A global search for novel transcription factors impacting the Neurospora crassa circadian clock

Abstract

Eukaryotic circadian oscillators share a common circuit architecture, a negative feedback loop in which a positive element activates the transcription of a negative one that then represses the action of the former, inhibiting its own expression. While studies in mammals and insects have revealed additional transcriptional inputs modulating the expression of core clock components, this has been less characterized in the model Neurospora crassa, where the participation of other transcriptional components impacting circadian clock dynamics remains rather unexplored. Thus, we sought to identify additional transcriptional regulators modulating the N. crassa clock, following a reverse genetic screen based on luminescent circadian reporters and a collection of transcription factors knockouts, successfully covering close to 60% of them. Besides the canonical core clock components WC-1 and WC-2, none of the tested transcriptional regulators proved to be essential for rhythmicity. Nevertheless, we identified a set of 23 transcription factors that when absent lead to discrete, but significant, changes in circadian period. While the current level of analysis does not provide mechanistic information about how these new players modulate circadian parameters, the results of this screen reveal that an important number of light and clock-regulated transcription factors, involved in a plethora of processes, are capable of modulating the clockworks. This partial reverse genetic clock screen also exemplifies how the N. crassa knockout collection continues to serve as an expedite platform to address broad biological questions.

A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

“…Our list of putative RNA-binding and RNA regulatory proteins was derived from bioinformatic databases and from the literature (Ray et al, 2013; Hogan et al, 2015; Zaveri et al, 2017; Basenko et al, 2018) to include proteins with nucleotide-binding functional annotation but exclude known transcriptional regulators. Multiple circadian period alterations were identified in a recent characterization of transcription factor knockouts (Muñoz-Guzmán et al, 2021), and Nutritional Compensation defects among transcription factors, in addition to CSP-1 and RCO-1, will be the subject of future study. Finally, a handful of manually selected candidate genes were included in the compensation screen.…”

“…Multiple circadian period alterations were identified in a recent characterization of transcription factor knockouts (Muñoz-Guzmán et al, 2021), and Nutritional Compensation defects among transcription factors, in addition to CSP-1 and RCO-1, will be the subject of future study. Finally, a handful of manually selected candidate genes were included in the compensation screen.…”

Cell-type specific circadian bioluminescence rhythms inDbpreporter mice

“…Rhythmically expressed reporter genes have been extremely important for demonstrating cell- autonomous circadian clocks and monitoring rhythmicity in several organisms, including plants (Millar et al, 1992), Neurospora (Morgan et al, 2003), cyanobacteria (Kondo et al, 1993), Drosophila (Brandes et al, 1996), zebrafish (Weger et al, 2013), cultured cells (Nagoshi et al, 2004; Hirota et al, 2010; Welsh et al, 2004; Zhang et al, 2009), rodent tissue explants (Abe et al, 2002; Maywood et al, 2013; Yamazaki et al, 2000; Yoo et al, 2004; Yoo et al, 2005), and rodent tissues in vivo (Saini et al, 2013; Tahara et al, 2012). Circadian reporter genes have been instrumental in screens to identify clock genes and modifiers in many of these systems (Cesbron et al, 2013; Chen et al, 2012; Hirota et al, 2010; Kondo et al, 1993; Millar et al, 1995; Muñoz-Guzmán et al, 2021; Stanewsky et al, 1998; Zhang et al, 2009). Circadian reporters have also been used to assess rhythmicity in peripheral tissues and the impact of alterations in experimental or environmental conditions (food availability, lighting cycles, glucocorticoid treatment) on peripheral oscillators, conducted by measuring bioluminescence rhythms in tissue explants monitored ex vivo (Davidson et al, 2008; Davidson et al, 2009; Nakamura et al, 2005; Pezuk et al, 2012; Sellix et al, 2012; Stokkan et al, 2001; Yamanaka et al, 2008; Yamazaki et al, 2000).…”

Cell-Type-Specific Circadian Bioluminescence Rhythms inDbpReporter Mice

“…Rhythmically expressed reporter genes have been extremely important for demonstrating cell-autonomous circadian clocks and monitoring rhythmicity in several organisms, including plants (Millar et al, 1992), Neurospora (Morgan et al, 2003), cyanobacteria (Kondo et al, 1993), Drosophila (Brandes et al, 1996), zebrafish (Weger et al, 2013), cultured cells (Nagoshi et al, 2004;Hirota et al, 2010;Welsh et al, 2004; E. E. Zhang et al, 2009), rodent tissue explants (Abe et al, 2002;Maywood et al, 2013;Yamazaki et al, 2000;Yoo et al, 2004Yoo et al, , 2005, and rodent tissues in vivo (Saini et al, 2013;Tahara et al, 2012). Circadian reporter genes have been instrumental in screens to identify clock genes and modifiers in many of these systems (Cesbron et al, 2013;Chen et al, 2012;Hirota et al, 2010;Kondo et al, 1993;Millar et al, 1995;Muñoz-Guzmán et al, 2021;Stanewsky et al, 1998;E. E. Zhang et al, 2009).…”

“…E. Zhang et al, 2009), rodent tissue explants (Abe et al, 2002; Maywood et al, 2013; Yamazaki et al, 2000; Yoo et al, 2004, 2005), and rodent tissues in vivo (Saini et al, 2013; Tahara et al, 2012). Circadian reporter genes have been instrumental in screens to identify clock genes and modifiers in many of these systems (Cesbron et al, 2013; Chen et al, 2012; Hirota et al, 2010; Kondo et al, 1993; Millar et al, 1995; Muñoz-Guzmán et al, 2021; Stanewsky et al, 1998; E. E. Zhang et al, 2009).…”