Biomedical subjects

Stephanie M Bartley

Publications and source records attributed to Stephanie M Bartley.

3 recordsLinked to original sources

PubMed2004

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.

Carrier Proteins↗

PubMed2003

Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy.

We have previously identified SU(Z)12 as an E2F target gene. Because many E2F target genes encode proteins that are critical for the control of cell proliferation, we have further characterized the regulation and expression of SU(Z)12. To understand the molecular mechanisms responsible for expression of SU(Z)12 mRNA, we have analyzed the promoter region. We found that the SU(Z)12 gene is controlled by dual promoters, one of which functions bidirectionally. In addition to the E2F binding site, we have identified two binding sites for T cell factor (TCF)/beta-catenin complexes. Using gel mobility shift assays, we demonstrated that both TCF sites can be bound by TCF4. TCF/beta-catenin complexes have been shown to be a critical regulator of gene expression in tumors of the colon, breast, and liver. Accordingly, we have used chromatin immunoprecipitation assays to confirm that TCF4/beta-catenin complexes are bound to the SU(Z)12 promoter in colon cancer cells but not in HeLa cells. We next adapted the chromatin immunoprecipitation assay for use with primary colon tumor samples, and, using matched pairs of normal and tumor tissue obtained from several different colon cancer patients, we demonstrate that levels of beta-catenin bound to the SU(Z)12 promoter are increased in colon tumors. Finally, we show that the SU(Z)12 mRNA is up-regulated in a number of different human tumors, including tumors of the colon, breast, and liver. Recent studies have found that SU(Z)12 is a component of the Drosophila ESC-E(Z) and the human EED-EZH2 Polycomb chromatin remodeling complexes. Therefore, we suggest that SU(Z)12, which may modulate the tumor phenotype by changing gene expression profiles, may be a logical target for the design of a new antitumor agent

Animals↗

PubMed2002

The identification of E2F1-specific target genes.

The E2F family of transcriptional regulators consists of six different members. Analysis of E2F-regulated promoters by using cultured cells suggests that E2Fs may have redundant functions. However, animal studies have shown that loss of individual E2Fs can have distinct biological consequences. Such seemingly conflicting results could be due to a difference in E2F-mediated regulation in cell culture vs. animals. Alternatively, there may be genes that are specifically regulated by an individual E2F which have not yet been identified. To investigate this possibility further, we have analyzed gene expression in E2F1 nullizygous mice. We found that loss of E2F1 did not cause changes in expression of known E2F target genes, suggesting that perhaps E2F1-specific promoters are distinct from known E2F target promoters. Therefore, we used oligonucleotide microarrays to identify mRNAs whose expression is altered on loss of E2F1. We demonstrate by chromatin immunoprecipitation that several of the promoters that drive expression of the deregulated mRNAs selectively recruit E2F1, but not other E2Fs, and this recruitment is via an element distinct from a consensus E2F binding site. To our knowledge, these are as yet undocumented examples of promoters being occupied in asynchronously growing cells by a single E2F family member. Interestingly, the E2F1-specific target genes that we identified encode proteins having functions quite different from the function of known E2F target genes. Thus, whereas E2F1 may share redundant functions in cell growth control with other E2F family members, it may also play an important biological role distinct from the other E2Fs.

Animals↗