Periodic Reporting for period 4 - CHROMATADS (Chromatin Packing and Architectural Proteins in Plants)
Periodo di rendicontazione: 2022-07-01 al 2023-12-31
The broad objective of this project is to investigate the biological meaning of chromatin packing in plants and to reveal functional regulatory elements hidden behind the linear genome. In metazoans, the hierarchy of the three-dimensional genome strongly correlates with local gene expression and many other genomic features. However, the functional and structural partition of chromatin domains and the structural proteins regulating local chromatin packing in plants remain unexplored. From a long-term perspective, such knowledge is indispensable for us to gain a good understanding of miscellaneous chromatin activities in living cells and to implement structure-informed plant genome engineering toward varieties with desired and consistent agronomic traits.
In the CHROMATADS project, we aim to understand the principles underlying functional demarcation of the plant genome, as well as the role of stable/dynamic local chromatin packing during stress adaptation. The CHROMATADS project contains several work packages which can be divided into two research areas. Firstly, it aims to profile plant chromatin domains from a three-dimensional perspective, for example, chromatin domains insulated from each other, or chromatin domains with preferential localization with respect to the nuclear envelope; secondly, it aims to identify the architectural protein(s) responsible for the formation of these chromatin domains, hence providing molecular insights into three-dimensional plant genome organization.
At the end, this project has accomplished most of its goals. Among the most notable discoveries is the identification of the PDS5A gene's pivotal role in regulating TAD formation in Arabidopsis, a finding that challenges previous assumptions and opens new avenues for understanding genome organization in plants. Furthermore, our investigations into Arabidopsis CRWN1 proteins and plant nuclear lamin proteins have provided novel insights into the mechanisms underlying nuclear organization and gene regulation in plants. These findings not only contribute to our understanding of plant nuclear structure and genome architecture but also have implications for agricultural research, offering potential targets for improving plant stress tolerance and crop productivity. In addition to the aforementioned achievements, our project has also yielded significant contributions to the understanding of Marchantia's 3D chromatin structure, whereby we discovered a novel type of TAD in Marchantia, demonstrating that plant TADs can be enriched with transcription factor proteins, defining a distinct nuclear compartment crucial for transcriptional regulation.
In the CHROMATADS project, we aim to understand the principles underlying functional demarcation of the plant genome, as well as the role of stable/dynamic local chromatin packing during stress adaptation. The CHROMATADS project contains several work packages which can be divided into two research areas. Firstly, it aims to profile plant chromatin domains from a three-dimensional perspective, for example, chromatin domains insulated from each other, or chromatin domains with preferential localization with respect to the nuclear envelope; secondly, it aims to identify the architectural protein(s) responsible for the formation of these chromatin domains, hence providing molecular insights into three-dimensional plant genome organization.
At the end, this project has accomplished most of its goals. Among the most notable discoveries is the identification of the PDS5A gene's pivotal role in regulating TAD formation in Arabidopsis, a finding that challenges previous assumptions and opens new avenues for understanding genome organization in plants. Furthermore, our investigations into Arabidopsis CRWN1 proteins and plant nuclear lamin proteins have provided novel insights into the mechanisms underlying nuclear organization and gene regulation in plants. These findings not only contribute to our understanding of plant nuclear structure and genome architecture but also have implications for agricultural research, offering potential targets for improving plant stress tolerance and crop productivity. In addition to the aforementioned achievements, our project has also yielded significant contributions to the understanding of Marchantia's 3D chromatin structure, whereby we discovered a novel type of TAD in Marchantia, demonstrating that plant TADs can be enriched with transcription factor proteins, defining a distinct nuclear compartment crucial for transcriptional regulation.
Work Package (WP) 1 in this project mainly covered the work concerning the first research area. In this WP, we discovered that the Arabidopsis CRWN1 proteins interacted directly with chromatin at the nuclear periphery to regulate perinuclear chromatin localization (Hu et al., 2019, doi: 10.1186/s13059-019-1694-3). Following this work, we discovered that the plant nuclear lamina was more dynamic than expected, disassembling under various abiotic stress conditions (Wang et al., 2023, doi: 10.1038/s41477-023-01457-2). In addition, we performed extended work on nuclear lamin proteins in Marchantia, which took advantage of the achievement on the Marchantia genome assembly (achieved in WP2). This work led to the functional characterization of the Marchantia CRWN1 homolog (Wang et al., 2021, doi:10.3389/fpls.2021.670306).
The work concerning the second research area consisted of three WPs (WP2-WP4). WP2 aimed to identify chromatin insulation in Marchantia, providing hints to WP3 to determine candidate insulator proteins for functional studies. In this WP, by collaborating with colleagues, we improved the Marchantia male genome assembly from a scaffold to a chromosomal level, allowing comprehensive profiling of the epigenomic landscape and 3D chromatin interaction patterns (Montgomery et al., 2020, doi:10.1016/j.cub.2019.12.015). Subsequently, we participated in collaborating projects to reveal imprinting by H3K27me3 during Marchantia reproduction (Montgomery et al., 2022, doi: 10.7554/eLife.79258) and to scaffold the female sex chromosome (Iwasaki et al., 2021, doi:10.1016/j.cub.2021.10.023).
In WP3, as initially proposed for this project, Marchantia was selected as the model species to investigate the role of plant-specific TCP transcription factors in 3D genome organization. Contrary to our initial hypothesis, our study provided novel insights by rejecting the notion of TCP factors functioning as architectural proteins in this context. However, our investigation yielded an unexpected discovery: the identification of a new type of Topologically Associating Domain (TAD) in Marchantia. This finding represents a significant departure from the established understanding of TADs, as we demonstrated for the first time that transcription factor proteins can densely bind plant TADs (Karaaslan et al., 2020, doi: 10.1038/s41477-020-00766-0).
In WP4, we characterized Marchantia transcription factors that might intensively interact with TADs as TCP. In total, eleven transcription factors’ protein-chromatin interactions have been profiled, which collectively suggests that Marchantia TADs function as hubs integrating multiple transcription factors. Besides, in a collaborating project, we discovered that Arabidopsis pds5a mutants developed prominent TAD structures across the Arabidopsis genome. We consider our finding that the Arabidopsis PDS5A protein suppresses TAD formation ground-breaking because it brings an end to the long-standing speculation about the absence of TAD structure in Arabidopsis; meanwhile, it marks the commencement of the efforts in the plant community to decipher the molecular mechanisms behind plant TAD regulation (Göbel et al., under review).
The work concerning the second research area consisted of three WPs (WP2-WP4). WP2 aimed to identify chromatin insulation in Marchantia, providing hints to WP3 to determine candidate insulator proteins for functional studies. In this WP, by collaborating with colleagues, we improved the Marchantia male genome assembly from a scaffold to a chromosomal level, allowing comprehensive profiling of the epigenomic landscape and 3D chromatin interaction patterns (Montgomery et al., 2020, doi:10.1016/j.cub.2019.12.015). Subsequently, we participated in collaborating projects to reveal imprinting by H3K27me3 during Marchantia reproduction (Montgomery et al., 2022, doi: 10.7554/eLife.79258) and to scaffold the female sex chromosome (Iwasaki et al., 2021, doi:10.1016/j.cub.2021.10.023).
In WP3, as initially proposed for this project, Marchantia was selected as the model species to investigate the role of plant-specific TCP transcription factors in 3D genome organization. Contrary to our initial hypothesis, our study provided novel insights by rejecting the notion of TCP factors functioning as architectural proteins in this context. However, our investigation yielded an unexpected discovery: the identification of a new type of Topologically Associating Domain (TAD) in Marchantia. This finding represents a significant departure from the established understanding of TADs, as we demonstrated for the first time that transcription factor proteins can densely bind plant TADs (Karaaslan et al., 2020, doi: 10.1038/s41477-020-00766-0).
In WP4, we characterized Marchantia transcription factors that might intensively interact with TADs as TCP. In total, eleven transcription factors’ protein-chromatin interactions have been profiled, which collectively suggests that Marchantia TADs function as hubs integrating multiple transcription factors. Besides, in a collaborating project, we discovered that Arabidopsis pds5a mutants developed prominent TAD structures across the Arabidopsis genome. We consider our finding that the Arabidopsis PDS5A protein suppresses TAD formation ground-breaking because it brings an end to the long-standing speculation about the absence of TAD structure in Arabidopsis; meanwhile, it marks the commencement of the efforts in the plant community to decipher the molecular mechanisms behind plant TAD regulation (Göbel et al., under review).
The unexpected results from this project turned out to be the most significant. Among them, the discovery of the PDS5A gene as a critical regulator of TAD (Topologically Associating Domain) formation in Arabidopsis is the most important one, although it was made in the last year of the funding period and the corresponding manuscript is still under peer review. In this work, we and our collaborators discovered that the Arabidopsis pds5a mutant formed TADs throughout the genome. While many plant species display TADs, they have been curiously sparse in the model plant Arabidopsis thaliana, which has puzzled the plant community for more than ten years. This finding likely marks the commencement of the plant community in deciphering the molecular mechanisms behind plant TAD regulation.
The next unexpected progress resulted from a strategic shift in one of the project’s work packages, which allowed us to discover that Arabidopsis CRWN1 proteins were required to tether chromatin at the nuclear periphery. This work unveiled the molecular mechanisms responsible for perinuclear chromatin anchoring in plants, a discovery made nearly twenty years following similar findings in animals. Later, we further discovered that the plant nuclear lamina was highly dynamic under abiotic stresses. Together, these findings represent a milestone in understanding the role of plant nuclear lamin proteins in regulating gene expression and genome organization.
The next unexpected progress resulted from a strategic shift in one of the project’s work packages, which allowed us to discover that Arabidopsis CRWN1 proteins were required to tether chromatin at the nuclear periphery. This work unveiled the molecular mechanisms responsible for perinuclear chromatin anchoring in plants, a discovery made nearly twenty years following similar findings in animals. Later, we further discovered that the plant nuclear lamina was highly dynamic under abiotic stresses. Together, these findings represent a milestone in understanding the role of plant nuclear lamin proteins in regulating gene expression and genome organization.