Understanding the Chromatin Regulatory Landscape Controlling Fibrinogen Gene Expression
ISTH Academy. Fish R. Jul 10, 2019; 274117; OC 74.2 Topic: Fibrinogen & Factor XIII
Richard Fish
Richard Fish
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OC 74.2

Understanding the Chromatin Regulatory Landscape Controlling Fibrinogen Gene Expression

R.J. Fish, C. Espitia Jaimes, C. Di Sanza, M. Neerman-Arbez
University of Geneva, Faculty of Medicine, Department of Genetic Medicine and Development, Geneva, Switzerland

Main Topic: Fibrinolysis and Proteolysis
Category: Fibrinogen & Factor XIII

Background: The human fibrinogen locus can adopt a looped chromatin configuration that contributes to fibrinogen expression. Within this loop are the three fibrinogen genes (FGA, FGB and FGG), their proximal promoters, and four liver cell enhancers. The functional importance of these enhancers and their regulatory hierarchy is unclear.
Aims: To understand the regulatory element hierarchy that coordinates expression of the fibrinogen gene cluster.
Methods: We used a CRISPR-Cas9-based method to direct an active regulatory element histone modification, acetylation of histone H3 lysine 27 (H3K27ac), to fibrinogen promoters and enhancers in non-fibrinogen-expressing HEK-293T cells. Transfection of combinations of plasmids for expression of single guide RNAs (sgRNAs) and a nuclease-deficient Cas9 nuclease fused to the histone acetyltransferase core domain of p300 (dCas9-p300), were used to direct p300 catalytic activity and H3K27ac to specific chromatin sites. Fibrinogen expression was measured by qRT-PCR.
Results: FGA and FGB transcripts were not detected in HEK-293T cells, FGG is expressed near the lower limit of qRT-PCR detection. dCas9-p300 and sgRNAs directed to the FGA, FGB and FGG proximal promoters induced expression of FGA and FGB and increased FGG expression, respectively. The FGG gene, with pre-existing basal expression, showed increased expression when targeting dCas9-p300 to any of the gene cluster enhancers. However, increased expression of the FGA and FGB genes, by directing dCas9-p300 to enhancers, was only measured when their proximal promoters were also targeted. By concurrently targeting a single fibrinogen promoter and individual enhancers for H3K27ac we measured similar gene expression increases for each enhancer on each gene, suggesting functional redundancy among the fibrinogen locus enhancer elements.
Conclusions: Active enhancer elements of the fibrinogen gene cluster can act on any of the fibrinogen genes, provided the proximal gene promoters are in a transcriptionally active state. This redundancy in enhancer activity can now be investigated by sequential deletion of enhancers in fibrinogen-expressing cells.

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