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Transcriptomic changes in P. falciparum upon knockdown of PfGCN5

ABSTRACT: This study is to analyze the overal transcriptomic alteration after knockdown of PfGCN5 by TetR-DOZI system. The transcriptomic changes of a parasite line (TetR-PfGCN5::GFP) after PfGCN5 knockdown (KD) by withdral of anhydrotetracycline (aTc) compared to its control (TetR-PfGCN5::GFP with aTc) were analyzed during asexual stage development fron ring, to schizont stage by RNAseq. Total RNA were harvested from four stage (ring, early trophozoite, late trophozoite, and schizont) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified over 5 thousands transcripts with high mapping rate at the range between 80% and 95. Differential expression analysis showed that PfGCN5 KD profoundly changed the global transcription pattern. GO enrichment analysis indicated that PfGCN5 was involved in general and parasite-specific gene transcription. Collectively, transcriptomic analysis of PfGCN5 KD shows that PfPfGCN5 plays important role in global gene regulation .

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE284301 | GEO | 2025/06/09

REPOSITORIES: GEO

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Project description:Purpose: In malaria parasite, PfGCN5, a histone acetyltansferase (HAT),forms a unique complex including a PHD domain containing protein named PfPHD1. To understand the function of this complex, the BrD in PfGCN5 and PHD in PfPHD1 were deleted. The transcritional changes after domain deletions were measured by RNA-seq. Methods: The mRNA profiles of parasite lines : 3D7 (WT), PfGCN5-ΔBrD and PfPHD1-ΔPHD, were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500 in Rapid Run mode using 100nt single read sequencing. Three replicates of total RNA from parasites at ring, early trophozoite, late trophozoite and schizont stages were harvested by using the ZYMO RNA purification kit, and used to generate the sequencing libraries using the KAPA Stranded mRNA Seq kit for the Illumina sequencing platform according to the manufacturer's protocol (KAPA biosystems). Quality trimming and adapter sequence was performed using Trimmomatic (v0.36). Low quality bases were trimmed using a sliding window approach with a minimum average quality of 18 in 4 base pair window. The Reads with length <35 base pairs were removed. The trimmed reads were mapped to the P. falciparum genome sequence (Genedb v3.1) using HISAT2 (Kim et al., 2015). The coverage was analyzed by using the bedtools (Quinlan, 2014). The expression levels and the differential expression were calculated by FeatureCounts and DESeq2 (Anders and Huber, 2010; Liao et al., 2014). Results: Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified 5643 transcripts with high mapping rate at the range between 80% and 95. PfGCN5 BrD deletion profoundly disturbed the global transcription pattern, causing 3533 (62.6%) genes to be differentially expressed in at least one of the four time points analyzed. Specifically, BrD deletion resulted in down-regulation of 997, 799, 861 and 902 genes, and up-regulation of 1127, 780, 846, and 368 genes at the ring, early trophozoite, late trophozoite, and schizont stage, respectively. PHD deletion in PfPHD1 caused a similar but more profound disturbance of gene expression during the IDC, with 3870 (68.6%) transcripts being differentially expressed in at least one of the four stages analyzed. The PfPHD1 PHD deletion resulted in the down-regulation of 872, 1021, 557, and 787 genes, and up-regulation of 1481, 1266, 648, and 1028 genes at the ring, early trophozoite, late trophozoite and schizont stage, respectively. Conclusions: Either domain deletion profoundly disturbed the global transcription pattern, causing altered expression in more than 60% of the genes including general cellular pathways such as DNA replication and translation, and parasite-specific pathways such as invasion, cytoadherence, and sexual development.

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Transcriptomic changes in P. falciparum upon knockdown of PfGCN5

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