Additional file 4: Movie S2. STAG2-GFP iFRAP

Aleksandar Kojic (14176872)

Ana Cuadrado (5563994)

Ana Losada (187338)

Daniel Giménez-Llorente (14176863)

Magali De Koninck (14176866)

Miguel Ruiz-Torres (14176869)

Miriam Rodríguez-Corsino (265470)

Epigenetics & Chromatin

PubMed

BACKGROUND: The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood. RESULTS: The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast. CONCLUSION: Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13072-022-00469-0

Cuadrado, Ana

Giménez-Llorente, Daniel

De Koninck, Magali

Ruiz-Torres, Miguel

Kojic, Aleksandar

Rodríguez-Corsino, Miriam

Losada, Ana

PubMed Central

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

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Additional file 4 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 3: Movie S1. STAG1-GFP iFRAP

Additional file 3 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 5: Movie S3. RAD21-GFP iFRAP

Additional file 5 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

BACKGROUND
The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood.
RESULTS
The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast.
CONCLUSION
Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.This work has been funded through grants from the Spanish Ministry of Science, Innovation and Universities and FEDER funds (Grants BFU2013-48481-R and BFU2016-79841-R to A.L.; BES-2014-069166 and BES-2012-055441 fellowships to M.D.K. and M.R-T) and from the Spanish Research Agency (AEI) (Grant PID2019-106499RB-I00 to AL). DG-Ll is currently supported by AECC (Spanish Association Against Cancer).S

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Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin.

https://repisalud.isciii.es/bitstream/20.500.12105/17655/1/Contributionofvariantsubunits_2022.pdf

Additional file 4 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

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