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Genome-wide analysis in Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy during the infectious cycle.

Rai R, Zhu L, Chen H, Gupta AP, Sze SK, Zheng J, Ruedl C, Bozdech Z, Featherstone M - BMC Genomics (2014)

Bottom Line: Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII.We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. zbozdech@ntu.edu.sg.

ABSTRACT

Background: Over the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.

Results: We exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.

Conclusions: The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.

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Statistically significant correlations between RNAPII binding and histone modification. Binding of the (A) unmodified (CTD), (B) Ser5-P and (C) Ser2/5-P forms of RNAPII was correlated (PCC) with 13 histone modifications present either 8 h earlier (-8 h) or 8 h later (+8 h) than RNAPII binding. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications were less or greater than the function of similar correlations after 8 h shift in RNAPII or histone modification profile. For each histone modification, a mode of correlation (mc) greater than 0.4 is shown in yellow, whereas an mc less than -0.4 is in blue. A white square denotes no correlation. The correlations derived from comparison within the same time point (0 h; see Figure 5) are included for convenience. Only statistically significant correlations are shown.
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Fig6: Statistically significant correlations between RNAPII binding and histone modification. Binding of the (A) unmodified (CTD), (B) Ser5-P and (C) Ser2/5-P forms of RNAPII was correlated (PCC) with 13 histone modifications present either 8 h earlier (-8 h) or 8 h later (+8 h) than RNAPII binding. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications were less or greater than the function of similar correlations after 8 h shift in RNAPII or histone modification profile. For each histone modification, a mode of correlation (mc) greater than 0.4 is shown in yellow, whereas an mc less than -0.4 is in blue. A white square denotes no correlation. The correlations derived from comparison within the same time point (0 h; see Figure 5) are included for convenience. Only statistically significant correlations are shown.

Mentions: To determine whether some histone modifications may precede or follow RNAPII recruitment, we calculated the PCC (r) between RNAPII occupancy (all three forms) and histone modification occurring 8 h before or after recruitment (Figure 6). For example, recruitment of each form of RNAPII at T2 (16 hpi) was assessed relative to histone modifications occurring 8 h earlier at T1 (8 hpi), and 8 h later at T3 (24 hpi), and so on for all six time points. For all forms of RNAPII, we found that some histone marks precede, accompany or follow RNAPII recruitment. Thus, the activating histone mark H3K9ac precedes (and accompanies) recruitment of all forms of RNAPII (Figure 6A-C). Other marks are specific for one or more of the RNAPII isoforms. Thus, H4K16ac, a mark of actively transcribed euchromatin [35], and H4K5ac which has been shown to “bookmark” genes for rapid activation [36], show clear positive correlations with subsequent recruitment of the Ser2/5-P form of RNAPII (Figure 6C). By contrast, both H3K4me3 and H4R3me2 show positive correlation with these histone modifications following recruitment of Ser2/5-P RNAPII. Conversely, H4R3me2, associated with repression of gene expression [37, 38], shows clear negative correlation with subsequent recruitment of all forms of RNAPII (Figure 6A-C), while H4K5ac was depleted 8 h following recruitment of the RNAPIISer2/5-P form (Figure 6C). Both the positive and negative correlations are highly statistically significant by the Kolmogorov-Smirnov test (p <0.001). Other histone modifications were not seen to anticipate or follow RNAPII recruitment (Additional files 6, 7 and 8: Figure S5, S6 and S7).Figure 6


Genome-wide analysis in Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy during the infectious cycle.

Rai R, Zhu L, Chen H, Gupta AP, Sze SK, Zheng J, Ruedl C, Bozdech Z, Featherstone M - BMC Genomics (2014)

Statistically significant correlations between RNAPII binding and histone modification. Binding of the (A) unmodified (CTD), (B) Ser5-P and (C) Ser2/5-P forms of RNAPII was correlated (PCC) with 13 histone modifications present either 8 h earlier (-8 h) or 8 h later (+8 h) than RNAPII binding. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications were less or greater than the function of similar correlations after 8 h shift in RNAPII or histone modification profile. For each histone modification, a mode of correlation (mc) greater than 0.4 is shown in yellow, whereas an mc less than -0.4 is in blue. A white square denotes no correlation. The correlations derived from comparison within the same time point (0 h; see Figure 5) are included for convenience. Only statistically significant correlations are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Statistically significant correlations between RNAPII binding and histone modification. Binding of the (A) unmodified (CTD), (B) Ser5-P and (C) Ser2/5-P forms of RNAPII was correlated (PCC) with 13 histone modifications present either 8 h earlier (-8 h) or 8 h later (+8 h) than RNAPII binding. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications were less or greater than the function of similar correlations after 8 h shift in RNAPII or histone modification profile. For each histone modification, a mode of correlation (mc) greater than 0.4 is shown in yellow, whereas an mc less than -0.4 is in blue. A white square denotes no correlation. The correlations derived from comparison within the same time point (0 h; see Figure 5) are included for convenience. Only statistically significant correlations are shown.
Mentions: To determine whether some histone modifications may precede or follow RNAPII recruitment, we calculated the PCC (r) between RNAPII occupancy (all three forms) and histone modification occurring 8 h before or after recruitment (Figure 6). For example, recruitment of each form of RNAPII at T2 (16 hpi) was assessed relative to histone modifications occurring 8 h earlier at T1 (8 hpi), and 8 h later at T3 (24 hpi), and so on for all six time points. For all forms of RNAPII, we found that some histone marks precede, accompany or follow RNAPII recruitment. Thus, the activating histone mark H3K9ac precedes (and accompanies) recruitment of all forms of RNAPII (Figure 6A-C). Other marks are specific for one or more of the RNAPII isoforms. Thus, H4K16ac, a mark of actively transcribed euchromatin [35], and H4K5ac which has been shown to “bookmark” genes for rapid activation [36], show clear positive correlations with subsequent recruitment of the Ser2/5-P form of RNAPII (Figure 6C). By contrast, both H3K4me3 and H4R3me2 show positive correlation with these histone modifications following recruitment of Ser2/5-P RNAPII. Conversely, H4R3me2, associated with repression of gene expression [37, 38], shows clear negative correlation with subsequent recruitment of all forms of RNAPII (Figure 6A-C), while H4K5ac was depleted 8 h following recruitment of the RNAPIISer2/5-P form (Figure 6C). Both the positive and negative correlations are highly statistically significant by the Kolmogorov-Smirnov test (p <0.001). Other histone modifications were not seen to anticipate or follow RNAPII recruitment (Additional files 6, 7 and 8: Figure S5, S6 and S7).Figure 6

Bottom Line: Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII.We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. zbozdech@ntu.edu.sg.

ABSTRACT

Background: Over the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.

Results: We exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.

Conclusions: The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.

Show MeSH
Related in: MedlinePlus