Elsevier

Genomics

Volume 86, Issue 3, September 2005, Pages 316-328
Genomics

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

https://doi.org/10.1016/j.ygeno.2005.05.006Get rights and content

Abstract

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression.

Section snippets

Intronic sequence conservation between human and mouse BRCA1 identifies nine potential regulatory regions

As an initial approach to identifying novel sequence elements involved in regulation of BRCA1 expression, we performed a comparative analysis of the human and mouse BRCA1 genomic sequences. Based on the observation that these elements are usually composed of long sequences (≥100 bp in length) and are highly conserved (≥70% identity) [14], nine CNS matching these criteria were identified in six different introns of the BRCA1 gene (unpublished data). A BLAST search of the conserved sequences

Discussion

Comparative genomics offers a potential strategy for identifying important regulatory elements on the basis that evolutionary conservation implies functional constraints. In this study, we utilized human–mouse genomic comparisons to identify potential noncoding regulatory elements in the BRCA1 gene. The evolutionary distance been humans and mice is reported to be sufficient for identification of functionally important elements [48]. This premise is supported by studies identifying functional

Cell culture

Cervical adenocarcinoma HeLa cells (ATCC CCL-2) and SV40 T-antigen-immortalized human mammary epithelial SVCT cells (ECACC 94122105) were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Invitrogen). SVCT growth medium was also supplemented with insulin (Sigma–Aldrich Pty. Ltd., Sydney, Australia; 10 μg/ml) and hydrocortisone (Sigma–Aldrich; 0.5 μg/ml). Cells were maintained at 37°C in a 5% CO2 humidified incubator.

Comparative sequence analysis

BRCA1 genomic DNA from the following species

Acknowledgments

The authors are grateful to Panayiotis Ioannou (Murdoch Institute, Melbourne) for the pGETrec plasmid, the ICRF (Cancer Research UK) for the use of the SVCT cell line, Matthew Wakefield (ANU, Canberra) for the marsupial genomic DNA, Sylvie Mayozer (IARC, Lyon) for primers and help with mapping BRCA1 deletions, Georgia Chenevix-Trench and Aaron Lewis (QIMR, Brisbane) for help with mutation analysis, and Ibtissam Abdul Jabbar (UQ, Brisbane) for MoFlo FACS. The authors thank the kConFab research

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