TRA-1 ChIP-seq reveals regulators of sexual differentiation and multilevel feedback in nematode sex determination

Abstract

How sexual regulators translate global sexual fate into appropriate local sexual differentiation events is perhaps the least understood aspect of sexual development. Here we have used ChIP followed by deep sequencing (ChIP-seq) to identify direct targets of the nematode global sexual regulator Transformer 1 (TRA-1), a transcription factor acting at the interface between organismwide and cell-specific sexual regulation to control all sex-specific somatic differentiation events. We identified 184 TRA-1-binding sites in Caenorhabditis elegans, many with temporal-and/or tissuespecific TRA-1 association. We also identified 78 TRA-1-binding sites in the related nematode Caenorhabditis briggsae, 19 of which are conserved between the two species. Some DNA segments containing TRA-1-binding sites drive male-specific expression patterns, and RNAi depletion of some genes adjacent to TRA-1-binding sites results in defects in male sexual development. TRA-1 binds to sites adjacent to a number of heterochronic regulatory genes, some of which drive male-specific expression, suggesting that TRA-1 imposes sex specificity on developmental timing. We also found evidence for TRA-1 feedback regulation of the global sex-determination pathway: TRA-1 binds its own locus and those of multiple upstream masculinizing genes, and most of these associations are conserved in C. briggsae. Thus, TRA-1 coordinates sexual development by reinforcing the sex-determination decision and directing downstream sexual differentiation events.

Sex‐specific regulation of aging in Caenorhabditis elegans

“…An unbiased TRA‐1‐specific ChIP‐seq study performed at four different developmental time points also identified TRA‐1 binding to daf‐16 (Berkseth et al., 2013), but the binding sites the authors determined in the daf‐16 locus are not the same as those we identified in this study. Here, we carried out ChIP‐qPCR (more sensitive and powerful than ChIP‐seq) on ultrasound fragmented chromatin to 500‐bp‐long fragments, prepared from mixed‐stage animals.…”

“…One of these consensus TRA‐1‐binding sites is located at 3‐kilobase (kb) upstream of the daf‐16d/f isoforms, while the other is located within the first exon of the daf‐16a isoform (exonic sequences often serve as binding elements for transcriptions factors; Stergachis et al., 2013). The ChIP‐seq analysis provided by Berkseth and colleagues also identified two potential TRA‐1‐binding sites in the daf‐16 coding region (Berkseth et al., 2013) which are however slightly diverged from the canonical one (Conradt & Horvitz, 1999; Hargitai et al., 2009) and the daf‐16 ‐specific TRA‐1‐binding sites identified by our present study (Figure 2a). To assess the functionality of these potential TRA‐1‐binding sites, we generated two transgenic strains expressing isoform‐specific gfp ‐ (green fluorescent protein) tagged daf‐16 reporter constructs, daf‐16d/f::gfp and daf‐16a::gfp (Figure 2a).…”

“…In other words, TRA‐1 strengthens the function of daf‐16 in aging control, explaining why hermaphrodites live significantly longer than males in populations containing both sexes. At first sight, these results were somewhat unexpected as TRA‐1 had previously been known as a transcriptional repressor rather than an activator (Berkseth et al., 2013; Chen & Ellis, 2000; Conradt & Horvitz, 1999; Hargitai et al., 2009; Mason et al., 2008; Schwartz & Horvitz, 2007; Szabó et al., 2009; Yi et al., 2000). In case of mammalian GLI proteins, however, both activation and inhibition functions were observed (Hui & Angers, 2011).…”

“…The sites were also found in the orthologous genomic regions of C. briggsae , a closely related Caenorhabditis species (Figure 2a). These data, together with results provided by a previous chromatin immunoprecipitation assay followed by deep sequencing (ChIP‐seq; Berkseth et al., 2013), raised the possibility of a direct regulatory interaction between TRA‐1 and daf‐16 isoforms involved in aging control. One of these consensus TRA‐1‐binding sites is located at 3‐kilobase (kb) upstream of the daf‐16d/f isoforms, while the other is located within the first exon of the daf‐16a isoform (exonic sequences often serve as binding elements for transcriptions factors; Stergachis et al., 2013).…”

“…Somatic sexual fates in C. elegans are specified by the global sex‐determination pathway, the terminal effector of which is the transcription factor TRA‐1 (sexual transformer) that exists in two major isoforms, A and B (Zarkower & Hodgkin, 1992). TRA‐1 is orthologous to mammalian GLI (glioma‐associated) and Drosophila Ci (Cubitus interruptus) proteins and determines hermaphrodite features by repressing the expression of male‐specific genes (Berkseth, Ikegami, Arur, Lieb & Zarkower, 2013; Chen & Ellis, 2000; Conradt & Horvitz, 1999; Hargitai et al., 2009; Mason, Rabinowitz & Portman, 2008; Schwartz & Horvitz, 2007; Szabó et al., 2009; Yi, Ross & Zarkower, 2000). In males, TRA‐1 appears to play only a minor role (Schvarzstein & Spence, 2006).…”