How does Structural Maintenance of Chromosomes (SMC) protein interact with DNA to organise bacterial chromosomes?
Lead Research Organisation:
JOHN INNES CENTRE
Abstract
DNA, the genetic blueprint of living organisms, uses a 4-letter code to define the instructions for organisms to grow and replicate. As the encoded information is very large, the DNA string is extremely long. To fit this string into the restricted space of the cell, the DNA must be folded in a specific way in 3-dimensional space. This folding has extremely important implications for the functioning of all living organisms and yet it is very poorly understood how this structure is established and maintained, especially in bacteria. This research proposal aims to understand how a protein called Structural Maintenance of Chromosomes (SMC) folds the chromosome in an aquatic bacterium called Caulobacter crescentus. Specifically, we aim to:
(i) understand the behaviour of individual SMC molecule in the cell when they are on or off DNA
(ii) understand how RNA polymerases move SMC around on the chromosome
(iii) discover new interacting partners that together with SMC organise the bacterial chromosome
The Caulobacter SMC protein is similar to human proteins called condensin and cohesin. Defective cohesin or condensin contributes to tumour formation. Indeed, in colorectal cancer, a significant fraction of cancerous tissues was found to have defects in cohesin or associated genes. In addition, defective cohesin was thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. I anticipate that, by employing a simpler living organism, my research into the role of SMC in Caulobacter will reveal an ancient, core function of the SMC family of proteins and thereby provide insights into the functions of human cohesin and condensin and how their malfunctions result in disease.
(i) understand the behaviour of individual SMC molecule in the cell when they are on or off DNA
(ii) understand how RNA polymerases move SMC around on the chromosome
(iii) discover new interacting partners that together with SMC organise the bacterial chromosome
The Caulobacter SMC protein is similar to human proteins called condensin and cohesin. Defective cohesin or condensin contributes to tumour formation. Indeed, in colorectal cancer, a significant fraction of cancerous tissues was found to have defects in cohesin or associated genes. In addition, defective cohesin was thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. I anticipate that, by employing a simpler living organism, my research into the role of SMC in Caulobacter will reveal an ancient, core function of the SMC family of proteins and thereby provide insights into the functions of human cohesin and condensin and how their malfunctions result in disease.
Technical Summary
The organisation of bacterial chromosome is profoundly influenced by structural DNA-binding proteins, including the highly conserved Structural Maintenance of Chromosomes protein (SMC). This research project aims to understand at the molecular level how SMC interacts with DNA to organise the global chromosome in bacteria, specifically in a bacterium called Caulobacter crescentus. The objectives will be addressed through a multidisciplinary programme incorporating cell biology, biochemistry, genetics, and "omics" techniques.
Objective 1: Determine diffusion characteristics of cytosolic and DNA-translocating SMC by Photo-activated Localization Microscopy (PALM)
Objective 2: Determine the molecular mechanism of RNA polymerase-dependent SMC redistribution in Caulobacter by testing the following non-mutually exclusive hypotheses:
a) A direct physical interaction with a large and fast-moving RNAP that push SMC along its path: the "physical collision" model
b) The supercoiling density generated during RNAP elongation, rather than collision that promotes or impedes SMC translocation: the "supercoiling" model
c) RNA transcripts emerge and move with RNAP, thereby drags SMC along: the "RNA dragging" model
Objective 3: Identify additional factors that interact genetically or physically with SMC to organise the chromosome by employing a high throughput lethal synthetic screen (Tn-seq), and by co-immunoprecipitation assays.
Objective 1: Determine diffusion characteristics of cytosolic and DNA-translocating SMC by Photo-activated Localization Microscopy (PALM)
Objective 2: Determine the molecular mechanism of RNA polymerase-dependent SMC redistribution in Caulobacter by testing the following non-mutually exclusive hypotheses:
a) A direct physical interaction with a large and fast-moving RNAP that push SMC along its path: the "physical collision" model
b) The supercoiling density generated during RNAP elongation, rather than collision that promotes or impedes SMC translocation: the "supercoiling" model
c) RNA transcripts emerge and move with RNAP, thereby drags SMC along: the "RNA dragging" model
Objective 3: Identify additional factors that interact genetically or physically with SMC to organise the chromosome by employing a high throughput lethal synthetic screen (Tn-seq), and by co-immunoprecipitation assays.
Planned Impact
WHO WILL BENEFIT FROM THIS RESEARCH? The outputs of this research will be of value to fundamental scientists, to biosynthetic engineers, to the pharmaceutical industry, and ultimately to the health sector and thus to patients.
HOW WILL THEY BENEFIT FROM THIS RESEARCH? The proposed research aims to understand the molecular mechanism on how the Structural Maintenance of Chromosomes (SMC) protein organises the chromosome in a bacterium called Caulobacter crescentus. The Caulobacter SMC protein is homologous to human cohesin and condensin. Cohesin and condensin are essential protein complexes for separating identical copies of the genome into daughter cells during cell division. Defective cohesin or condensin contributes to tumour formation. In colorectal cancer, a significant fraction of cancerous tissues was found to have defects in cohesin or associated genes. Mutations in human cohesin have also been implicated in myeloid leukemogenesis. In addition, defective cohesin was thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. From my preliminary research, we have shown that Caulobacter SMC shares significant similarity in molecular mechanism to eukaryotic cohesin and condensin, and can be thought of as an ancestral SMC. I anticipate that, in the near future, this research may not only elucidate the pathology underlying some of these diseases, but may also provide insights leading to new treatment.
Furthermore, my research on SMC is very relevant in the dawn of total synthetic chromosomes (for example, synthetic yeast Sc 2.0 project) and designed minimal genome (for example, M. mycoides JCVI-syn1.0, Craig Venter 2016. SMC-encoding gene is one of 473 essential genes in this synthetic bacterium). My research into a fundamental component of the chromosome organisation will inform researchers of rules for designing synthetic chromosomes.
Overall, the proposed work is highly relevant to the Bioscience for Health, and Industrial Biotechnology and Bioenergy of the Strategic Research Priority 2 and 3 in the current BBSRC Strategic Plan, and will contribute significantly to our knowledge-based economy.
WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? Please see Pathways to Impact statement for more details. Briefly, outputs with potential commercial impact will be identified during regular reviews of progress (Month 12, 24 and 30). Discoveries with potential commercial implications will be discussed (with a view to patenting) with Plant Biosciences Ltd (PBL), a technology transfer company owned by the JIC. The purpose of PBL is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC.
Tung Le will train scientists employed on this project on latest "omics" techniques such as chromosome conformation capture (Hi-C), ChIP-seq, Tn-seq and bioinformatics. Recently, Tung was invited to an INSERM workshop (France, 2016) as one of the instructors on the practicality and applications of Hi-C.
JIC has an excellent External Relations Department (http://www.jic.ac.uk/corporate/media-and-public/index.htm) and, where appropriate, we will work proactively with them to approach and interact with the press and broadcast media to publicise this scientific area in general and the outputs of the grant.
HOW WILL THEY BENEFIT FROM THIS RESEARCH? The proposed research aims to understand the molecular mechanism on how the Structural Maintenance of Chromosomes (SMC) protein organises the chromosome in a bacterium called Caulobacter crescentus. The Caulobacter SMC protein is homologous to human cohesin and condensin. Cohesin and condensin are essential protein complexes for separating identical copies of the genome into daughter cells during cell division. Defective cohesin or condensin contributes to tumour formation. In colorectal cancer, a significant fraction of cancerous tissues was found to have defects in cohesin or associated genes. Mutations in human cohesin have also been implicated in myeloid leukemogenesis. In addition, defective cohesin was thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. From my preliminary research, we have shown that Caulobacter SMC shares significant similarity in molecular mechanism to eukaryotic cohesin and condensin, and can be thought of as an ancestral SMC. I anticipate that, in the near future, this research may not only elucidate the pathology underlying some of these diseases, but may also provide insights leading to new treatment.
Furthermore, my research on SMC is very relevant in the dawn of total synthetic chromosomes (for example, synthetic yeast Sc 2.0 project) and designed minimal genome (for example, M. mycoides JCVI-syn1.0, Craig Venter 2016. SMC-encoding gene is one of 473 essential genes in this synthetic bacterium). My research into a fundamental component of the chromosome organisation will inform researchers of rules for designing synthetic chromosomes.
Overall, the proposed work is highly relevant to the Bioscience for Health, and Industrial Biotechnology and Bioenergy of the Strategic Research Priority 2 and 3 in the current BBSRC Strategic Plan, and will contribute significantly to our knowledge-based economy.
WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? Please see Pathways to Impact statement for more details. Briefly, outputs with potential commercial impact will be identified during regular reviews of progress (Month 12, 24 and 30). Discoveries with potential commercial implications will be discussed (with a view to patenting) with Plant Biosciences Ltd (PBL), a technology transfer company owned by the JIC. The purpose of PBL is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC.
Tung Le will train scientists employed on this project on latest "omics" techniques such as chromosome conformation capture (Hi-C), ChIP-seq, Tn-seq and bioinformatics. Recently, Tung was invited to an INSERM workshop (France, 2016) as one of the instructors on the practicality and applications of Hi-C.
JIC has an excellent External Relations Department (http://www.jic.ac.uk/corporate/media-and-public/index.htm) and, where appropriate, we will work proactively with them to approach and interact with the press and broadcast media to publicise this scientific area in general and the outputs of the grant.
Organisations
People |
ORCID iD |
| Tung Le (Principal Investigator) |
 http://orcid.org/0000-0003-4764-8851
|
Publications
Jalal AS
(2021)
A CTP-dependent gating mechanism enables ParB spreading on DNA.
in eLife
Jalal AS
(2020)
ParB spreading on DNA requires cytidine triphosphate in vitro.
in eLife
Jalal ASB
(2020)
Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.
in Cell reports
Le TBK
(2019)
Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the Structural Maintenance of Chromosomes Complex In Vivo.
in Methods in molecular biology (Clifton, N.J.)
| Description | The structural maintenance of chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome-partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription. More recently, we reconstituted a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and showed that CTP is required for spreading. We further showed that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC. |
| Exploitation Route | The outputs of our proposed research have the potential to deliver impact at four levels: 1. the advancement of our fundamental knowledge of bacterial chromosome organisation, the molecular mechanism of bacterial SMC and their essential homologs in humans such as cohesin and condensin. 2. providing insights into the functions of human cohesin and condensin and how their malfunctions result in disease. Defective cohesin, condensin or associated genes contributes to tumour formation such as in colorectal cancer. Defective cohesin was also thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. In the near future, this research may not only elucidate the pathology underlying some of these diseases, but may also provide insights leading to new treatment. 3. informing synthetic biologists and relevant industries of rules to design total synthetic chromosome (for example, synthetic yeast Sc 2.0 project) and minimal genome (for example, SMC-encoding gene is one of 473 essential genes in the synthetic bacterium M. mycoides JCVI-syn1.0, Craig Venter 2016). 4. training of next-generation scientists and postdoctoral researcher directly employed in this project. |
| Sectors | Chemicals Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
| Description | Research Fellows Enhanced Research Expenses |
| Amount | £167,949 (GBP) |
| Funding ID | RF\ERE\210039 |
| Organisation | The Royal Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 01/2022 |
| End | 12/2023 |
| Description | Royal Society University Research Fellowship Renewal |
| Amount | £483,734 (GBP) |
| Funding ID | URF\R\201020 |
| Organisation | The Royal Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 01/2021 |
| End | 12/2023 |