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Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum.

Gupta AP, Chin WH, Zhu L, Mok S, Luah YH, Lim EH, Bozdech Z - PLoS Pathog. (2013)

Bottom Line: While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution.In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription.Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, Plasmodium falciparum. To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the P. falciparum intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the P. falciparum IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5' ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.

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Histone modification patterns on ectopically integrated promoters.(A) Cloning strategy for ectopic integration of promoter regions. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods). P. falciparum strain Dd2attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.
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ppat-1003170-g005: Histone modification patterns on ectopically integrated promoters.(A) Cloning strategy for ectopic integration of promoter regions. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods). P. falciparum strain Dd2attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.

Mentions: Presently, very little is known about the mechanisms of chromatin remodeling in Plasmodium parasites. Given the highly dynamic character of histone modifications observed by this as well as previous studies [13], [20], [22], these mechanisms are likely to be highly evolutionarily diverse. Transcription factors bound to promoter and upstream regions are known to recruit chromatin modifiers in other species. We therefore investigated the role of promoter regions in the recruitment of H4K8ac that we found mainly in the upstream regions of active genes. In particular, we wanted to assess the presence of any DNA elements which help to establish histone marks in promoter regions. Four promoters (1.5–2 Kb upstream of the ATG) marked by H4K8ac were selected and cloned into luciferase reporter constructs including upstream regions of ring-specific (MAL13P1.122), trophozoite-specific (PF14_0705), schizont-specific (PFD0240c) and sporozoite-specific (PFC0210c) genes. Here, we made use of the strain Dd2attB[32] in which transgenes can be integrated at the cg6 locus (Figure 5A). We found that the occupancy profile of H4K8ac was recapitulated on three of the four ectopic promoters (Figure 5B). These profiles override an existing profile of the endogenous cg6 gene (dashed line) that is normally characterized by high levels in rings and gradually declines through trophozoites and schizonts. The luciferase activity profiles were also similar to the acetylation patterns of all transfected promoters (data not shown). For one of the promoters (PF14_0705), there was an incomplete “carry-over” of the H4K8ac occupancy profile that was matched only in the ring stage. This may be due to unknown factors like insufficient promoter length. Overall our data suggest that the promoter regions of P. falciparum genes carry DNA regulatory elements that establish H4K8ac independently of their endogenous chromatin environment.


Dynamic epigenetic regulation of gene expression during the life cycle of malaria parasite Plasmodium falciparum.

Gupta AP, Chin WH, Zhu L, Mok S, Luah YH, Lim EH, Bozdech Z - PLoS Pathog. (2013)

Histone modification patterns on ectopically integrated promoters.(A) Cloning strategy for ectopic integration of promoter regions. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods). P. falciparum strain Dd2attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585154&req=5

ppat-1003170-g005: Histone modification patterns on ectopically integrated promoters.(A) Cloning strategy for ectopic integration of promoter regions. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods). P. falciparum strain Dd2attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.
Mentions: Presently, very little is known about the mechanisms of chromatin remodeling in Plasmodium parasites. Given the highly dynamic character of histone modifications observed by this as well as previous studies [13], [20], [22], these mechanisms are likely to be highly evolutionarily diverse. Transcription factors bound to promoter and upstream regions are known to recruit chromatin modifiers in other species. We therefore investigated the role of promoter regions in the recruitment of H4K8ac that we found mainly in the upstream regions of active genes. In particular, we wanted to assess the presence of any DNA elements which help to establish histone marks in promoter regions. Four promoters (1.5–2 Kb upstream of the ATG) marked by H4K8ac were selected and cloned into luciferase reporter constructs including upstream regions of ring-specific (MAL13P1.122), trophozoite-specific (PF14_0705), schizont-specific (PFD0240c) and sporozoite-specific (PFC0210c) genes. Here, we made use of the strain Dd2attB[32] in which transgenes can be integrated at the cg6 locus (Figure 5A). We found that the occupancy profile of H4K8ac was recapitulated on three of the four ectopic promoters (Figure 5B). These profiles override an existing profile of the endogenous cg6 gene (dashed line) that is normally characterized by high levels in rings and gradually declines through trophozoites and schizonts. The luciferase activity profiles were also similar to the acetylation patterns of all transfected promoters (data not shown). For one of the promoters (PF14_0705), there was an incomplete “carry-over” of the H4K8ac occupancy profile that was matched only in the ring stage. This may be due to unknown factors like insufficient promoter length. Overall our data suggest that the promoter regions of P. falciparum genes carry DNA regulatory elements that establish H4K8ac independently of their endogenous chromatin environment.

Bottom Line: While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution.In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription.Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, Plasmodium falciparum. To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the P. falciparum intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the P. falciparum IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5' ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.

Show MeSH
Related in: MedlinePlus