Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter

Johan Auwerx, Kristina Schoonjans, Walter Wahli

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

1995

The role and transcriptional regulation of FAM46A in adipocyte differentiation

Ann-Kristin Hov

Obesity is currently a leading global health concern. Treatments include undergoing a weight-loss regimen. However, successful weight-loss is variable, where the heritability of obesity is considered part of the cause. This patient variability to weight-loss was addressed by Carayol et al. (2017), where protein plasma level changes was associated to genetic variability and changes in BMI under a low-calorie diet intervention. A highly significant association region was found to regulate downstream gene FAM46A; coding for a nucleotidyl-transferase protein. Loss of function mutations in FAM46A have previously been discovered in multiple human diseases, such as osteogenesis imperfecta. However, there is a lack of understanding of FAM46Aâs function, especially in the context of adipose tissue and metabolism. This thesis aimed to follow up on the Carayol et al. study by investigating the transcriptional regulation of FAM46A and how the associated region is influencing this regulation, as well as how FAM46A is involved in adipose tissue function. When mapping the physical DNA interaction network around the associated region using chromosome conformation capture (3C) in human subcutaneous adipocytes (SGBS cells), no interactions between the FAM46A promoter and selected loci of the associated region were found. Interestingly, significant interaction peaks downstream of the promoter overlapped with regions of open chromatin and were enriched for transcription factor (TF) motifs that are involved in stem cell differentiation and TGFÎ² signalling. SMAD4, part of the TGFÎ² signalling pathway, binds to this region, indicating a potential regulator of FAM46A. To address FAM46Aâs potential role in adipocyte function, FAM46A expression was disrupted in vitro in differentiating adipocytes. Reduction of FAM46A expression in SGBS cells resulted in decreased expression of PPARG and CEBPA, the master regulators of adipogenesis, complemented by a reduction in lipid accumulation. Follow up studies in murine mesenchymal stem cells where Fam46a was over-expressed were controversial as this led to a decrease in adipogenic markers (Adipoq and PPARðŸ). Overall, disruption of FAM46A expression led to opposing changes in adipogenic potential, suggesting the requirement for a more stable cell system to study FAM46A in vitro. To further characterise the role of Fam46a, metabolic phenotyping of a Fam46a knockout (KO) mouse model under hyper-caloric stress was performed in collaboration with the IMG in Prague. This model revealed a significantly smaller mouse, and increased plasma alkaline phosphatase, as previously observed. When fed a high-fat diet, KO mice had reduced overall fat mass, improved glucose clearance and reduced adipocyte size in both subcutaneous and visceral white adipose tissue (subWAT/visWAT) depots. Transcriptomic analysis of these two depots revealed an up-regulation of genes expressed in ribosome related pathways in the subWAT, whereas the visWAT showed the up-regulation of fatty acid metabolism pathways, suggesting that the KO adipose tissue is more metabolically active. Other down-regulated pathways involved stem cell differentiation and BMP/TGFÎ²-signalling. In conclusion, this study found the possible regulation of the FAM46A promoter by TFs involved in adipogenesis regulation, such as SMAD proteins, as well as strengthening its possible functional involvement in TGFÎ² signalling by enrichment of this pathway in KO studies.

EPFL2021

Genomic Context and KRAB/KAP1-mediated Transcriptional Repression

Sylvain Meylan

Chromatin structure and its impact on transcriptional activity represent one of the major revolutions in the biomedical field over the passed decade. The implications concern as much fundamental as clinically-oriented research; diseases and therapeutical strategies are being approached from a new point of view (Mackay et al., 2008; Richman et al., 2009). The current work dissects the different susceptibilities of genes in the human genome and how these react to the changes in chromatin landscape upon the recruitment of a transcriptional corepressor. The KRAB-Zinc finger proteins are the largest family of transcriptional repressors in the human genome. These repressors use the KRAB domain as a docking site for KAP-1 which in turn recruits a macromolecular chromatin-remodelling complex leading to heterochromatin formation. Yet current knowledge of their impact on gene regulation is limited. By artificially recruiting a KRAB-repressor in the context of genes, our lab has previously shown that this repression can act over several tens of kilobases (Groner et al., 2010). Specifically, transcriptional silencing depends on the spreading of repressive marks to the upstream promoter. However, such spreading to the trapped promoter is not systematic, suggesting potential counter-acting influences. To identify the determinants of repression, we designed a high-throughput approach of our experimental system to accumulate thousands of KRAB docking sites for which the trapped promoter is regulable and docking sites for which the promoter is immune to repression. Firstly, our data indicate that KRAB/KAP-1-repression can act efficiently over distances of 20 kb albeit in a very variable fashion, but is limited beyond that. Second, propagation of repression in the transcribed regions was favoured in most open chromatin. Third, we observed that the broader context of genes sustaining repression was that of most euchromatic regions, recapitulating the genetic context of natural targets of KAP1 repression. On the other hand, we have no evidence that barrier elements such as CTCF counteract this KRAB-mediated repression within genes. Finally, work on cases where repression acts over distances greater than 40 kb hints at the regulation of enhancers by our KRAB repressor rather than promoter. In conclusion, this work broadens the knowledge in the emerging field of gene regulation and provides further insight on the complexity of chromatin architecture and its meaning at a functional level.