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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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Related in: MedlinePlus

Distribution of RNAPII along the gene length and enriched pathways. (A) The plots represent the average distribution along the gene of probes (with p <0.05) associated with each form of RNAPII. The start codon is taken as “0”. Data are organized into bins ranging from -500 bp upstream of the ATG to +4000 bp downstream and plotted against the percentage of total probes falling into each bin. The position of a probe along the x axis is the average of the positions of all probes within a given bin. While all genes are aligned at the ATG (“0”), they terminate at a wide range of positions downstream with an average at +2.5 kb. The plots on the right show the distribution of early/positive and late/positive loci (r >0.4), while those on the left plot the distribution of early/negative and late/negative loci (r <0.4). (B-C) The plots represent the average distribution of the genetic loci associated with each form of RNAPII as a function of gene length. Position distributions were plotted as histograms for each gene group with intervals of 500 bp from -2 kb to +5 kb. Histograms were plotted to display the binding positions in 500 bp intervals from -2 kb to 5 kb flanking the translational start codon at 0. A goodness-of-fit test was performed within each bin to calculate the probability of that region being equally bound by RNAPII for different gene groups. Regions with binding bias by group were assigned p <0.05 using the chi-square test. Data were assessed in this manner for (B) early/positive and late/positive and (C) early/negative and late/negative subsets. (D) Functionally enriched pathways (p <0.05) for each of the four gene groups. Colors refer to the three distinct databases that were used for pathway enrichment analysis.
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Fig4: Distribution of RNAPII along the gene length and enriched pathways. (A) The plots represent the average distribution along the gene of probes (with p <0.05) associated with each form of RNAPII. The start codon is taken as “0”. Data are organized into bins ranging from -500 bp upstream of the ATG to +4000 bp downstream and plotted against the percentage of total probes falling into each bin. The position of a probe along the x axis is the average of the positions of all probes within a given bin. While all genes are aligned at the ATG (“0”), they terminate at a wide range of positions downstream with an average at +2.5 kb. The plots on the right show the distribution of early/positive and late/positive loci (r >0.4), while those on the left plot the distribution of early/negative and late/negative loci (r <0.4). (B-C) The plots represent the average distribution of the genetic loci associated with each form of RNAPII as a function of gene length. Position distributions were plotted as histograms for each gene group with intervals of 500 bp from -2 kb to +5 kb. Histograms were plotted to display the binding positions in 500 bp intervals from -2 kb to 5 kb flanking the translational start codon at 0. A goodness-of-fit test was performed within each bin to calculate the probability of that region being equally bound by RNAPII for different gene groups. Regions with binding bias by group were assigned p <0.05 using the chi-square test. Data were assessed in this manner for (B) early/positive and late/positive and (C) early/negative and late/negative subsets. (D) Functionally enriched pathways (p <0.05) for each of the four gene groups. Colors refer to the three distinct databases that were used for pathway enrichment analysis.

Mentions: According to one model of CTD function, phosphorylation of Ser5 residues predominates near the promoter, whereas toward the end of the gene RNAPII is extensively phosphorylated on Ser2 residues [25, 26]. To explore the phosphorylation status of the CTD at different positions along the transcription unit, we examined the distribution of the three forms of RNAPII along the gene for each of the four classes, namely early and late binders that positively correlated with the mRNA levels (Figure 4A, right panels) and early and late binders that negatively correlated with mRNA levels (Figure 4A, left panels). We observed very distinct profiles for genes that show a positive correlation between mRNA levels and peak RNAPII binding either early or late in the IDC. Promoter regions in P. falciparum are predicted to lie between 300 and 1650 bp upstream of the ATG [27]. RNAPII accumulates in the promoter region of early binders, whereas for late binders RNAPII is enriched in the gene body (Figure 4A, right panel). Likewise, the Ser2/5-P form of RNAPII was concentrated within upstream presumptive promoter regions (–1,000 to –500) of genes expressed early and negatively correlating with mRNA levels (Figure 4A, left panel). On the other hand, the unmodified (CTD) and Ser5-P forms did not obviously follow this distribution. A goodness-of-fit test assigns a significant p value (<0.01) to the difference in RNAPII distribution (all three forms) between early and late binders (Figure 4B,C) suggesting that the transcriptional process in early vs late phases may be distinguished by mechanistic differences. However, it is possible that this has more to do with the transcription of shorter vs longer genes or the coupling of the splicing machinery to the CTD, since “late” genes are longer and have fewer introns, as noted above.


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)

Distribution of RNAPII along the gene length and enriched pathways. (A) The plots represent the average distribution along the gene of probes (with p <0.05) associated with each form of RNAPII. The start codon is taken as “0”. Data are organized into bins ranging from -500 bp upstream of the ATG to +4000 bp downstream and plotted against the percentage of total probes falling into each bin. The position of a probe along the x axis is the average of the positions of all probes within a given bin. While all genes are aligned at the ATG (“0”), they terminate at a wide range of positions downstream with an average at +2.5 kb. The plots on the right show the distribution of early/positive and late/positive loci (r >0.4), while those on the left plot the distribution of early/negative and late/negative loci (r <0.4). (B-C) The plots represent the average distribution of the genetic loci associated with each form of RNAPII as a function of gene length. Position distributions were plotted as histograms for each gene group with intervals of 500 bp from -2 kb to +5 kb. Histograms were plotted to display the binding positions in 500 bp intervals from -2 kb to 5 kb flanking the translational start codon at 0. A goodness-of-fit test was performed within each bin to calculate the probability of that region being equally bound by RNAPII for different gene groups. Regions with binding bias by group were assigned p <0.05 using the chi-square test. Data were assessed in this manner for (B) early/positive and late/positive and (C) early/negative and late/negative subsets. (D) Functionally enriched pathways (p <0.05) for each of the four gene groups. Colors refer to the three distinct databases that were used for pathway enrichment analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232647&req=5

Fig4: Distribution of RNAPII along the gene length and enriched pathways. (A) The plots represent the average distribution along the gene of probes (with p <0.05) associated with each form of RNAPII. The start codon is taken as “0”. Data are organized into bins ranging from -500 bp upstream of the ATG to +4000 bp downstream and plotted against the percentage of total probes falling into each bin. The position of a probe along the x axis is the average of the positions of all probes within a given bin. While all genes are aligned at the ATG (“0”), they terminate at a wide range of positions downstream with an average at +2.5 kb. The plots on the right show the distribution of early/positive and late/positive loci (r >0.4), while those on the left plot the distribution of early/negative and late/negative loci (r <0.4). (B-C) The plots represent the average distribution of the genetic loci associated with each form of RNAPII as a function of gene length. Position distributions were plotted as histograms for each gene group with intervals of 500 bp from -2 kb to +5 kb. Histograms were plotted to display the binding positions in 500 bp intervals from -2 kb to 5 kb flanking the translational start codon at 0. A goodness-of-fit test was performed within each bin to calculate the probability of that region being equally bound by RNAPII for different gene groups. Regions with binding bias by group were assigned p <0.05 using the chi-square test. Data were assessed in this manner for (B) early/positive and late/positive and (C) early/negative and late/negative subsets. (D) Functionally enriched pathways (p <0.05) for each of the four gene groups. Colors refer to the three distinct databases that were used for pathway enrichment analysis.
Mentions: According to one model of CTD function, phosphorylation of Ser5 residues predominates near the promoter, whereas toward the end of the gene RNAPII is extensively phosphorylated on Ser2 residues [25, 26]. To explore the phosphorylation status of the CTD at different positions along the transcription unit, we examined the distribution of the three forms of RNAPII along the gene for each of the four classes, namely early and late binders that positively correlated with the mRNA levels (Figure 4A, right panels) and early and late binders that negatively correlated with mRNA levels (Figure 4A, left panels). We observed very distinct profiles for genes that show a positive correlation between mRNA levels and peak RNAPII binding either early or late in the IDC. Promoter regions in P. falciparum are predicted to lie between 300 and 1650 bp upstream of the ATG [27]. RNAPII accumulates in the promoter region of early binders, whereas for late binders RNAPII is enriched in the gene body (Figure 4A, right panel). Likewise, the Ser2/5-P form of RNAPII was concentrated within upstream presumptive promoter regions (–1,000 to –500) of genes expressed early and negatively correlating with mRNA levels (Figure 4A, left panel). On the other hand, the unmodified (CTD) and Ser5-P forms did not obviously follow this distribution. A goodness-of-fit test assigns a significant p value (<0.01) to the difference in RNAPII distribution (all three forms) between early and late binders (Figure 4B,C) suggesting that the transcriptional process in early vs late phases may be distinguished by mechanistic differences. However, it is possible that this has more to do with the transcription of shorter vs longer genes or the coupling of the splicing machinery to the CTD, since “late” genes are longer and have fewer introns, as noted above.

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