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Chromatin Packing and Architectural Proteins in Plants

Periodic Reporting for period 3 - CHROMATADS (Chromatin Packing and Architectural Proteins in Plants)

Reporting period: 2021-01-01 to 2022-06-30

When fully stretched, the DNA of a cell might span meters. In reality, the DNA is folded and packed inside a nucleus, which is just a few micrometers in diameter – a micrometer being a millionth of a meter. The packed DNA together with other associated molecules, such as proteins and RNAs, is called chromatin. Upon checking how the chromatin is organized in a living cell, one can realize that it has a three-dimensional structure: Some regions are tightly packed, while some are relatively loose and accessible to many regulatory factors. Besides, some regions can be in physical contact with each other, regardless of the distance in the linear DNA. These three-dimensional conformations of chromatin are highly relevant to the transcriptional profile of cells, that is which genes are active at a given moment, which in turn substantially determine how cells function. Recently, research in the animal field has greatly advanced the researchers’ understanding of 3D chromatin structures. In contrast, relevant work in plants has been much delayed, which is due to the lack of our knowledge of plant-specific factors regulating chromatin folding and organization.
The main aim of this project, CHROMATADS, is to fill this knowledge gap with state-of-the-art methods in both Molecular Biology and Computing. Specifically, this project has four parts. Part 1 aims to identify and characterize the stability and plasticity of functional chromatin domains in plant nuclei. Part 2 and Part 3 aim to identify insulator elements and other structural features of chromatin packing in the Marchantia polymorpha genome from a structural genomics approach. Part 4 aims to generate functional insights into the molecular mechanism by which plants shape the three-dimensional genome.
This project intends to achieve a significant advancement in plant functional genomics and to open many new directions of fundamental research related to chromatin structure and transcriptional regulation in plant science. It will also represent a critical step in genetic engineering of crops, which aims to generate new varieties with stable agronomic traits.
WP1 (Dynamic chromatin structures in rice under temperature stress)
As planned, tagging lines of rice putative lamin proteins, as well as CRISPR-Cas knock-out mutants, have been created. When this work was ongoing, an independent project in the PI’s lab, which is highly relevant to WP1, led to a discovery, that the CRWNs protein were the long-sought functional nuclear lamin (Hu et al., 2019, Genome Biology, DOI:10.1186/s13059-019-1694-3). Following this discovery, by taking advantage of the newly established Marchantia platform (generated in WP3), the team started working on candidate lamin proteins in Marchantia. The functional studies of the newly identified lamins under the scope of “dynamic chromatin structure” in different plant species (rice, Marchantia, and Arabidopsis in the near future) will be perfectly serving the main aim of this project, which is “to identify and characterize architectural proteins involved in shaping three-dimensional plant genomes”.

WP2 (Genome-wide identification of chromatin regions showing insulating behavior in Marchantia polymorpha)
As planned, Hi-C maps of Marchantia thalli have been produced, and the chromatin contact patterns were analysed with various genomic and epigenomic datasets. TADs, which appeared as one of the predominant features in local Marchantia local chromatin organization, have been identified. Features associated with chromatin regions forming TADs and regions overlapping with TAD borders have been analyzed intensively. All proposed work in WP2 has been completed on time. The conclusion derived from WP2 provides critical information for the implementation of experiments in WP3.
Additionally, WP2 has benefited greatly from collaboration with Dr. Frédéric Berger’s lab in Gregor Mendel Institute, Austria. This collaboration aimed to generate a complete Marchantia genome (Tak-1) assembly to promote Marchantia research substantially. This collaboration was extremely successful: it led to not only publication in a decent journal (Montgomery et al., 2020, Current Biology) but also largely extended the PI’s networking from the vascular to the non-vascular plant research community.

WP3 (Identification of plant insulators and insulator element binding proteins)
Results in WP2 supported our hypothesis concerning the role of TCP proteins in plant TADs formation. All planed work in WP3 has been carried out according to the timetable described in the Grant agreement deed. The team has successfully established a platform to conduct Marchantia work, which was not available in the host institute. Essentially, mutant lines of the candidate factors identified in WP2 have been generated. As MpTCP1 is the top candidate, antibodies recognizing this protein have been acquired ahead of schedule, and ChIP-seq experiment haven been completed. Pattern analysis further supports the role of this protein in regulating Marchantia TADs formation. We managed to establish a CRISPR-Cas9 genome editing system in the lab to generate Marchantia TCP1 loss-of-function mutant. Functional analyses on tcp1 mutant did not support the hypothesis that Marchantia TCP1 proteins play structural roles in regulating TADs formation. However, surprising association patterns between Marchantia TCP1 and TADs have been unveiled. In brief, the team discovered a new type of TADs that displayed intensive interactions with TCP1 proteins (which was named as “TCP1-rich”). This type of association between transcription factor/co-factor and TADs has never been reported in animals or plants.
Besides, data analyses in WP2 revealed additional candidate genes linked to TADs formation and/or function. Thus, work in WP3 has been expanded to these candidates as well. Following our discovery of TCP1-rich TADs, we have also broadened the scope of WP3 by assaying protein-DNA interactions more transcription factors that potentially exhibit intensive binding with TADs.

WP4 (Functional studies on roles of plant architectural proteins)
Some planned work has been started ahead of schedule. We successfully performed TCP1 Co-IP and identified TCP1-interacting proteins by MS/MS. Also, we generated CRISPR-Cas9 edited lines to study TCP1-chromatin interactions. Surprisingly, our data indicate that TCP1-chromatin interactions do not rely on the presence of the DNA motif recognized by TCP1. This result directed us to design and generate more molecular and genetic tools to perform in-depth study to understand how this transcription factor executes its function.

WP5 (Dissemination of results)
So far, this project has generated more peer-reviewed publications than planned. Publications, which are directly resulted from this project, are listed below.

Publications directly resulted from this project (the PI has corresponding authorship)

S. A. Montgomery et al. (2020) Current Biology 30:573-588.

N. Wang and C. Liu. (2020) Methods in Molecular Biology 2093, 115-127.

F. Pontvianne and C. Liu. (2020) Current Opinion in Plant Biology 54:1-10.

N. Wang and C. Liu. (2019) Current Opinion in Genetics and Development 55:59-65.

B. Hu et al. (2019) Genome Biology 20:87.

E. Doğan and C. Liu. (2018) Nature Plants 4, 521-229.
• The confirmation of CRWN proteins as plant lamin analog paves the foundation for future studies concerning perinuclear chromatin positioning in plants.

• The completion of a chromosome-scale Marchantia genome assembly and annotation provide the growing Marchantia research community an essential resource.

• The ongoing work related to three-dimensional chromatin organization is expected to bring significant advance to plant functional genomics. It will be of broad interest to researchers studying genome architecture, chromatin folding, and transcriptional regulation.
overview of 3D chromatin organization