Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target

by Selleckchem | Oct 15 2023

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

Comments:

Your hypothesis and experimental findings are intriguing and provide valuable insights into the regulation of the tissue factor pathway inhibitor (TFPI) gene expression. By conducting a knockdown screen of chromatin binding and regulatory proteins (CBRPs) in zebrafish, you have identified several novel regulators of tfpia mRNA levels, namely brd7, ing2, ing3, ing4, and suz12b. Furthermore, the simultaneous knockdown of these five genes resulted in increased tfpia mRNA levels, suggesting their cooperative effect on TFPI regulation.

To further investigate the functional relevance of these regulators, you performed individual and simultaneous knockdowns of the identified genes in zebrafish larvae. The results indicated that after laser injury, the time taken for the formation of a thrombus to occlude the caudal vessel was prolonged compared to control larvae. This suggests that the downregulation of these genes impairs the normal clotting process, likely due to the decreased levels of TFPI.

Additionally, you treated zebrafish larvae and adults with UNC6852, a chemical known to degrade the polycomb repressor complex 2, which includes SUZ12 as a member. The treatment resulted in both prolonged time to occlude the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. These findings suggest that targeting the polycomb repressor complex 2 with UNC6852 enhances TFPI expression and delays thrombus formation.

The discovery of these novel epigenetic regulators for tfpia and the identification of UNC6852 as a potential antithrombotic drug represent significant advancements. These findings lay the foundation for further investigations into the therapeutic potential of UNC6852 and its application as an antithrombotic drug. Moreover, your approach of studying chromatin remodeling and gene regulation can be extended to explore the regulation of other plasma proteins involved in coagulation, including both coagulant and anticoagulant factors.

Overall, your research provides valuable insights into the regulatory mechanisms of TFPI expression and offers potential avenues for the development of new therapeutic strategies in the field of thrombosis and hemostasis.