of three helix–turn–helix motifs. Through various combinations of its DNA-binding motifs, Pax-6 may achieve specific and modular transcription regulation. Although it is likely that there are many target genes for Pax-6, few have been identified. In these few Pax-6 target genes, the sequences of the identified Pax-6 binding sites are quite different from the consensus Pax-6 PD binding sites (Epstein et al., 1994). The latter was determined by in-vitro selection of high-binding-affinity DNA fragments to the Pax-6 PD from a synthetic degenerate oligonucleotide pool. Since the natural and consensus Pax-6 binding sites identified so far are all relatively long, it is possible to isolate Pax-6 binding sequences from genomic DNA fragments by in-vitro experiments and identify the candidate Pax-6 target genes by database searching for genes containing the Pax-6 binding sites.
We are interested in using an in-vitro approach to isolate potential PAX6 binding sites in the human genome, thus predicting putative PAX6 target genes for further in-vivo studies. Through characterization of PAX6 binding sites, we are especially interested in searching for PAX6 binding sites that have a high similarity with repeated elements and in the evolutionary mechanisms of gene recruitment.
Section snippets
Construction of a human MboI single-copy DNA (sc-DNA) library
Human genomic DNA was isolated from human cell line K562. DNA (0.4 mg) was digested completely with MboI. Fragmented DNA was denatured and reassociated according to Saunders et al. (1972) with modifications. After digestion, DNA was denatured by boiling for 16 min, and reassociated at 65°C in 240 mM NaP. The sc-DNA was separated from repetitive DNA based on DNA reassociation rates. High-copy (hc) and middle-copy (mc) DNA were reassociated to about Cot 56 and separated from unassociated sc-DNA by
A novel PAX6 binding sequence
Our isolation of PAX6 binding sequences involved several rounds of screening, utilizing a procedure of cyclic amplification of protein binding DNA sequences (CAPBS). This process has been widely used for in-vitro PCR-based randomized selection of transcription factor binding sites. In our experiments, CAPBS was used to screen PAX6 binding sequences from human genomic DNA fragments. In order to identify the possible functional PAX6 binding sites, we screened PAX6 binding sequence from a human Mbo
Discussion
We have identified a new type of Pax-6 binding sequence that has a high similarity to a region in rodent B1 repetitive elements. Our sequence and phylogenetic analyses showed that such sequences might exist prior to the divergence of the rodent, but it became amplified with the amplification of B1 repeats in the rodent genome. Our in-vitro experimental data showed that the sequences in majority B1 repeats do not bind Pax-6, suggesting that they lost their Pax-6 binding ability before amplified
Acknowledgements
This work was supported in part by the National Research Service Award (NRSA) Fellowship 1F32 EY06949-1, NIH Grants EY09675, EY10608, GM57721, and GM55759; and the Texas Advanced Research Program (011618-061, 000015-046). We are indebted to Dr Hongmin Sun for providing her constructs of the human PD of PAX6 and the mouse PD of Pax-1, Pax-2, and Pax-3. We thank Dr Sheng Zhao for fruitful discussions and help in using GCG.
References (31)
- et al.
The characterization of novel Pax genes of the sea urchin and Drosophila reveal an ancient evolutionary origin of the Pax2/5/8 subfamily
Mech. Dev.
(1997) - et al.
Searching for patterns in genomic data
Trends Genet.
(1997) - et al.
Identification of a Pax paired domain recognition sequence and evidence for DNA-dependent conformational changes
J. Biol. Chem.
(1994) - et al.
PAX 8 regulates human WT1 transcription through a novel DNA binding site
J. Biol. Chem.
(1997) - et al.
Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon
Mech. Dev.
(1997) Evolution and role of Pax genes
Curr. Opin. Genet. Dev.
(1993)- et al.
Populations of repeated DNA sequences in the human genome
J. Mol. Biol.
(1972) - et al.
PAX genes
Curr. Opin. Genet. Dev.
(1994) - et al.
Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region
Cell
(1991) - et al.
Pax: genes for mice and men
Pharmacol. Ther.
(1994)
In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product
Development
DNA sequence insertion and evolutionary variation in gene regulation
Proc. Natl. Acad. Sci. USA
PAX-6 in development and evolution
Annu. Rev. Neurosci.
DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5)
Mol. Cell. Biol.
PHYLIP-phylogenetic inference package (version 3.2)
Cladistics
Cited by (21)
Pax6: A multi-level regulator of ocular development
2012, Progress in Retinal and Eye ResearchCitation Excerpt :These studies revealed novel binding sites that resemble the consensus rodent B1 repetitive element (Zhou et al., 2000) and the consensus primate Alu element (Zhou et al., 2002), both completely divergent from the P6CON site. These sequences were shown to be bound by Pax6 in vitro using EMSA (Zhou et al., 2000, 2002). The existence of Pax6-binding sites on short interspersed sequences (SINEs) such as Alu and B1 offers a possible evolutionary scenario in which Pax6 “took advantage” of retro-transposons to recruit new targets (Zhou et al., 2000).
Temporal regulation of Ath5 gene expression during eye development
2009, Developmental BiologyStructure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene
2008, Fish and Shellfish ImmunologyCitation Excerpt :The high similarity of above described domains and residues with that of other species suggested that the role of carp SLP-76 domains and residues may be similar to that of human and mice SLP-76s. Analysis of the transcription factor binding sites in carp SLP-76 gene promoter suggests that at least five potential transcription factors could regulate SLP-76 transcription: Pax-6, few of whose target genes have been identified including carp SLP-76 gene [47]; FOXJ2, a fork head transcriptional activator, whose function could be important for the very early phases of the embryonic development and some basic cellular function [48]; Bcd, which acts by stimulating spatially restricted transcription of the zygotic gap gene hunchback and other potential targets, including the head-specific genes orthodenticle, empty spiracles, and buttonhead and the gap gene Kriippel [49]; H4TF, a factor that binds the human histone H4 promoter, which also has been implicated as an important regulatory element in the promoters of human eosinophil peroxidase, and human pyruvate dehydrogenase-β [50]; JCV repeat, a sequence specific to human polyoma virus which is highly neuro-oncogenic in hamsters [51]. Except for Pax-6 which has been reported in zebrafish, the other four transcriptional factors have not been reported in teleosts yet.
Novel target sequences for Pax-6 in the brain-specific activating regions of the rat aldolase C gene
2002, Journal of Biological ChemistryCitation Excerpt :Binding sites for Pax-6 were found within its own promoter (49), in the promoters and enhancers of several crystalline genes (50), in the promoters of the genes encoding the transcription factors c-Maf and SIX3, also involved in lens development (51, 52), and in the promoter and enhancer of the glucagon, insulin, and somatostatin genes (14, 47). Other genes containing a Pax-6 binding site have been identified after selection of genomic sites (53), and potential lens target genes were selected recently using cDNA microarrays (54). For some of them, mutation of the Pax-6 binding site alters the functionality of their promoter/enhancer in transgenic mice (38, 55).
The paired homeodomain transcription factor Pax-2 is expressed in the endocrine pancreas and transactivates the glucagon gene promoter
2000, Journal of Biological ChemistryCitation Excerpt :We therefore conclude that Pax-2 transactivates the glucagon gene promoter but may have no role in insulin gene transcription. The functional role of Pax-2 in organogenesis has been extensively studied during kidney and brain development (15, 18, 20, 21, 23); however, its molecular role is still poorly characterized. Only recently, a few Pax-2 in vivo target genes have been described.