Periodic Reporting for period 2 - LoopMechRegFun (Mechanism, regulation and functions of genome folding through loop extrusion by cohesin-NIPBL)
Reporting period: 2023-04-01 to 2024-09-30
The organization of genomic DNA into chromatin fibers and their regulation by histone modifications is well understood, as is the relevance of these mechanisms for gene regulation. However, chromatin fibers are also folded over long distances into loops or topologically associating domains (TADs). TADs and loops are thought to play important roles in processes in which distant genomic regions need to be brought into proximity, such as enhancer-promoter interactions and the assembly of immunoglobulin genes by V(D)J recombination.
Chromatin loops and TADs depend on cohesin and are anchored at sites occupied by CTCF, a sequence specific DNA binding protein. Cohesin is a member of the SMC family of protein complexes, which are multi-subunit ATPases present in all kingdoms of life. Cohesin was initially discovered for its ability to mediate sister chromatid cohesion by connecting replicated DNA molecules in trans but is also able to form long-range cis-interactions by a process called loop extrusion. However, the mechanism of cohesin-mediated DNA loop extrusion, the regulation of this process and its cellular functions are incompletely understood. To address these questions, this project pursues the following objectives:
1. What is the mechanism of DNA loop extrusion by cohesin-NIPBL?
2. How is DNA loop extrusion by cohesin-NIPBL regulated?
3. Are chromatin fibers in cells folded by loop extrusion?
4. What determines whether cohesin mediates loop extrusion or sister chromatid cohesion?
5. What are the cellular functions of loop formation by cohesin-NIPBL?
Relevance and impact.
The results of this project will be of broad relevance for understanding genome architecture, gene regulation and recombination, including the generation of a diverse repertoire of B cell receptors and antibodies in the mammalian immune system, and for understanding how SMC complexes function as molecular motors. Since cohesin subunit genes are among the most frequently mutated genes found in human cancers, mutations in NIPBL and cohesin are the cause of rare congenital ‘cohesinopathies’ and cohesin malfunctioning is thought to be a major cause of trisomy 21 and spontaneous human abortions, the research proposed here may also provide insight into the etiology of these disorders.
Chromatin loops and TADs depend on cohesin and are anchored at sites occupied by CTCF, a sequence specific DNA binding protein. Cohesin is a member of the SMC family of protein complexes, which are multi-subunit ATPases present in all kingdoms of life. Cohesin was initially discovered for its ability to mediate sister chromatid cohesion by connecting replicated DNA molecules in trans but is also able to form long-range cis-interactions by a process called loop extrusion. However, the mechanism of cohesin-mediated DNA loop extrusion, the regulation of this process and its cellular functions are incompletely understood. To address these questions, this project pursues the following objectives:
1. What is the mechanism of DNA loop extrusion by cohesin-NIPBL?
2. How is DNA loop extrusion by cohesin-NIPBL regulated?
3. Are chromatin fibers in cells folded by loop extrusion?
4. What determines whether cohesin mediates loop extrusion or sister chromatid cohesion?
5. What are the cellular functions of loop formation by cohesin-NIPBL?
Relevance and impact.
The results of this project will be of broad relevance for understanding genome architecture, gene regulation and recombination, including the generation of a diverse repertoire of B cell receptors and antibodies in the mammalian immune system, and for understanding how SMC complexes function as molecular motors. Since cohesin subunit genes are among the most frequently mutated genes found in human cancers, mutations in NIPBL and cohesin are the cause of rare congenital ‘cohesinopathies’ and cohesin malfunctioning is thought to be a major cause of trisomy 21 and spontaneous human abortions, the research proposed here may also provide insight into the etiology of these disorders.
1. What is the mechanism of DNA loop extrusion by cohesin-NIPBL?
We and others discovered previously that cohesin extrudes DNA into loops (Davidson, Science 2019) by undergoing large-scale conformational changes (Bauer, Cell 2021). In this project, we identified the force-generating step among these conformational changes (Pobegalov, Nat. Comms. 2023; in collaboration with Maxime Molodtsov; Crick Inst.), and in collaboration with Cees Dekker’s group (Univ. Delft) provided evidence that cohesin does not have to entrap DNA inside its ring structure to extrude loops (Pradhan, Cell Rep. 2022; Barth, Sci. Rep. 2022), showed that cohesin frequently changes the direction of DNA loop extrusion (Barth, bioRxiv 2023) and discovered that cohesin negatively supercoils DNA during loop extrusion (Davidson, bioRxiv 2024, Janissen, bioRxiv 2024).
2. How is DNA loop extrusion by cohesin-NIPBL regulated?
CTCF was known to have an important role in specifying the location of cohesin loops, but the mechanistic basis of this functions was incompletely understood. In this project we discovered that CTCF is sufficient to stop loop extruding cohesin molecules in an orientation and tension dependent manner (Davidson, Barth, Nature 2023; in collaboration with the Dekker lab), and in collaboration with the Tachibana (MPIB) and Mirny labs (MIT) we found that the replicative helicase MCM and active genes can also function as boundaries for cohesin-mediated loop extrusion (Dequeker, Nature 2022; Banigan, PNAS 2023).
3. Are chromatin fibers in cells folded by loop extrusion?
It is unknown whether cohesin can not only fold DNA but also chromatin fibers in cells by loop extrusion. We have now found that a cohesin mutant that is reduced in DNA loop extrusion in vitro is also reduced in chromatin looping in cells, supporting the hypothesis that cohesin also forms chromatin loops by extrusion in cells (Davidson, bioRxiv 2024). Furthermore, in collaboration with the Dekker and Ellenberg laboratories (EMBL), we found that SMC complexes can bypass nucleosomes during loop extrusion in vitro (Pradhan, Cell Rep., 2021) and that cohesin forms loops in cells in a manner that is consistent with loop extrusion (Beckwith, bioRxiv 2021).
4. What determines whether cohesin mediates loop extrusion or sister chromatid cohesion?
How cohesin’s functions in loop extrusion and sister chromatid cohesion are specified is unknown. We discovered a partial separation-of-function mutant of cohesin, which is still able to extrude DNA but unable to mediate cohesion, suggesting that cohesin uses at least partially distinct mechanisms for these two functions (Nagasaka, Mol. Cell 2023).
5. What are the cellular functions of loop formation by cohesin-NIPBL ?
The role of cohesin in gene regulation is controversial since cohesin depletion experiments lead only to limited gene regulation defects in cell culture models. One potential explanation for this conundrum is that cohesin is only required for specific gene activation events during development. Support for this hypothesis comes from the observation that mutations in the activating cohesin subunit NIPBL cause the congenital disease Cornelia de Lange Syndrome (CdLS), which is thought to be caused by developmental gene regulation defects. We found that some of the NIPBL mutations associated with CdLS cause DNA loop extrusion defects in vitro, consistent with the possibility that cohesin-mediated loop extrusion is required for developmental gene regulation (Panarotto, PNAS 2022). In collaboration with the Busslinger lab (IMP) we also found that cohesin mediated loop extrusion is essential for recombination events in developing B cells for the generation of a diverse antibody repertoire (Hill, Nature 2020; Hill, Nat. Comms. 2023).
We and others discovered previously that cohesin extrudes DNA into loops (Davidson, Science 2019) by undergoing large-scale conformational changes (Bauer, Cell 2021). In this project, we identified the force-generating step among these conformational changes (Pobegalov, Nat. Comms. 2023; in collaboration with Maxime Molodtsov; Crick Inst.), and in collaboration with Cees Dekker’s group (Univ. Delft) provided evidence that cohesin does not have to entrap DNA inside its ring structure to extrude loops (Pradhan, Cell Rep. 2022; Barth, Sci. Rep. 2022), showed that cohesin frequently changes the direction of DNA loop extrusion (Barth, bioRxiv 2023) and discovered that cohesin negatively supercoils DNA during loop extrusion (Davidson, bioRxiv 2024, Janissen, bioRxiv 2024).
2. How is DNA loop extrusion by cohesin-NIPBL regulated?
CTCF was known to have an important role in specifying the location of cohesin loops, but the mechanistic basis of this functions was incompletely understood. In this project we discovered that CTCF is sufficient to stop loop extruding cohesin molecules in an orientation and tension dependent manner (Davidson, Barth, Nature 2023; in collaboration with the Dekker lab), and in collaboration with the Tachibana (MPIB) and Mirny labs (MIT) we found that the replicative helicase MCM and active genes can also function as boundaries for cohesin-mediated loop extrusion (Dequeker, Nature 2022; Banigan, PNAS 2023).
3. Are chromatin fibers in cells folded by loop extrusion?
It is unknown whether cohesin can not only fold DNA but also chromatin fibers in cells by loop extrusion. We have now found that a cohesin mutant that is reduced in DNA loop extrusion in vitro is also reduced in chromatin looping in cells, supporting the hypothesis that cohesin also forms chromatin loops by extrusion in cells (Davidson, bioRxiv 2024). Furthermore, in collaboration with the Dekker and Ellenberg laboratories (EMBL), we found that SMC complexes can bypass nucleosomes during loop extrusion in vitro (Pradhan, Cell Rep., 2021) and that cohesin forms loops in cells in a manner that is consistent with loop extrusion (Beckwith, bioRxiv 2021).
4. What determines whether cohesin mediates loop extrusion or sister chromatid cohesion?
How cohesin’s functions in loop extrusion and sister chromatid cohesion are specified is unknown. We discovered a partial separation-of-function mutant of cohesin, which is still able to extrude DNA but unable to mediate cohesion, suggesting that cohesin uses at least partially distinct mechanisms for these two functions (Nagasaka, Mol. Cell 2023).
5. What are the cellular functions of loop formation by cohesin-NIPBL ?
The role of cohesin in gene regulation is controversial since cohesin depletion experiments lead only to limited gene regulation defects in cell culture models. One potential explanation for this conundrum is that cohesin is only required for specific gene activation events during development. Support for this hypothesis comes from the observation that mutations in the activating cohesin subunit NIPBL cause the congenital disease Cornelia de Lange Syndrome (CdLS), which is thought to be caused by developmental gene regulation defects. We found that some of the NIPBL mutations associated with CdLS cause DNA loop extrusion defects in vitro, consistent with the possibility that cohesin-mediated loop extrusion is required for developmental gene regulation (Panarotto, PNAS 2022). In collaboration with the Busslinger lab (IMP) we also found that cohesin mediated loop extrusion is essential for recombination events in developing B cells for the generation of a diverse antibody repertoire (Hill, Nature 2020; Hill, Nat. Comms. 2023).
This project made so far the following progress beyond state of the art:
1. We provided first evidence that defects in cohesin-mediated DNA loop extrusion might be the root cause of the human congenital disease Cornelia de Lange Syndrome (CdLS; Panarotto et al., PNAS 2022).
2. We showed that single CTCF molecules are sufficient to function as boundaries for cohesin mediated DNA loop extrusion and discovered that the tension of DNA modulates CTCF’s boundary function (Davidson, Barth et al., Nature 2023).
3. We identified a partial separation-of-function mutant of human cohesin and provided evidence that cohesin complexes mediate their DNA loop extrusion and sister chromatid cohesion functions through at least partially distinct mechanisms (Nagasaka et al., Mol. Cell 2023).
4. We discovered that cohesin complexes extrude DNA asymmetrically but frequently switch the direction of this process. This finding unifies different observations made for cohesin and condensin, supports the notion that these SMC complexes extrude DNA in similar ways and has important implications for the mechanism of cohesin mediated DNA loop extrusion (Barth et al., bioRxiv 2023).
5. We discovered that cohesin supercoils DNA during loop extrusion. These findings have important implications for understanding the mechanism of loop extrusion and for interpreting genome architecture maps obtained by the Hi-C technique (Davidson et al., bioRxiv 2024).
This project made so far the following technical beyond state of the art:
1. We developed an assay in which the boundary function of CTCF can be reconstituted in vitro and its effect on loop extruding cohesin complexes can be analyzed in real time at the single-molecule level (Davidson, Barth et al., Nature 2023).
2. We developed an approach with which endogenous proteins can be replaced by mutants in human cell lines (Nagasaka et al., Mol. Cell 2023). This approach is based on inducible degradation of endogenous proteins and inducible expression of their mutant counterparts.
Expected results until end of the project:
Current projects in the laboratory focus on the role of PDS5 proteins in regulating cohesin-mediated DNA loop extrusion, are testing whether topoisomerases have a role in this process and are exploring previously unknown functions of cohesin loops. We are expecting results in these three areas until end of the project.
1. We provided first evidence that defects in cohesin-mediated DNA loop extrusion might be the root cause of the human congenital disease Cornelia de Lange Syndrome (CdLS; Panarotto et al., PNAS 2022).
2. We showed that single CTCF molecules are sufficient to function as boundaries for cohesin mediated DNA loop extrusion and discovered that the tension of DNA modulates CTCF’s boundary function (Davidson, Barth et al., Nature 2023).
3. We identified a partial separation-of-function mutant of human cohesin and provided evidence that cohesin complexes mediate their DNA loop extrusion and sister chromatid cohesion functions through at least partially distinct mechanisms (Nagasaka et al., Mol. Cell 2023).
4. We discovered that cohesin complexes extrude DNA asymmetrically but frequently switch the direction of this process. This finding unifies different observations made for cohesin and condensin, supports the notion that these SMC complexes extrude DNA in similar ways and has important implications for the mechanism of cohesin mediated DNA loop extrusion (Barth et al., bioRxiv 2023).
5. We discovered that cohesin supercoils DNA during loop extrusion. These findings have important implications for understanding the mechanism of loop extrusion and for interpreting genome architecture maps obtained by the Hi-C technique (Davidson et al., bioRxiv 2024).
This project made so far the following technical beyond state of the art:
1. We developed an assay in which the boundary function of CTCF can be reconstituted in vitro and its effect on loop extruding cohesin complexes can be analyzed in real time at the single-molecule level (Davidson, Barth et al., Nature 2023).
2. We developed an approach with which endogenous proteins can be replaced by mutants in human cell lines (Nagasaka et al., Mol. Cell 2023). This approach is based on inducible degradation of endogenous proteins and inducible expression of their mutant counterparts.
Expected results until end of the project:
Current projects in the laboratory focus on the role of PDS5 proteins in regulating cohesin-mediated DNA loop extrusion, are testing whether topoisomerases have a role in this process and are exploring previously unknown functions of cohesin loops. We are expecting results in these three areas until end of the project.