Alternative pre-mRNA Splicing in Mammary Epithelial Cells

Institution: Lawrence Berkeley National Laboratory
Investigator(s): John Conboy, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9IB-0186 Award: $119,968
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2003)
Normal development, including generation of a properly functioning breast with the ability to synthesize milk proteins, requires cells to turn individual genes on and off at the appropriate times. In some cases, a single gene can make two products with different functions, by splicing in (retaining) or splicing out (removing) an internal part of the coding sequence. This splicing choice can be regulated to allow the cell to make predominantly one spliced product at one stage of development, and the alternate spliced form at another stage of development. Our proposal will study how this splicing process works in specialized breast "epithelial" cells, and will investigate whether such splicing events are common among epithelial genes.

We hypothesize that mammary epithelial cells make splicing factor protein(s) whose very specialized function is to decide which of the two spliced products will be made by a gene at a particular stage of development. Further, we propose that changes in the master splicing proteins will lead to changes in other genes' splicing, and ultimately to development of a growing immature cell into a specialized nondividing mammary epithelial cell. We believe this process is essential to prevent unregulated cell growth, i.e. cancer.

We will use cloning techniques to study several important genes during the process of mammary epithelial cell maturation in culture. For each gene we will ask whether its major product is similar in less vs. more mature cells, i.e., whether changes in splicing have occurred. For one case in which we have already identified a change in splicing as the immature cells develop into specialized mammary epithelial cells, we will make mutations in the spliced portion of the gene to identify regions necessary for control of the splicing decision. The goal will be to understand why the spliced region is "spliced out" in immature cells and "spliced in" in more mature cells i.e., to understand the mechanism of this splicing "switch". In the final portion of the proposal, we will test the function of a candidate splicing factor. We will ask whether this factor is directly involved in the splicing switch, by increasing or decreasing the level of this protein and observing whether changes in the splicing switch occur.

Although the role of splicing is well established as an important mechanism in normal general cell differentiation, little is known about this process in mammary epithelial cells. Furthermore, it has been shown that disruption of splicing occurs in several disease models and in various types of cancers. We propose that our studies represent a beginning towards understanding a critical mechanism for gene regulation in normal mammary cell biology. In the future, understanding the key splicing factor proteins that function in mammary cells may provide targets for cancer therapy.


Final Report (2006)
Normal development, including generation of a properly functioning breast with the ability to synthesize milk proteins, requires cells to turn individual genes on and off at the appropriate times. In many cases, a single gene can make multiple products that sometimes exhibit different functions, by splicing in (retaining) or splicing out (removing) an internal part(s) of the gene's coding sequence known as "exons". Exon splicing can be regulated to allow the cell to make predominantly one spliced product at one stage of development, and an alternate spliced form at another stage of development. Our proposal was designed to study how this splicing process works in specialized breast "epithelial" cells, and to ask whether alternative splicing is common among epithelial genes. Proper splicing is essential to prevent unregulated cell growth, i.e. cancer. We hope these studies will provide new insights into deranged gene expression in breast cancer.

We studied a gene called "protein 4.1" as a model for these splicing studies. We already knew that this gene makes one version of its protein in proliferating mammary epithelial cells, and a different version, containing an extra spliced exon "17B", in more differentiated cells. We investigated specific DNA sequences in the 4.1 gene that regulate splicing of exon 17B and thereby control which form of protein 4.1 is synthesized in mammary epithelial cells.

We identified three potential regulatory elements. Within exon 17B, there is one "enhancer" sequence that stimulates splicing, and another "silencer" sequence that inhibits splicing. In addition, there is a sequence in a region adjacent to exon 17B that represents a second potential enhancer to help differentiated cells splice exon 17B. In a related development, we are taking advantage of recent progress in DNA sequencing for the genomes of humans, mice, rats, and chickens. Following the general logic that sequences preserved in all of these species are likely to have a specific important function, and are using the computer to compare the sequences of exon 17B and its adjacent regions in these genomes. At least one of the elements mentioned above seems well conserved in all of these species.

We are using advanced genome DNA techniques to investigate whether changes in splicing occur in other genes in these mammary epithelial cells. Our recent experiments in a related project demonstrate proof of principle that these new genome analysis methods work successfully to identify differences in splicing in different model cell types. Now we are ready to apply these methods to study whether changes in splicing control proteins play a role in aberrant splicing in breast cancer cells.

Although the role of splicing is well established as an important mechanism in general cell differentiation, little is known about this process in normal mammary epithelial cells or about aberrations in this process in breast cancer. Our studies represent a beginning towards understanding a critical mechanism for gene regulation in normal mammary cell biology. In the future, understanding the key splicing factor proteins that function in mammary cells may provide targets for cancer therapy.