GSE136123
A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment
Purpose: T. gondii has a complex life cycle typified by an asexual development taking place in vertebrate and a sexual reproduction occurring exclusively in felids and thereby is less studied. The developmental transitions rely on changes in gene expression patterns, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific programs for a given stage. Here, we identified T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. MORC, in partnership with Apetala (AP2) transcription factors, was shown to recruit the histone deacetylase HDAC3, thereby impeding the chromatin accessibility of the genes predestined to be exclusively expressed in sexual stages. As such, MORC-depleted cells underwent marked transcriptional changes, resulting in the expression of a specific repertoire of genes, thus revealing a shift from asexual proliferation to sexual differentiation. MORC acts as a master regulator that directs the hierarchical expression of secondary AP2 factors, with these latter potentially contributing to the unidirectionality of the life cycle. Thus, MORC plays a cardinal role in the T. gondii life cycle, and its conditional depletion offers a way to study the parasite's sexual development in vitro, and proposes an alternative to the requirement of cat infections. Overall design: Methods: RNA profiles were generated from HFF infected cells that were left untreated or treated with IAA (500 µM) or FR235222 (50 nM). Biological duplicates were performed for each condition. Total RNAs were extracted and purified using RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was assessed using an Agilent RNA ScreenTape Analysis.  Illumina TruSEQ RNA library prep of selected polyA RNAs and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2?×?150 bp) on the Illumina Hiseq platform and generated at least 25 million reads for each sample. The RNA-Seq reads (FASTQ) were processed and analyzed using the Lasergene Genomics Suite version 15 (DNASTAR, Madison, WI, USA) using default parameters. The paired-end reads were uploaded onto the SeqMan NGen (version 15, DNASTAR. Madison, WI, USA) platform for reference-based assembly using the Toxoplasma Type II ME49 strain (ToxoDB-40, ME49 genome) as reference template. The ArrayStar module (version 15, DNASTAR. Madison, WI, USA) was used for normalization, differential gene expression and statistical analysis of uniquely mapped paired-end reads using the default parameters. The expression data quantification and normalization were calculated using the RPKM (Reads Per Kilobase of transcript per Million mapped reads) normalization method.