The switch from gamma to beta globin gene expression during development is controlled by a complex regulatory network that includes autonomous regulation of gamma gene expression mediated by sequences near the gene and cis competition between gamma and beta for interaction with a Locus Control Region (LCR), located upstream of the cluster. The goal of this proposal is to exploit documented differences in the timing of the gamma to beta switch among primate globin clusters as a tool to dissect these complex regulatory influences: the galago gamma to beta switch occurs at the end of embryonic life, the capuchin monkey exhibits a mid- fetal switch, while the human switch occurs at birth. Three hypotheses are tested: 1) Specific cis differences linked to the gamma genes themselves control the different patterns of autonomous regulation of the human, capuchin and galago gamma gene in transgenic mice. Chimeric galago/human or capuchin/human gamma genes will be analyzed in transgenic mice using a cosmid construct, LCR-epsilongamma, to identify elements that confer stage-specific control of gamma expression and silencing. 2) The expression of human globin genes will be altered (via changes in competitive interactions) when galago globin sequences are substituted into an intact human globin locus. In the context of a human beta cluster YAC, human sequences will be replaced by 4- 10 kb segments of othologous galago sequences (from gamma or beta gene regions or LCR regions), and expression of the remaining human genes will be assessed in transgenic mice. These experiments will allow dissection of the sequences involved in competitive interactions under conditions in which the beta cluster remains intact. 3) Knowledge of in vivo globin gene expression patterns in extant primates will allow reconstruction of the evolutionary pathway that led to their different characteristic gamma to beta switch patterns. To construct an evolutionary model that can explain the generation of the different patterns of globin gene expression observed in extant primates requires careful documentation of those gene expression patterns in vivo. Thus, globin protein and mRNA will be examined in extant primates to document the patterns of gamma to beta switching; this information will be integrated into an evolutionary model that correlates cis sequence changes with alterations in the detailed switching pattern characteristic of each species. This comparative functional analysis of primate globin genes in transgenic mice, combined with studies in extant primates will provide new insights into mechanisms of hemoglobin switching.
