NSF Award
9304793
9304793 McCarrey The goal of the proposed studies is to understand how a mammalian gene evolves a tissue-specific mode of regulation. The mammalian phosphoglycerate kinase (Pgk) gene system provides an ideal system for such studies, since we have now amassed sufficient information about the expression, regulation, and evolution of these genes to formulate testable hypotheses about how different modes of transcriptional regulation have evolved in different members of this particular gene family. The autosomal Pgk-2 gene evolved as a duplication of the X-linked Pgk-1 gene by retroposition or gene processing during mammalian evolution, prior to the divergence of metatherians (marsupials) and eutherians (placental mammals) about 120-150 million years ago. In eutherian mammals, the Pgk-1 gene is expressed ubiquitously, while expression of the Pgk-2 gene is tissue-specific, occurring only in spermatogenic cells. The tissue-specificity of Pgk-2 expression apparently evolved after its origin, since ubiquitous expression of Pgk-2 can still be observed in certain marsupial species. We believe the evolution of tissue-specific expression of the Pgk-2 gene in eutherian mammals has resulted from a combination of changes leading to: 1) the enhancement of Pgk-2 expression in spermatogenic cells, and 2) the repression of Pgk-2 expression in somatic cells. Our recent studies have revealed several characteristics of transcriptional regulation that distinguish the specifically expressed Pgk-2 gene from the ubiquitously expressed Pgk-1 gene in the mouse and human, including the absence or presence of a CpG island in the promoter region, single or multiple transcription start sites, tissue-specific or ubiquitous protein- DNA interactions, and differential or constitutive DNA hypomethylation in expressing tissues, respectively. We believe these differences represent examples of molecular changes that have lead to the evolution of tissue-specific regulation of the Pgk-2 gene in eutherians. Thus in this application, we propose to examine these same characteristics in metatherian and eutherian species that show intermediate degrees of tissue-specificity of Pgk-2 expression to better understand the evolution of these characteristics and to distinguish those associated primarily with enhancement of Pgk-2 expression in spermatogenic cells from those associated with repression of Pgk-2 expression in somatic cells. %%% Organic evolution occurs by various genetic mechanisms, including the appearance or loss of entire genes, and changes in the structure of the proteins encoded by specific genes. However changes in another mechanism, gene regulation, which determines whether or not, or when or where (ie- in what organs or tissues) a particular gene is used (or expressed) also play a major role in the evolutionary process, but this area has received inordinantly little attention to date. This lack of attention has occurred primarily because appropriate hypotheses and informative model systems in which to test those hypotheses have not been readily available. The mammalian phosphoglycerate kinase (Pgk) gene system now provides an ideal system for such studies, because we have learned enough about it that we can develop and test appropriate hypotheses. The mammalian Pgk gene system includes two functional genes, Pgk-1 and Pgk-2, each of which encodes a similar metabolic enzyme called phosphoglycerate kinase. The Pgk-1 gene is used ubiquitously in almost every part of the body, however the Pgk-2 gene is used in only one type of cell, the spermatogenic cells that give rise to sperm in the male. We know that the Pgk-2 gene evolved as a duplicate copy of the Pgk-1 gene early in mammalian evolution, and we have evidence that when it first evolved it was used in all parts of the body just like the Pgk-1 gene. Thus we believe that it was not until after it originally appeared as a duplication of the Pgk-1 gene that the Pgk-2 gene evolved its tis sue-specific mode of regulation such that it is only used in one type of cell. What is more, we believe that by studying the right species of mammals, we can recapitulate how this evolution of tissue-specific expression occurred. Thus we know that in some species of marsupials that exist today, the Pgk-2 gene is still expressed in all parts of the body, while in other marsupial species the Pgk-2 gene is expressed in several, but not all, parts of the body. It is only in placental mammals that expression of the Pgk-2 gene is strictly limited to the spermatogenic cells. We have already carried out several experiments at the molecular level to determine what mechanisms are involved in the ubiquitous expression of the Pgk-1 gene and the tissue-specific expression of the Pgk-2 gene in the mouse and the human. Thus in this project, we plan to investigate the status of these same molecular mechanisms in certain marsupial species that show intermediate degrees of tissue-specificity of Pgk-2 expression. In this way, we hope to learn more about which molecular mechanisms were directly involved in the transition from ubiquitous to tissue-specific expression of the Pgk-2 gene, and also to learn more about how these mechanisms changed during mammalian evolution. ***
