Dataset Information

GCN5 associated complex in human malaria Plasmodium falciparum

ABSTRACT: The histone acetyltransferase (HAT) GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. To study GCN5 associated complex in human malaria parasite Plasmodium falciparum, PfGCN5 was tagged with PTP tag for a tandem affinity purification (TAP). We performed the TAP procedure using nuclear extracts from synchronized trophozoites of the PfGCN5::PTP parasite, which was followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) for accurate protein identification. The MS data were subjected to Significance Analysis of INTeractome (SAINT) using a threshold of probability above 94% and false discovery rate (FDR) below 1% (Teo et al., 2014). Nine proteins were identified as core components of PfGCN5 complex including a plant apetela2 (AP2)-domain transcription factor (PF3D7_0802100), three hypothetical proteins with unknown function (PF3D7_1019700, PF3D7_1364400 and PF3D7_1402800) and two PHD domain containing proteins (PF3D7_1008100 and PF3D7_1433400, named as PfPHD1 and PfPHD2). Among them, only PfGCN5 and PfADA2 are known as the components of SAGA complex, indicating PfGCN5 complex is highly divergent from SAGA complex. To confirm the subunits identified by TAP, PfPHD1 and PF3D7_1019700 were tagged with C-myc and GFP tag, respectively. Immuno-precipitations (IPs) from parasites with PfPHD1-myc and PF3D7_1019700-GFP were conducted with myc- or GFP-trap beads followed by LC-MS/MS. Indeed, the core complex were identified by these reciprocal pulldowns. To study the function of PfGCN5 complex, Bromodomain (BrD) in PfGCN5 and PHD in PfPHD1 at the C-termini of PfGCN5 and PfPHD1 were deleted and meanwhile tag the C-termini of these truncated proteins with a GFP tag for sorting parasites with truncated PfGCN5 or PfPHD1. The parasite lines with these deletions grew significantly slower with substantial defect in merozoite invasion. IPs by GFP-trap beads from these parasites were performed to reveal the integrity of PfGCN5 complex after domain deletion. IPs with the PfGCN5-∆BrD::GFP parasites confidently purified the nine core subunits of the PfGCN5 complex, suggesting the deletion of the BrD from PfGCN5 did not affect the integrity of the complex, whereas only four major components of the PfGCN5 complex (PfGCN5, PfADA2, PfPHD1, and PF3D7_1402800) were detected from the PfPHD1-∆PHD::GFP parasites. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including the AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Malaria

SUBMITTER: JUN MIAO

LAB HEAD: Jun Miao

PROVIDER: PXD023389 | Pride | 2021-08-06

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20191010_Jun_0079-GFP-1_1__F160308_.mzid_20191010_Jun_0079-GFP-1_1__F160308_.MGF	Mzid
	20191010_Jun_0079-GFP-1_2__F160309_.mzid_20191010_Jun_0079-GFP-1_2__F160309_.MGF	Mzid
	20191010_Jun_0079-GFP-1_3__F160310_.mzid_20191010_Jun_0079-GFP-1_3__F160310_.MGF	Mzid
	20191010_Jun_0079-GFP-2_1__F160311_.mzid_20191010_Jun_0079-GFP-2_1__F160311_.MGF	Mzid
	20191010_Jun_0079-GFP-2_2__F160312_.mzid_20191010_Jun_0079-GFP-2_2__F160312_.MGF	Mzid

Items per page:

1 - 5 of 76

Similar Datasets

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.

Project description:In order to identify epigenetic protein complexes recruited to histone PTMs in the deadly human malaria parasite Plasmodium falciparum, we set out to perform histone peptide pulldowns with various histone peptides. Biotinylated peptides corresponding to P. falciparum histone variant H2A.Z, H2B.Z and H3.3, canonical P. falciparum histone H4 or human histone H4 tail sequences (differing on position 21 from the P. falciparum peptide) bearing multiple histone post-translational modifications (acetylations and/or methylations) as well as the unmodified control peptides were coupled to beads and incubated with native nuclear extract obtained from mixed-stage asexual cultures. Proteins preferentially binding to the modified histone peptide were identified by quantitative proteomics using di-methyl labelling. This yielded 6 out of 7 P. falciparum bromo-domain proteins preferentially binding to the acetylated histone peptides, as well as many putative bromo-domain protein interactors. In addition, a PHD-domain containing protein was found to be recruiting a PfSAGA-like complex to H3K4 di- and tri-methylated peptides. 7 reader-domain containing proteins (BDP1, BDP2, BDP4, TAF1, GCN5, PHD1, PHD2) and 5 putative interactors (PF3D7_0306100, PF3D7_1124300, PF3D7_1128000, PF3D7_1225200, PF3D7_1451200) were endogenously tagged by GFP or HA and used in quantitative GFP-/HA-IP-MS/MS experiments to further characterize epigenetic reader-complex composition. As negative control, GFP- and HA-IPs were performed on native nuclear extract not encoding tagged protein. Most HPP experiment employed a 3-label setup, while few only relied on “light” and “heavy”-label sample pools (labels and conditions listed below). GFP- and HA-IP experiments were using a 2-label “light” and “heavy” di-methyl setup only. For all experiments technical replicates were performed using a label-swap approach (called F (forward) and R (reverse)) and independent biological replicates were performed for each. For GFP- and HA-IP forward reactions are set-up as: GFP-/HA-beads = “heavy” di-methyl label, control-beads = “light” di-methyl label. Reverse reactions are set-up as: GFP-/HA-beads = “light” di-methyl label, control-beads = “heavy” di-methyl label. For GFP-/HA-IP raw files the bait and tag used for fishing are included in the file name.

Dataset Information

GCN5 associated complex in human malaria Plasmodium falciparum

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets