Victoria Harris and Dr. Chin-Yo Lin, Microbiology and Molecular Biology
Estrogen is a steroid hormone that acts in the body in both beneficial and detrimental ways. It plays a crucial role in many human processes including coagulation, bone formation, and development of sexual organs. However, estrogen is also associated with the proliferation of breast cancer. Estrogen activates the estrogen receptor (ER), a ligand-dependent transcription factor that regulates gene expression by binding to specific DNA elements and promoting or repressing transcription.
By understanding the specific mechanisms of gene regulation by ER, we can design more effective therapies for ER-positive breast cancers. The process of up-regulation by ER has been heavily studied, whereas the mechanisms of down-regulation have yet to be elucidated. A key step in the process of understanding down-regulation is the identification of ER binding sites that promote down-regulation of genes. A recent genome-wide analysis identified over 1000 ER binding sites in MCF-7 breast cancer cells (ER+) using chromatin immunoprecipitation (ChIP) technology1.In ChIP experiments, proteins and DNA are cross-linked by formaldehyde, and then ER-specific antibodies are added to precipitate out the ER-DNA complexes. Subsequently, the DNA segments can be isolated and sequenced to reveal ER binding sites.
Using that data, we have identified ER binding sites (~500 bp each) which can be linked to five known down-regulated genes: ZNRF3, PSCA, NFIA, MME, and SLC35A1 (see Figure 1). The figure shows a target transcript, or regulated gene, and the relative locations of the ER binding site for each gene: upstream, downstream, or within an intron of the transcript.
ER binds to a specific palindromic sequence, 5’-GGTCAnnnTGACC-3’, also known as the estrogen response element (ERE). ER can bind directly to this sequence to regulate transcription, or bind indirectly through tethering to another DNA-bound nuclear protein, such as transcription factors AP-1 or Sp1 1. The mechanism of tethering requires that the binding site of another transcription factor be present within the ER-bound region. Figure 2 shows the PSCA binding region and other transcription factor binding sites contained therein. The presence of these binding sites presents an opportunity for study of the tethering mechanism and understanding how this affects down regulation.
In order to study these binding sites we must first confirm that they are responsible for down regulation. To do this I have inserted the binding site sequence into plasmid constructs upstream of a firefly luciferase reporter gene. This process included isolating the binding sites from genomic DNA using specific primer sets, cutting the fragment and vector with specific restriction enzymes, and then ligating the two sections together to create a unique plasmid. The luciferase gene is controlled by a SV40 promoter which constitutively expresses the gene in order to test subsequent down regulation, and the levels of the luciferase gene can be monitored by the light emitted following the addition of the appropriate substrate. After confirming the estrogen dependent down regulation relative to these binding sites, I will conduct site-directed mutagenesis to determine exactly which region of the binding site is actively responsible for gene regulation. These studies will be coupled with re-ChIP assays (applying a second antibody precipitation step) to determine if ER forms a complex with other transcription factors. As we compile data from the five different binding sites, we will be able to recognize patterns in ER binding mechanisms and design further research projects to determine the effects on cell proliferation.
My participation in this research has allowed me to understand the research process firsthand. I have learned the difficulties associated with research as I have had unreliable results, but also the excitement of research as I have turned in new directions. I have learned many techniques in molecular biology such as cell culture, RNA extraction, real time PCR, and cloning. This mentored experience has had a great impact on me and my future as a research scientist.
References
- Lin CY, Vega VB, Thomsen JS, Zhang T, Kong SL, Xie M, Chiu KP, Lipovich L, Barnett DH, Stossi F, Yeo A, George J, Kuznetsov VA, Lee YK, Charn TH, Palanisamy N, Miller LD, Cheung E, Katzenellenbogen BS, Ruan Y, Bourque G, Wei CL, Liu ET. Whole-genome cartography of estrogen receptor alpha binding sites. PLoS Genetics 2007; 3(6):e87.