Glucocorticoid-Responsive Transcription Factor Krüppel-Like Factor 9 Regulates fkbp5 and Metabolism

Abstract

Krüppel-like factor 9 (Klf9) is a feedforward regulator of glucocorticoid receptor (GR) signaling. Here we show that in zebrafish klf9 is expressed with GR-dependent oscillatory dynamics in synchrony with fkbp5, a GR target that encodes a negative feedback regulator of GR signaling. We found that fkbp5 transcript levels are elevated in klf9–/– mutants and that Klf9 associates with chromatin at the fkbp5 promoter, which becomes hyperacetylated in klf9–/– mutants, suggesting that the GR regulates fkbp5 via an incoherent feedforward loop with klf9. As both the GR and Fkbp5 are known to regulate metabolism, we asked how loss of Klf9 affects metabolic rate and gene expression. We found that klf9–/– mutants have a decreased oxygen consumption rate (OCR) and upregulate glycolytic genes, the promoter regions of which are enriched for potential Klf9 binding motifs. Our results suggest that Klf9 functions downstream of the GR to regulate cellular glucocorticoid responsivity and metabolic homeostasis.

Glucocorticoid-Mediated Developmental Programming of Vertebrate Stress Responsivity

“…Experiments with the knockout line revealed that the defensive/pro-inflammatory transcriptomic response to chronic cortisol that we had previously reported in wildtype larvae was absent in klf9 −/− mutants (Gans et al, 2020), which in contrast responded by upregulating genes involved in glycolysis and related metabolic pathways (Gans et al, 2021). Furthermore, we found that klf9 expression is both oscillatory and synchronous with that of fkpb5, which appears to be directly repressed by Klf9 (Gans et al, 2021). Together these results suggest that klf9 is a key node in the gene regulatory network regulating GC responsivity (Figure 5), both by co-regulating fkbp5 in an incoherent feedforward loop with the GR, and by repressing glycolytic gene expression (possibly also via incoherent feedforward regulation) in a way that is predicted to favor flux through the pentose phosphate pathway (Gans et al, 2021).…”

“…Given the ease of adding treatments to water, zebrafish are increasingly used in screening for toxicant effects. In a screening of steroid hormones, modest decrease in activity level was found in larvae treated with GCs, along with a significant increase in trough expression level of circadian genes including per1a and nr1d2a (Zhao et al, 2018), two genes we have also found increased by chronic cortisol exposure (Gans et al, 2020(Gans et al, , 2021. Another group found that environmentally relevant concentrations of cortisol and the synthetic prescription GC clobetasol propionate impacted larval muscle and heart function, as well as expression of genes involved in immunity, glucose metabolism, development, and the circadian clock (Willi et al, 2018).…”

“…Maternal GR (nr3c1) mRNA and cortisol are deposited in the embryo but are largely depleted by the time of hatching, (~48 h post-fertilization, hpf), at which point embryonic transcription is well underway and tissues of the HPI axis are established and functional (Alsop and Vijayan, 2008a,b). We have found that the expression of GR targets fkbp5 and klf9 parallels that of nr3c1, being relatively high immediately after fertilization, then decreasing to a minimal level on days 2-3 post-fertilization before rebounding again by day 4 (Gans et al, 2021). Though embryos can produce a basal level of cortisol by 48 hpf, a measurable cortisol response to stress is not mounted until roughly 4 days post-fertilization (4 dpf).…”

“…Moreover, motif enrichment analysis of the ATAC-seq peaks scoring highest for differential accessibility in response to developmental cortisol exposure revealed significant enrichment for consensus KLF binding motifs, including for Klf9 (Hartig et al, 2020). To further interrogate the role of Klf9 in GC signaling, we used CRISPR both to create a klf9 knockout in zebrafish (Gans et al, 2020), and to introduce a C-terminal epitope tag into the endogenous locus (Gans et al, 2021). Experiments with the knockout line revealed that the defensive/pro-inflammatory transcriptomic response to chronic cortisol that we had previously reported in wildtype larvae was absent in klf9 −/− mutants (Gans et al, 2020), which in contrast responded by upregulating genes involved in glycolysis and related metabolic pathways (Gans et al, 2021).…”

“…To further interrogate the role of Klf9 in GC signaling, we used CRISPR both to create a klf9 knockout in zebrafish (Gans et al, 2020), and to introduce a C-terminal epitope tag into the endogenous locus (Gans et al, 2021). Experiments with the knockout line revealed that the defensive/pro-inflammatory transcriptomic response to chronic cortisol that we had previously reported in wildtype larvae was absent in klf9 −/− mutants (Gans et al, 2020), which in contrast responded by upregulating genes involved in glycolysis and related metabolic pathways (Gans et al, 2021). Furthermore, we found that klf9 expression is both oscillatory and synchronous with that of fkpb5, which appears to be directly repressed by Klf9 (Gans et al, 2021).…”