Role of Estrogen Response Element in the Human Prolactin Gene:Transcriptional Response and Timing

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located −1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17β-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The −1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the −1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding

Metabolic rate changes proportionally to circadian frequency in tau mutant Syrian hamsters

The tau mutation in Syrian hamsters (Mesocricetus auratus) is phenotypically expressed in a period of the circadian rhythm of about 20 h in homozygotes (SS) and about 22 h in heterozygotes (S+). The authors investigate whether this well-defined model for variation in circadian period exhibits associated changes in energy metabolism. In hamsters of the three genotypes (SS, S+, and wild type [WT]), oxygen consumption measurements were performed at 28 degrees C (thermoneutral), 18 degrees C, and (after acclimatization) 10 degrees C. After correction for body mass, SS tau mutant hamsters had a higher overall metabolic rate (average oxygen consumption per hour over 24 h) and a higher resting metabolic rate (the lowest 30-min oxygen consumption in the subjective day) than did WT hamsters at all ambient temperatures. S+ hamsters were intermediate in both after taking body mass into account. The differences in metabolism among the three genotypes indicate that the increase in metabolic rate was statistically indistinguishable from a proportional increase in circadian frequency. The oxygen consumption totals per circadian cycle (24 h for WT, 22 h for S+, and 20 h for SS mutants) were not statistically different among the genotypes after correcting for body mass. The possible roles of pleiotropic effects, of linkage to genes involved in growth and metabolism and of early ontogenetic influences are briefly discussed

Temporal Organization of Feeding in Syrian Hamsters with a Genetically Altered Circadian Period

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/− and tau −/−) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation.

Metabolic Rate Changes Proportionally to Circadian Frequency in tau Mutant Syrian Hamsters

The tau mutation in Syrian hamsters (Mesocricetus auratus) is phenotypically expressed in a period of the circadian rhythm of about 20 h in homozygotes (SS) and about 22 h in heterozygotes (S+). The authors investigate whether this well-defined model for variation in circadian period exhibits associated changes in energy metabolism. In hamsters of the three genotypes (SS, S+, and wild type [WT]), oxygen consumption measurements were performed at 28°C (thermoneutral), 18°C, and (after acclimatization) 10°C. After correction for body mass, SS tau mutant hamsters had a higher overall metabolic rate (average oxygen consumption per hour over 24 h) and a higher resting metabolic rate (the lowest 30-min oxygen consumption in the subjective day) than did WT hamsters at all ambient temperatures. S+ hamsters were intermediate in both after taking body mass into account. The differences in metabolism among the three genotypes indicate that the increase in metabolic rate was statistically indistinguishable from a proportional increase in circadian frequency. The oxygen consumption totals per circadian cycle (24 h for WT, 22 h for S+, and 20 h for SS mutants) were not statistically different among the genotypes after correcting for body mass. The possible roles of pleiotropic effects, of linkage to genes involved in growth and metabolism, and of early ontogenetic influences are briefly discussed.