REGULATORY LINKAGE AND COORDINATED EXPRESSION OF THE CHKB AND CPT1B GENES: IMPLICATIONS FOR AN ELEVATED METABOLIC CAPACITY AND THE ORIGIN OF BROWN ADIPOSE TISSUE

Abstract

The emergence of endothermy was an important event in the evolution of vertebrates. Specifically, the acquisition of BAT was a key achievement for the radiation and diversification of eutherian (placental) mammals. However, the molecular mechanism behind the development of this thermogenic tissue has not been thoroughly characterized. The theory of the UCP1 gene being the key impetus for BAT formation has been challenged considering the UCP1 gene has been found in several ray-finned fish species that diverged early in the timescale of vertebrate evolution. Additionally, the UCP1 gene has been found in several non-placental mammals from the marsupial and monotreme clades, however finding BAT depots in these species has proven unsuccessful. Mitochondrial fatty acid oxidation (FAO) contributes to the proton motive force that drives ATP synthesis in many mammalian tissues. In eutherian mammals, brown adipose tissue (BAT) can also dissipate this proton gradient through uncoupling protein 1 (UCP1) to generate heat. An essential step in FAO is the transport of cytoplasmic long-chain acyl coenzyme A (acyl-CoA) into the mitochondrial matrix, which requires the action of carnitine palmitoyltransferase 1B (CPT1B) in striated muscle and BAT. In eutherians, the CPT1B gene is closely linked to the choline kinase beta (CHKB) gene, which is transcribed from the same DNA strand and terminates just upstream of CPT1B. CHKB is a rate-limiting enzyme in the synthesis of phosphatidylcholine (PC), a predominant mitochondrial membrane phospholipid, suggesting that the coordinated expression of CHKB and CPT1B may cooperatively enhance mitochondrial FAO. The present findings show that transcription of the eutherian CHKB and CPT1B genes is linked within a unitary epigenetic domain targeted to the CHKB gene, and that that this regulatory linkage appears to have resulted from an intergenic deletion in eutherians that significantly altered the distribution of CHKB and CPT1B expression. Informed by the timing of this event relative to the emergence of BAT, the phylogeny of CHKB-CPT1B synteny, and the insufficiency of UCP1 to account for eutherian BAT, these data support a mechanism for the emergence of BAT based on the acquisition of a novel capacity for adipocyte FAO in a background of extant UCP1. Additionally, this dissertation shows that Crispr-Cas9 mediated deletion of the Chkb 5' region, where a variety of chromatin modifications and transcriptional initiation is localized, has a drastic perturbance on Cpt1b expression indicating that the co expression and regulation of both genes is facilitated by the one Chkb 5' CpG island promoter. The condensation of the intergenic region between CHKB and CPT1B through vertebrate evolution coincided with elevated basal metabolic rate and body temperature as well as the acquisition of BAT in placental mammals.