Phylogenetic comparisons suggest that distance from the locus control region guides developmental expression of primate beta-type globin genes

Abstract

Phylogenetic inferences drawn from comparative data on mammalian beta-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related beta-type globin loci (5'-LCR-epsilon-gamma-psieta-delta-beta-3'), with epsilon and gamma expressed solely during embryonic life. A gamma locus tandem duplication (5'-gamma(1)-gamma(2)-3') triggered gamma's evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, gamma(1) at the ancestral distance from epsilon is down-regulated, whereas gamma(2) at increased distance is up-regulated. Catarrhine gamma(1) and gamma(2) acquired longer distances from epsilon (14 and 19 kb, respectively), and both are up-regulated throughout fetal life with gamma(1)'s expression predominating over gamma(2)'s. On enlarging the platyrrhine expression data, we find Aotus gamma is embryonic, Alouatta gamma is inactive at term, and in Callithrix, gamma(1) is down-regulated fetally, whereas gamma(2) is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal beta-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (<10 kb) between epsilon and the adjacent gamma is a plesiomorphic character that allows the LCR to drive embryonic expression of both genes, whereas a longer distance (>10 kb) impedes embryonic activation of the downstream gene.

Fig. 1.

Major events in the proposed evolution of β-globin cluster gene expression in primates. The linkage order shown in the primate ancestor is retained in strepsirrhines, represented by the bush baby (Otolemur sp.), along with predominantly ε and γ expression during embryonic life and δ and β during fetal and adult developmental stages. Based on the existence of only one γ gene in tarsiers (12), this pattern likely persisted until the duplication of the γ gene in stem anthropoids. In the anthropoid ancestor, γ2 was relatively silent because of its separation from the LCR by two intervening embryonically expressed genes and to the retention of postembryonic cis-acting silencer elements, such as those found in the bush baby promoter (13). In its silent condition, γ2 was free to acquire the base changes that inactivated those cis elements that bound fetal repressors. Gene conversions from γ2 to γ1 then conferred the capacity for fetal expression on γ1. In platyrrhines, the retained proximity of γ1 to ε (and, by extension, to the LCR) promotes predominantly embryonic expression of γ1; in this clade, represented by the marmoset genus Callithrix, γ2 is the primary fetally expressed gene. In contrast, in stem catarrhines, LINE element insertions between ε and γ1 weakened the LCR’s ability to promote embryonic expression of γ1; in the catarrhine clade, represented here by humans (Homo sapiens), γ1 is the primary fetally expressed gene. E, F, and A indicate embryonic, fetal, and adult stage gene expression, respectively. Where a gene is expressed in more than one stage, the stage of predominant expression, if any, is indicated by double letters. Stages shown in parentheses indicate trace levels of expression.

Fig. 2.

Representative time course of digestion of amplicons from Callithrix γ globin genes. PCR of RNA from a C. penicillata fetus was performed by using a primer set that complements sequences that are identical in C. jacchus γ¹ and γ², but which enclose a region with a base pair difference between γ¹ and γ² that produces an EcoICRI cleavage site in γ¹. The mixture of amplified fragments from fetal mRNA was digested with EcoICRI, yielding two fragments of 136 and 24 bp from the γ¹ amplicon. To ensure that digestion was complete, a time course across four time points (1 h, 2 h, 3 h, and 4 h) was run. A 10-bp ladder is in lane 1. The mean γ²/γ¹ ratio for three digestions was 6.6 ± 0.7.