Transcriptional and structural outcomes of genome-wide CTCF depletion in B cells

Trabajo Fin de Máster en Bioinformática y Biología ComputacionalCTCF is involved in establishing long-range interactions that define chromatin architecture and regulate transcriptional programs. In B cells, CTCF participates during VDJ recombination and class switch recombination, both critical processes for the immune response. However, to date, the relationship between CTCF-mediated contacts and their transcriptional implications in mature B cells is not completely understood. Here we used a conditional mouse model where CTCF is eliminated specifically in mature B cells and found a subset of CTCF-binding sites that are resistant to protein depletion. These "retained" CTCF sites have a higher proportion of consensus like CTCF motifs and are preferentially localized at topologically associating domains (TADs) boundaries. In addition, we found that CTCF deletion causes few transcriptional changes in mature B cells. To link CTCF differential binding with changes in gene expression we studied CTCF binding to promoter regions and the formation of CTCF-mediated loops. With that aim, we developed an algorithm that identifies regions that can be transcriptionally regulated by CTCF-dependent loops. We consider that this approach represents a step forward in the understanding of transcriptional regulation mediated by CTCF loops, which will be further strengthen with the experimental validation of the regions identified in our study

Assessing the impact of an antigen-specific antibody response on atherosclerosis development in mice.

The antibody immune response plays a critical role in atherosclerosis. Here, we present a protocol for assessing the impact of an antigen-specific germinal center antibody response on atherosclerosis development, using a pro-atherogenic mouse model deficient for the production of germinal-center-derived antibodies. We describe steps for bone marrow transfer from donor mice into irradiated recipient mice. We then detail immunization of mouse chimeras with atheroprotective malondialdehyde low-density lipoprotein during high-fat diet feeding and atherosclerosis burden analysis. For complete details on the use and execution of this protocol, please refer to Martos-Folgado et al. (2022).1.We thank all members of the B Cell Biology Laboratory for useful discussions. A.D.M.M. is funded by ‘‘la Caixa’’ Foundation HR17-00247, I.M.-F. was a fellow of the research training program funded by Ministerio de Economía y Competitividad (SVP-2014-068216), A.R-R. is a fellow of the research training program funded by Ministerio de Ciencia, Innovacio´ n y Universidades (PRE2020-091873), and A.R. is supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from ‘‘la Caixa’’ Foundation under the project code HR17-00247 and HR22-0253 and from SAF2016-75511-R and PID2019-106773RB-I00/AEI/ 10.13039/501100011033 grants to A.R.R. (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016 Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigación: Proyectos I + D + i 2016, Ministerio de Economía, Industria y Competitividad) and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa institute (CEX2020-001041-S grant funded by MCIN/AEI/ 10.13039/501100011033).S

Low-affinity CTCF binding drives transcriptional regulation whereas high-affinity binding encompasses architectural functions.

CTCF is a DNA-binding protein which plays critical roles in chromatin structure organization and transcriptional regulation; however, little is known about the functional determinants of different CTCF-binding sites (CBS). Using a conditional mouse model, we have identified one set of CBSs that are lost upon CTCF depletion (lost CBSs) and another set that persists (retained CBSs). Retained CBSs are more similar to the consensus CTCF-binding sequence and usually span tandem CTCF peaks. Lost CBSs are enriched at enhancers and promoters and associate with active chromatin marks and higher transcriptional activity. In contrast, retained CBSs are enriched at TAD and loop boundaries. Integration of ChIP-seq and RNA-seq data has revealed that retained CBSs are located at the boundaries between distinct chromatin states, acting as chromatin barriers. Our results provide evidence that transient, lost CBSs are involved in transcriptional regulation, whereas retained CBSs are critical for establishing higher-order chromatin architecture.We thank all the members of the B lymphocyte Biology lab for helpful suggestions, Sonia Mur for technical assistance, Ana Losada and Ana Cuadrado for helpful discussions on experimental design, Roma´ n Pe´ rez Santalla for his help on algorithm design, Simon Bartlett for English editing, and the CNIC Genomics Unit for ChIP-seq and RNA-seq. We also thank Rafael Casellas and Erez Aiden for sharing HiC data with us and Jing Luan and Gerd Blobel for their help to access their datasets. This work was supported by grants from the Spanish Ministerio de Economı´a, Industria y Competitividad and ERDF, A way of making Europe (SAF2016-75511-R), the Spanish Ministerio de Ciencia e Innovacio´ n (PID2019-106773RB-I00/AEI/10.13039/ 501100011033) and the ‘‘la Caixa’’ Banking Foundation under the project code HR17-00247 to A.R.R. F.S.-C. received support from the Spanish Ministerio de Economı´a y Competitividad (RTI2018-102084-B-I00). E.M.-Z. and A.R.-R. are fellows of the research training program (FPI) funded by the Ministerio de Economı´a y Competitividad (BES-2014-069525) and Ministerio de Ciencia e Innovacio´ n (PRE2020-091873). M.J.G., F.S.-C., and A.R.R. are supported by CNIC. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacio´ n (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence, CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033.S