Identification of Novel Binding Elements and Gene Targets for the Homeodomain Protein BARX2

Abstract

BARX2 is a homeobox transcription factor that influences cellular differentiation in various developmental contexts. To begin to identify the gene targets that mediate its effects, chromatin immunoprecipitation (ChIP) was used to isolate BARX2 binding sites from the human MCF7 breast cancer cell line. Cloning and sequencing of BARX2-ChIP-derived DNA fragments identified 60 potential BARX2 target loci that were proximal to or within introns of genes involved in cytoskeletal organization, cell adhesion, growth factor signaling, transcriptional regulation, and RNA metabolism. The sequences of over half of the fragments showed homology with the mouse genome, and several sequences could be mapped to orthologous human and mouse genes. Binding of BARX2 to 21 genomic loci examined was confirmed quantitatively by replicate ChIP assays. A combination of sequence analysis and electrophoretic mobility shift assays revealed homeodomain binding sites within several fragments that bind to BARX2 in vitro. The majority of BARX2 binding fragments tested (14/19), also affected transcription in luciferase reporter gene assays. Mutation analyses of three fragments showed that their transcriptional activities required the HBS, and suggested that BARX2 regulates gene expression by binding to DNA elements containing paired TAAT motifs that are separated by a poly(T) sequence. Inhibition of BARX2 expression in MCF7 cells led to reduced expression of eight genes associated with BARX2 binding sites, indicating that BARX2 directly regulates their expression. The data suggest that BARX2 can coordinate the expression of a network of genes that influence the growth of MCF7 cells.

BARX2 and estrogen receptor-α (ESR1) coordinately regulate the production of alternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion

“…Functional interaction between BARX2 and E 2 might explain the dichotomous observation that ectopic BARX2 expression inhibits invasion of OAW42 ovarian cancer cells (Sellar et al, 2001), whereas increased BARX2 expression promotes invasion of MCF7 cells. OAW42 cells do not express ESR1 (O'Doherty et al, 2002;Stevens et al, 2004), thus one factor controlling the effect of BARX2 on cancer cell invasion may be ESR1 expression status.…”

“…Human BARX2 has been described as a potential tumor suppressor gene in ovarian cancer, where its loss correlates with invasiveness, and ectopic expression inhibits invasion (Sellar et al, 2001). Recently, we found that BARX2 is expressed in estrogen-dependent (MCF7 and T47D) but not estrogen-independent (MDA-MB-231) breast cancer cell lines (Stevens et al, 2004). Moreover, chromatin immunoprecipitation (ChIP) and RNAmediated inhibition (RNAi) of BARX2 identified the estrogen receptor-a gene (ESR1) as a direct target of BARX2 in MCF7 cells (Stevens et al, 2004).…”

“…BARX2 and estrogen coordinately regulate gene expression A number of previously identified BARX2 target genes (Stevens et al, 2004) are also E 2 responsive as shown by Serial Analysis of Gene Expression (see http://science park.mdanderson.org/ggeg). Quantitative RT-PCR was used to examine whether BARX2 and E 2 might coordinately regulate the expression of a selected number of these genes (Figure 4).…”

“…The earlier observation that BARX2 binds to the TIMP4 gene in MCF7 cells (Stevens et al, 2004) prompted us to examine the effect of BARX2 on TIMP mRNA levels using RT-PCR. On plates and in Matrigel, MCF7-pcBARX2 cells had 1.5-2-fold higher TIMP4 expression than control cells (Figure 7c).…”

“…Recently, we found that BARX2 is expressed in estrogen-dependent (MCF7 and T47D) but not estrogen-independent (MDA-MB-231) breast cancer cell lines (Stevens et al, 2004). Moreover, chromatin immunoprecipitation (ChIP) and RNAmediated inhibition (RNAi) of BARX2 identified the estrogen receptor-a gene (ESR1) as a direct target of BARX2 in MCF7 cells (Stevens et al, 2004).…”