Pathways of BP1: (1) Role of BP1 in Estrogen Signaling (2) Prediction of Phosphorylation Sites in BP1 and its Effect on DNA-Binding
Open AccessBP1 is a transcription factor belonging to the family of homeobox genes.BP1 protein (pBP1) appears to play a crucial role in cell survival, aggressiveness and metastasis. It has been shown that pBP1 is expressed in 80% of invasive ductal breast tumors, and that 100% of estrogen receptor (ER) negative tumors are BP1 positive. The aim of this study was to determine the pathways regulated by BP1. The first part deals with the role of BP1 in estrogen signaling, while in the second part, we attempt to predict phosphorylation sites in BP1 and elucidate the role of this Post Translational Modification on DNA binding. BRCA1 is a tumor suppressor gene that expresses the protein breast cancer susceptibility 1 protein and is responsible for DNA repair, and it plays a crucial role in Estrogen signaling by inhibiting ER-alpha at the protein level and prevents the transcription of ER-alpha target genes. Mutation in BRCA1 or loss of its expression increases the risk of breast cancer. One of the pathways by which the risk of breast cancer is increased is by the loss of ability of BRCA1 to inhibit ER-alpha. It has been shown that BP1 overexpression results in decreased expression of wild-type BRCA1. p300 is a Histone Acetyl Transferase (HAT) known to acetylate ER-alpha and is known to increase its hormone sensitivity, transactivation and stability. In our study, Western blotting showed that the overexpression of pBP1 in MCF-7 cells grown in complete media leads to the activation and stabilization of the ER-Alpha protein. Further, our RT-PCR analysis on the genes positively regulated by ER-Alpha (eg. pS2) confirms the presence of biologically active ER-alpha protein. In this study we found that, in the presence of serum, pBP1 over-expression leads to an increase in the expression of biologically active ER-alpha; protein and increased expression of the Histone Acetyl Transferase (HAT) p300 protein. Through ChIP, we showed that pBP1 binds to the first intron of the p300 gene (EP300). Our real-time PCR data shows 80% increase in the mRNA expression levels of ER-Alpha and 50% increase in p300 mRNA expression levels when the cells over-expressing BP1 were compared with those containing the empty vector. Such a small increase in mRNA levels was enough to show significant changes in the protein levels, as verified by Western blots. In previous studies by Sanket Awate in our lab, it was shown that ER-Alpha binds to the ERE sequence present in the first intron of the BP1 gene by a ChIP. It was also shown that BP1 expression increases after treatment with 10 nM Estradiol (E2). Piecing together the data previously generated and obtained in this study, we have designed a pathway that may describe the role of BP1 in estrogen signaling. Transcription factors undergo Post-Translational Modifications (PTM), such as phosphorylation, glycosylation, etc. Thus, these proteins are tightly regulated within the cell. Homeobox proteins, in general, and Distal-less proteins, in particular, have been shown to be phosphorylated by kinases. Basic sequence analysis shows possible phosphorylation sites in BP1 protein. Further study to narrow down the most likely sites of phosphorylation showed three possible sites in the homeodomain region of BP1 that were highly conserved in all Distal-less proteins in mammals. Further, using Homology modeling, we designed and verified the model of the BP1 homeodomain region. We plan to introduce the phosphate groups at these predicted sites in silico and determine the effects of the PTM on DNA-binding. We plan to carry out molecular experiments to verify that BP1 is indeed phosphorylated. BP1 is an important transcription factor and is shown to play a role in breast cancer. Here we try to establish its pathway in estrogen signaling and speculate on the regulatory mechanisms involved to regulate BP1 and effects on its target genes.
- All rights reserved
Notice to Authors
If you are the author of this work and you have any questions about the information on this page, please use the Contact form to get in touch with us.
| Thumbnail | Title | Date Uploaded | Visibility | Actions |
|---|---|---|---|---|
|
|
Kirolikar_gwu_0075M_11133.pdf | 2018-01-16 | Open Access |
|