CTCF knockout reveals an essential role for this protein during the zebrafish development

Abstract

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans

“…How strategies to demarcate chromatin domains have evolved in eukaryotes remain unclear. In vertebrates, CTCF has a central role in defining TAD boundaries and is essential for development [11,59,60]. In D. melanogaster, CTCF is essential but does not appear to define TAD boundaries, and instead acts as a barrier insulator [61][62][63].…”

Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans

“…How strategies to demarcate chromatin domains have evolved in eukaryotes remain unclear. In vertebrates, CTCF has a central role in defining TAD boundaries by mediating DNA loops and is essential for development [11,57,58]. In D. melanogaster, CTCF is essential but does not appear to define TAD boundaries, and instead acts as a barrier insulator [59][60][61].…”

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

“…For immunofluorescence, embryos were fixed overnight at 4°C with 4% paraformaldehyde, washed in PBT (PBS supplemented with 0.2% Triton-X100) and blocked in this solution with 2% goat serum and 2 mg/ml BSA for 1 h at RT. Then, they were incubated overnight at 4°C with primary antibody specific for zebrafish CTCF 49 (used in 1:500 dilution). After extensive washings with PBT, embryos were incubated overnight at 4°C with goat anti-rabbit Alexa Fluor 488 secondary antibody (used 1:800 dilution, A27034 Invitrogen).…”

“…The recovered supernatant, which contained soluble chromatin, was used for ChIP or frozen at −80°C after checking the size of the sonicated chromatin. Four 250 μ l aliquots of sonicated chromatin were used for each independent ChIP experiment, and each aliquot incubated with 2 μ g of anti-CTCF antibody 49 and rotated overnight at 4°C. Next day, 20 μ l of protein G Dynabeads (Invitrogen) per aliquot were washed twice with ChIP dilution buffer and resuspended in 50 μ l/aliquot of the same solution.…”