Abstract Number: PB0134
Meeting: ISTH 2021 Congress
Background: Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa. It has been shown that FOXP3 is a TFPI repressor at the transcriptional level. At present, there are limited studies on the control of TFPI expression.
Aims: To identify the Chromatin Binding and Regulatory Proteins (CBRPs) involved in tfpia gene expression using the zebrafish model.
Methods: The RNAs encoding CBRPs were selected from zebrafish thrombocyte Single-cell RNASeq data. Piggyback knockdowns of these CBRPs using ASOs and the quantitative Real Time PCR (qRT-PCR) were performed in adult zebrafish. Knockdowns and laser-induced thrombosis to measure TTO were performed on larvae.
Results: To identify the CBRPs involved in tfpia gene expression, we selected 103 CBRP mRNAs. We eliminated 34 mRNAs encoding histone-modifying enzymes. Thus, we targeted 69 mRNAs and performed 3-mRNA knockdowns using 3 ASOs each in 23 sets. After 48 hours, qRT-PCR results showed knockdown of brd7, brd9, ccdc61, ing2, ing3, ing4, supt16h, suz12a, and suz12b increased tfpia gene expression. In the secondary screen, we found knockdown of 5 of these genes, brd7, ing2, ing3, ing4, and suz12b showed an increase in tfpia mRNA levels. We confirmed these 5 genes by a tertiary knockdown. We then examined the effects of simultaneous knockdown of all these 5 genes and found increased tfpia gene expression but was not greater than that shown by individual gene knockdowns. We also confirmed the above adult knockdown results of 5 genes in larvae by TTO studies.
Conclusions: Our results revealed that proteins encoded by brd7, ing2, ing3, ing4, and suz12b are negatively regulating tfpia gene transcription and the reduction in their gene expression led to increased tfpia mRNA levels. This information should be useful in designing novel drugs that inhibit these proteins such that Tfpia levels could be increased to treat patients with thrombotic risk.
To cite this abstract in AMA style:Raman R, Fallatah W, Al Qaryoute A, Jagadeeswaran P. Genome-wide Knockdown of Chromatin Binding and Regulatory Protein Genes and Regulation of Zebrafish tfpia Gene Expression [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/genome-wide-knockdown-of-chromatin-binding-and-regulatory-protein-genes-and-regulation-of-zebrafish-tfpia-gene-expression/. Accessed January 24, 2022.
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