Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity

Abstract

The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR 369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR 369larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously. The vertebrate hypothalamus-pituitary-adrenal (HPA) axis orchestrates physiological, behavioral, and metabolic adjustments required for homeostasis, by dynamically regulating production and secretion of adrenal steroids known as glucocorticoids. In humans the primary glucocorticoid is cortisol, the biological activity of which is mediated by two regulatory proteins in the nuclear receptor family, the ubiquitous glucocorticoid receptor (GR) and the more tissue-restricted mineralocorticoid receptor (MR). The GR binds cortisol less avidly than the MR and is thus more dynamically regulated over the normal physiological range of cortisol fluctuations 1,2. The GR and MR function both as transcription factors and as non-nuclear signaling proteins, including in the central nervous system where both proteins are highly expressed 1-5. Given that the GR is more widely expressed and more dynamically regulated by cortisol, it is generally thought to be the principal mediator of cortisolinduced genomic responses to circadian rhythms and acute stress 5. An important question for understanding GR function is what downstream transcriptional regulatory genes does it regulate, and to what end? Answering this question is not only important for understanding the physiological function and regulation of the GR, but also for deciphering the gene regulatory networks that orchestrate adaptive developmental programming in response to chronic glucocorticoid exposure such as occurs with chronic early life stress 6. The zebrafish has recently emerged as a model system well-suited to investigating the developmental functions of glucocorticoid signaling and mechanisms underlying stress-induced developmental programming 7-...

Glucocorticoid receptor activities in the zebrafish model: a review

“…Another key point of Gr function is related to reproduction, since GCs have been proposed to play a crucial role in this process both in mammals (Whirledge & Cidlowski 2017) and in fish (Faught & Vijayan 2018b). Zebrafish gr homozygous mutants are genetically viable, but their fertility is significantly reduced, in particular with aging (Facchinello et al 2017), a result confirmed also by Gans et al (2020). As hypothesised by Faught & Vijayan (2018b), our analysis on gr ia30/ia30 females confirms that GCs and Gr control ovarian maturation and the ovulation process.…”

“…The VBA assay is based on the neuroendocrine response determining the shrinkage of zebrafish melanocytes when larvae are exposed to bright background (Kurrasch et al 2009). Nowadays, this test is used in strains defective in the synthesis of GCs for the preliminary selection of homozygous recessive larvae, in which this response is lost (Griffiths et al 2012, Griffin et al 2016, Eachus et al 2017, Facchinello et al 2017, Weger et al 2018, Gans et al 2020, Marchi et al 2020.…”

“…Recently, Gans et al (2020), analyzed the transcriptomes of WT and gr mutants (gr 369/369 ) upon chronic cortisol treatment. The outcome of a transcriptional activity in WT larvae similar to that of gr 369/369 when chronically treated with cortisol, is explained by the authors with the development of Gr resistance.…”

“…The outcome of a transcriptional activity in WT larvae similar to that of gr 369/369 when chronically treated with cortisol, is explained by the authors with the development of Gr resistance. Moreover, they identified the transcription factor Kruppel-like factor 9 (Klf9), for which they generated a mutant line, as a crucial player in the regulation of the transcriptional response to GCs (Gans et al 2020).…”

Single-cell multi-omics reveals elevated plasticity and stem-cell-like blasts relevant to the poor prognosis ofKMT2A-rearranged leukemia

“…NR3C1 encodes the glucocorticoid receptor and previous studies have reported that NR3C1 is important for glucocorticoid treatment response in various types of acute leukemia (Haowen et al, 2018;Wandler et al, 2020). KLF9 has been reported as a gene that responds to glucocorticoid signals (Gans et al, 2020;Reddy et al, 2009). It is still unknown how NR3C1 mediates glucocorticoid treatment response in different age groups of infant KMT2A-r ALL.…”