Mechanisms of chromatin binding by the MORC2 ATPase
(2024) MOBN03 20232Degree Projects in Molecular Biology
- Abstract
- MORC2 (Microorchidia CW type Zinc Finger protein 2) is a GHKL (gyrase, heat shock protein 90, histidine kinase, MutL) ATPase involved in chromatin remodeling and is highly conserved in eukaryotes. Mutations in the ATPase domain of MORC2 are known to cause neurodevelopmental disorders. MORC2 is recruited by the HUSH complex to target long, intronless transcription units such as LINE-1 retrotransposons, and genes such as protocadherins and Zinc Finger Proteins as well as to newly integrated transgenes. Until now, the mechanisms of MORC2 recruitment and domain requirements for its function have focused largely on transgene silencing. By understanding MORC2 recruitment to genomic targets we can grasp the mechanisms involved in gene regulation.... (More)
- MORC2 (Microorchidia CW type Zinc Finger protein 2) is a GHKL (gyrase, heat shock protein 90, histidine kinase, MutL) ATPase involved in chromatin remodeling and is highly conserved in eukaryotes. Mutations in the ATPase domain of MORC2 are known to cause neurodevelopmental disorders. MORC2 is recruited by the HUSH complex to target long, intronless transcription units such as LINE-1 retrotransposons, and genes such as protocadherins and Zinc Finger Proteins as well as to newly integrated transgenes. Until now, the mechanisms of MORC2 recruitment and domain requirements for its function have focused largely on transgene silencing. By understanding MORC2 recruitment to genomic targets we can grasp the mechanisms involved in gene regulation. For this reason, we performed a series of genetic complementation assays by re-expressing MORC2 variants in MORC2 knockout SH-SY5Y neuroblastoma cells. We validated our experimental model with Western blot and RT-qPCR experiments. We employed CUT&RUN to profile MORC2 binding regions and assessed the domain dependencies for MORC2 recruitment. We found that MORC2 occupies two types of binding sites: H3K9me3-marked retrotransposons and H3K4me3 or H3K27Ac-marked gene promoters/enhancers. Our results suggest that dimerization, ATPase activity and DNA binding via the Coiled coil 1 domain are critical for MORC2 binding to LINE1 retrotransposons but an alternative mechanism is involved in MORC2 recruitment to gene promoters. The uncharacterized CD domain of MORC2 is essential for this mechanism. Furthermore, we identified that patient mutation S87L relocalizes MORC2 from retrotransposons to gene promoters of ZNF genes. Our insights advance our understanding of domain requirements for MORC2 recruitment. The discovery of S87L mutant binding to ZNF genes opens up new fields for exploring mechanisms involved in MORC2 mutant binding and neurodevelopmental disorders. (Less)
- Popular Abstract
- MORC2’s shape shifting arms and its quest across the genome library.
Our genome, containing genetic information, is organised in a way that can change and adapt. This organization helps in controlling genes by turning them on or off. There are two ways in which the chromosome is organised to regulate this process. Think of the genome as a library where some books (genes) are more accessible (open structure) and others are locked away (closed structure).
MORC proteins are a family of proteins that are like the staff of the genomic library that manage accessibility of various books (genes). One of these proteins, called MORC2 is particularly important. MORC2 can change its shape by binding to a molecule called ATP, like a transformer... (More) - MORC2’s shape shifting arms and its quest across the genome library.
Our genome, containing genetic information, is organised in a way that can change and adapt. This organization helps in controlling genes by turning them on or off. There are two ways in which the chromosome is organised to regulate this process. Think of the genome as a library where some books (genes) are more accessible (open structure) and others are locked away (closed structure).
MORC proteins are a family of proteins that are like the staff of the genomic library that manage accessibility of various books (genes). One of these proteins, called MORC2 is particularly important. MORC2 can change its shape by binding to a molecule called ATP, like a transformer toy. When ATP is chemically broken down, MORC2 goes back to its original shape. Mutations in the part of MORC2 that binds to ATP have been linked to brain development disorders, showing how significant this protein is. MORC2 binds to various genomic regions using different arms in its structure, we still don’t fully understand how its structure assists it with this function.
In this study, we mutated different arms (domains) of MORC2 and tested whether it can bind to different genomic regions. For this, we removed the intact MORC2 protein from cells and then added MORC2 with its mutated arms. We ensured that the intact MORC2 protein was removed by performing additional tests with Western blot and RTqPCR. We then performed CUT&RUN experiment to see the regions in the genome that MORC2 binds to. Our experiment showed that MORC2 binds to two kinds of regions: repetitive DNA and to the start of genes (promoters). MORC2 could not bind to repetitive DNA (transposons) after Y18A, S87L, 3XRE arm mutations, stating that those arms are essential for its function. But, we saw that despite mutating those arms, MORC2 could still bind to promoters. This means that MORC2 uses different arms in its structure to bind to various genomic regions. Interestingly, we also saw that when we deleted the CD arm of MORC2, it could not bind to these promoter regions.
We also explored the function of S87L mutated arm of MORC2 that is known to cause brain development disorders. It looks like this mutated arm helps MORC2 to bind tightly to some genes called Zinc finger proteins. This is important as understanding how this disease mutated arm works could help us in getting closer to finding the cure for brain disorders.
Overall, our study provides a clearer picture of how the different arms of MORC2 work to manage the genomic library.
Master’s thesis Project in Molecular Biology,
Genetics & Biotechnology, 60 credits
Supervisor: Dr. Christopher Douse
Laboratory of Epigenetics and Chromatin Dynamics
Department of Experimental medical Science, Section of neurobiology. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9176100
- author
- Sivani, Bala Mohan
- supervisor
- organization
- course
- MOBN03 20232
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9176100
- date added to LUP
- 2024-10-04 11:30:29
- date last changed
- 2024-10-04 11:30:29
@misc{9176100, abstract = {{MORC2 (Microorchidia CW type Zinc Finger protein 2) is a GHKL (gyrase, heat shock protein 90, histidine kinase, MutL) ATPase involved in chromatin remodeling and is highly conserved in eukaryotes. Mutations in the ATPase domain of MORC2 are known to cause neurodevelopmental disorders. MORC2 is recruited by the HUSH complex to target long, intronless transcription units such as LINE-1 retrotransposons, and genes such as protocadherins and Zinc Finger Proteins as well as to newly integrated transgenes. Until now, the mechanisms of MORC2 recruitment and domain requirements for its function have focused largely on transgene silencing. By understanding MORC2 recruitment to genomic targets we can grasp the mechanisms involved in gene regulation. For this reason, we performed a series of genetic complementation assays by re-expressing MORC2 variants in MORC2 knockout SH-SY5Y neuroblastoma cells. We validated our experimental model with Western blot and RT-qPCR experiments. We employed CUT&RUN to profile MORC2 binding regions and assessed the domain dependencies for MORC2 recruitment. We found that MORC2 occupies two types of binding sites: H3K9me3-marked retrotransposons and H3K4me3 or H3K27Ac-marked gene promoters/enhancers. Our results suggest that dimerization, ATPase activity and DNA binding via the Coiled coil 1 domain are critical for MORC2 binding to LINE1 retrotransposons but an alternative mechanism is involved in MORC2 recruitment to gene promoters. The uncharacterized CD domain of MORC2 is essential for this mechanism. Furthermore, we identified that patient mutation S87L relocalizes MORC2 from retrotransposons to gene promoters of ZNF genes. Our insights advance our understanding of domain requirements for MORC2 recruitment. The discovery of S87L mutant binding to ZNF genes opens up new fields for exploring mechanisms involved in MORC2 mutant binding and neurodevelopmental disorders.}}, author = {{Sivani, Bala Mohan}}, language = {{eng}}, note = {{Student Paper}}, title = {{Mechanisms of chromatin binding by the MORC2 ATPase}}, year = {{2024}}, }