Project description:ChIP-seq experiments were performed for the putative telomere repeat-binding factor (PfTRF) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRF at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRF was enriched at seven additional, intra-chromosomal sites and called in the PfTRF-HA ChIP-seq only. Plasmodium falciparum 3D7 parasites were generated with -GFP or -3xHA C-terminal tagged TRF (PF3D7_1209300). Nuclei were isolated from formaldehyde cross-linked schizont-stage transgenic parasites and used to prepare chromatin. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001) or rat anti-HA 3F10 (Roche Diagnostics, #12158167001). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.
Project description:In this study we have investigated PfAP2-HC (PF3D7_1456000), a protein that was identified by co-immunoprecipitation with PfHP1 coupled with liquid chromatography-tandem mass spectrometry. PfAP2-HC belongs to the ApiAP2 family, the main transcription factor family in Apicomplexan parasites. We have confirmed that AP2-HC colocalises with HP1 with the use of immunofluorescence assays and chromatin immunoprecipitation-sequencing. We show that PfAP2-HC is not required for heterochromatin maintenance and inheritance with the use of PfAP2-HC Kock out and knockdown. We show with transcriptome-wide microarray time course analysis that PfAP2-HC does not act as a transcription factor in blood stage parasites. We demonstrate that the AP2 domain is dispensable for heterochromatin targeting by introducing a premature stop codon before the AP2 domain. We show that PfAP2-HC binding to heterochromatin dependents on PfHP1. and PfAP2-HC is likely not involved in de novo heterochromatin formation
Project description:Differentiation from asexual blood stages to sexual gametocytes is required for transmission of malaria parasites from the human to the mosquito host. Preventing gametocyte commitment and development would block parasite transmission, but the underlying molecular mechanisms behind these processes remain poorly understood. Here, we report that the ApiAP2 transcription factor, PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes. PfAP2-G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2-G2 within the gene body of almost 3000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2-G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption of pfap2-g2 does not impact asexual development, parasite multiplication rate, or commitment to sexual development but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence of pfap2-g2 leads to overexpression of 28% of the genes bound by PfAP2-G2 and none of the PfAP2-g2 bound are downregulated, suggesting that it is a repressor. We also find that PfAP2-G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2-G2 is an important transcription factor that establishes an essential gametocyte maturation program in association with other chromatin-related proteins.
Project description:Differentiation from asexual blood stages to sexual gametocytes is required for transmission of malaria parasites from the human to the mosquito host. Preventing gametocyte commitment and development would block parasite transmission, but the underlying molecular mechanisms behind these processes remain poorly understood. Here, we report that the ApiAP2 transcription factor, PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes. PfAP2-G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2-G2 within the gene body of almost 3000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2-G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption of pfap2-g2 does not impact asexual development, parasite multiplication rate, or commitment to sexual development but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence of pfap2-g2 leads to overexpression of 28% of the genes bound by PfAP2-G2 and none of the PfAP2-g2 bound are downregulated, suggesting that it is a repressor. We also find that PfAP2-G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2-G2 is an important transcription factor that establishes an essential gametocyte maturation program in association with other chromatin-related proteins.
Project description:To determine the genome-wide occupancy of the Plasmodium falciparum transcriptional regulator of invasion PfAP2-I (PfDd2_100013100/PF3D7_1007700), we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Synchronized, schizont stage, 40 hours post-invasion, cultures of parasites expressing the AP2-I-GFP fusion protein were treated with formaldehyde to crosslink proteins to DNA and harvested. After shearing the DNA, the chromatin was incubated with anti-GFP antibody or IgG (as control) for immunoprecipitation. This material was used to generate Illumina sequencing libraries. The final libraries were multiplexed with fourteen barcoded samples per lane on an Illumina HiSeq 2500 system to generate 150 base pair single-end reads.
Project description:ChIP-seq using anti-GFP antibody to map genomic binding of Prdm4-EGFP in stably transfected mouse embryonic stem cells Two independent ChIP-seq replicates for each of two independent Prdm4-EGFP expressing ES cell clones resulting in four anti-GFP ChIP-seq experiments. These samples have paired mouse IgG control.