Elsevier

Gene

Volume 245, Issue 2, 21 March 2000, Pages 319-328
Gene

A novel Pax-6 binding site in rodent B1 repetitive elements: coevolution between developmental regulation and repeated elements?

https://doi.org/10.1016/S0378-1119(00)00019-6Get rights and content

Abstract

Pax-6 encodes a transcription factor that is important in the development of eye and CNS. Identification of Pax-6 target genes is crucial for understanding the gene regulatory network in these developmental processes. Using an in-vitro approach of cyclic amplification of the protein binding sequences (CAPBS), we isolated a PAX6 binding sequence from a human single-copy (sc) DNA library. Characterization of this PAX6 binding sequence revealed a 15 bp region (hGCα1BLs5) that is sufficient for PAX6 specific binding. From a homology search in the GenBank, we found that an hGCα1BLs5-like Pax-6 binding site exists in 21 genes (16 from rodent), 15 of which were shown to be able to bind Pax-6 in vitro. Interestingly, some of these sites occur in B1 repetitive elements. Although hGCα1BLs5 is highly similar to a region in B1 repetitive elements, PAX6 does not bind to the consensus sequence in B1. However, a single-step mutation in some B1 elements can lead to a gain of function for PAX6 binding. This experimental evidence and phylogenetic analysis raise an interesting speculation for the coevolution between PAX6 regulation and repeat elements. Since a (Pax-6-binding) null B1 element can be re-activated by even a single-step mutation, it has the potential to recruit gene targets for Pax-6 if it is inserted into the regulatory region, and therefore may play a role for evolutionary modification of Pax-6 regulation.

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.

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