The chromosomes of all living organisms must be compacted nearly three orders of magnitude to fit within cells. Moreover, DNA must be packaged in a way that is compatible with a myriad of DNA-based processes, including replication, segregation, transcription, repair, and recombination. This challenge is particularly acute in bacteria as chromosome segregation occurs concomitantly with DNA replication rather than being separated temporally, as in eukaryotes. How chromosome organization and its integration with biological processes are achieved in vivo remains poorly understood. To fill this knowledge gap, our lab aims to:
Elucidate the molecular mechanisms underlying chromosome organization and segregation
Unravel the relationship between spatial chromosome organization (locally and globally) and important biological processes in the cell
In particular, we focus our resources to investigate how the Structural Maintenance of Chromosomes (SMC) protein interacts with DNA to organize the bacterial chromosome, specifically in an aquatic bacterium Caulobacter crescentus. The investigation of SMC is a perfect starting place for our new lab since SMC plays a central role in chromosome organization and segregation in most organisms, and is highly conserved from bacteria to humans. Furthermore, there is a great synergy between the bacterial and eukaryotic SMC research fields, and our research has benefited tremendously from the recent explosion of interest in, and technological advances from the eukaryotic chromosome organization field. The combination of genetics, cell biology, and genome-wide techniques (ChIP-seq and Hi-C) has proved to be very effective in investigating SMC in our lab. In the long term, we aim to apply similar approaches to investigate other classes of chromosome-structuring proteins. The ultimate aim of our lab is to understand the integration of actions of different classes of architecture proteins in organizing the chromosome.
SMC and its accessory proteins
How does Structural Maintenance of Chromosomes (SMC) protein interact with DNA to organize bacterial chromosomes?
SMC proteins, also known as cohesins and condensins in eukaryotes, are crucial for chromosome organization in all living organisms. But how SMC translocates on a protein-laden chromosome is poorly investigated. Whether SMC impacts DNA-translocating proteins such as RNA polymerase and is, in turn, influenced by such proteins, is not well understood. How different cellular processes share the same DNA but avoid, or resolve, conflicts is a question that arises in all cells and all domains of life. Our recent study provided experimental evidence that the translocation of bacterial SMC on the chromosome is strongly influenced by RNA polymerase. We showed that highly-transcribed genes oriented to collide head-on with translocating SMC slow down and potentially stop SMC translocation. This might have contributed to the evolutionary selection for highly-transcribed genes to be co-oriented with the direction of DNA replication and SMC movement. Broadly, our work demonstrated a tight interdependence of bacterial chromosome organization and the highly non-random global pattern of transcription. Furthermore, we demonstrated that the translocation of bacterial SMC is directional, starting at the bacterial centromere site parS and moving progressively towards the replication terminus, extruding out DNA in a loop as a result of this directional movement. The finding that bacterial SMC translocation is directional has significant implications for how eukaryotic cohesins extrude DNA loops (Rao et al 2015 Cell, Sanborn et al 2015 PNAS, Fudenberg et al 2016 Cell Rep).
A model for the loading and translocation of bacterial SMC
Caulobacter crescentus as model organism
Our findings were greatly aided by using Caulobacter as a model system. Caulobacter is easily synchronized, enabling us to generate genome-wide data for a homogenous population of G1-phase cells that each contain a single chromosome. As there is no active DNA replication in the G1 cells, we were able to isolate and specifically study the effect of transcription on SMC translocation and global chromosome organization, without confounding effects from replication-transcription conflicts.
How does the centromere-binding protein ParB interact with DNA to recruit SMC?
Stemming directly from our work on SMC, we discovered that the centromere-binding protein ParB recruits SMC onto DNA by a direct protein-protein interaction. In this strand of research, we are investigating the properties of ParB to understand how SMC is first recruited onto the DNA to initiate folding of the chromosome. We investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of ChIP-seq and IDAP-seq to discover at least five ParB-binding parS sites that closely cluster ~8 kb from the origin of replication, and defined these sites to single-nucleotide resolution.
We showed that ParB is also capable of binding DNA non-specifically to form a “daisy chain” of ParB molecules emanating outwards from its parS nucleation site, and this is mediated by a protein-protein “handshaking” between neighbouring ParB dimers. We hypothesized that this unusual “spreading” property of ParB is important to load enough SMC molecules onto the chromosome at a given time. This is a research area that we are actively pursuing in the lab. The biological significance is that the rate of SMC loading might dictate how large the DNA loop being formed is, and how compacted the chromosome is. If correct, this would fit well with recent findings that altering the unloading rate of cohesin dramatically impacts DNA loop formation in eukaryotes (Haarhuis et al 2017 Cell, Gassler et al 2017 EMBOJ, and Wutz et al 2017 EMBOJ).
A model of ParB spreading and bridging
The cell cycle of Caulobacter crescentus
The co-crystal structure of ParB (DNA-binding domain) in complex with parS
Evolution of protein-DNA interfaces
In living organisms, hundreds of DNA-binding proteins carry out a plethora of roles in homeostasis, transcriptional regulation in response to stress, and in maintenance and transmission of genetic information. These DNA-binding proteins do so faithfully due to their distinct DNA-binding specificity towards their cognate DNA sites. Yet it remains unclear how related proteins, sometimes with a very similar DNA-recognition motif, can recognize entirely different DNA sites. What were the changes at the molecular level that brought about the diversification in DNA-binding specificity? As proteins evolved, did the intermediates in this process drastically switch DNA-binding specificity or did they transit gradually through promiscuous states that recognized multiple DNA sequences? In this strand of research, we employ evolutionarily-related DNA-binding proteins (that are important for bacterial chromosome maintenance) as model systems to answer these questions. We employ X-ray crystallography, molecular dynamics simulations, and deep-sequencing techniques to characterize in-depth protein-DNA interfaces of interest. Our goal is to identify common biophysical mechanisms that underlie specific/promiscuous DNA recognition, and thereby understand biophysical requirements/constraints to evolve a new DNA-binding specificity. The methodologies and concepts that we develop might be applicable to studying the evolutions of other DNA-binding protein families or protein-protein interfaces.