PfMORC protein regulates chromatin accessibility and transcriptional repression in the human malaria parasite, P. falciparum
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eLife assessment
This valuable study underscores the significance of PfMORC in shaping chromatin and guiding transitions in the malaria parasite, Plasmodium falciparum, that are essential for its survival. Solid evidence reveals PfMORC's influence on genes related to antigenic variation and the parasite's lifecycle, marking PfMORC as a key regulator of parasite heterochromatin.
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Abstract
The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light into the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggest that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but is also most likely a key modulator of stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.
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Author Response
Reviewer #1 (Public Review):
Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.
Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. …
Author Response
Reviewer #1 (Public Review):
Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.
Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.
We greatly appreciate the overall positive feedback . Our application of CRISPR/Cas9 genome editing tools coupled with complementary cellular and functional approaches shed light on the importance ofPfMORC in maintaining chromatin structural integrity in the parasite and highlight this protein as a promising target for novel therapeutic intervention.
Weaknesses: Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation.
Our conclusions were made on the basis of multiple, unbiased molecular and functional assays that point to the relevance of the PfMORC protein in maintaining the parasite’s chromatin landscape. Although we do not claim to have precise evidence on the step-by-step pathway to which PfMORC is involved, we bring forth first-hand evidence of its overall function in heterochromatin binding and gene-regulation, its association with major TF regulatory players, and essentiality for parasite survival. We however agree with the comment regarding the lack of direct effects of PfMORC KD and will provide additional evidence by performing ChIP-seq experiments against additional histone marks in WT and PfMORC KD lines.
Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.
We do agree with the reviewer's comment. Validation of the identified interacting partners is critical and most likely essential to understanding their role in directing MORC to its targets. However, our protein pull down experiments have been done using biological replicates. Several of the interacting partners have also been identified and published by other labs. A direct comparison of our work together with previous published work will be incorporated in a revised version of the manuscript to further validate the identified interacting partners and the accuracy of the data we obtained in this manuscript. Molecular validation of all proteins identified in our protein may take a few more years and will be submitted for publication in futur manuscripts.
Reviewer #2 (Public Review):
Summary: This paper, titled "Regulation of Chromatin Accessibility and Transcriptional Repression by PfMORC Protein in Plasmodium falciparum," delves into the PfMORC protein's role during the intra-erythrocytic cycle of the malaria parasite, P. falciparum. Le Roch et al. examined PfMORC's interactions with proteins, its genomic distribution in different parasite life stages (rings, trophozoites, schizonts), and the transcriptome's response to PfMORC depletion. They conducted a chromatin conformation capture on PfMORC-depleted parasites and observed significant alterations. Furthermore, they demonstrated that PfMORC depletion is lethal to the parasite.
Strengths: This study significantly advances our understanding of PfMORC's role in establishing heterochromatin. The direct consequences of the PfMORC depletion are addressed using chromatin conformation capture.
We appreciate the Reviewer’s comments and reflection on the importance of our work.
Weaknesses: The study only partially addressed the direct effects of PfMORC depletion on other heterochromatin markers.
Here again, we agree with the reviewer’s comment and intend to perform additional experiments to delve deeper into the multifaceted roles of PfMORC. We have begun to explore the effects of PfMORC depletion on heterochromatin marks using ChIP-seq experiments at distinct stages of parasite development. We hope our new results will shed light on the direct implications of PfMORC in heterochromatin regulation.
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eLife assessment
This valuable study underscores the significance of PfMORC in shaping chromatin and guiding transitions in the malaria parasite, Plasmodium falciparum, that are essential for its survival. Solid evidence reveals PfMORC's influence on genes related to antigenic variation and the parasite's lifecycle, marking PfMORC as a key regulator of parasite heterochromatin.
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Reviewer #1 (Public Review):
Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.
Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq …
Reviewer #1 (Public Review):
Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.
Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.
Weaknesses: Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation. Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.
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Reviewer #2 (Public Review):
Summary: This paper, titled "Regulation of Chromatin Accessibility and Transcriptional Repression by PfMORC Protein in Plasmodium falciparum," delves into the PfMORC protein's role during the intra-erythrocytic cycle of the malaria parasite, P. falciparum. Le Roch et al. examined PfMORC's interactions with proteins, its genomic distribution in different parasite life stages (rings, trophozoites, schizonts), and the transcriptome's response to PfMORC depletion. They conducted a chromatin conformation capture on PfMORC-depleted parasites and observed significant alterations. Furthermore, they demonstrated that PfMORC depletion is lethal to the parasite.
Strengths: This study significantly advances our understanding of PfMORC's role in establishing heterochromatin. The direct consequences of the PfMORC …
Reviewer #2 (Public Review):
Summary: This paper, titled "Regulation of Chromatin Accessibility and Transcriptional Repression by PfMORC Protein in Plasmodium falciparum," delves into the PfMORC protein's role during the intra-erythrocytic cycle of the malaria parasite, P. falciparum. Le Roch et al. examined PfMORC's interactions with proteins, its genomic distribution in different parasite life stages (rings, trophozoites, schizonts), and the transcriptome's response to PfMORC depletion. They conducted a chromatin conformation capture on PfMORC-depleted parasites and observed significant alterations. Furthermore, they demonstrated that PfMORC depletion is lethal to the parasite.
Strengths: This study significantly advances our understanding of PfMORC's role in establishing heterochromatin. The direct consequences of the PfMORC depletion are addressed using chromatin conformation capture.
Weaknesses: The study only partially addressed the direct effects of PfMORC depletion on other heterochromatin markers.
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