Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB325

Poster Communications

The altered gene expressions induced by the lysine deacetylase (KDAC) inhibitor trichostatin A in HAP1 cells is unaffected by knockout of KDAC8 or KDAC1

I. A. Ghouri1, N. Ossai2, G. N. Europe-Finner1, M. Taggart1

1. Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom. 2. Newcastle University, Newcastle upon Tyne, United Kingdom.

It is now apparent, partly through the use of class I lysine deacetylase (KDAC) inhibitors such as trichostatin A (TSA), that regulation of the acetylation status of protein lysine (K) residues is an important determinant of smooth muscle (SM) function (1). Although nuclear-resident KDACs, such as KDAC1, are thought to regulate the transcriptional machinery controlling much gene expression, another class I KDAC, KDAC8, is localised predominantly outside the nucleus in SM and regulates acetylation of non-nuclear proteins. However, it is uncertain if KDAC8 influences global gene expression changes. This study utilised a model system of human cells with selective knockout (KO) of KDAC8 or KDAC1 to compare the relative influences that absence of each protein had on basal and TSA-mediated gene expression. Control (WT), and 3 separate clones each of KDAC8 KO and KDAC1 KO HAP1 cells, were produced using CRISPR-Cas9 technology (Horizon Discovery, Vienna). The presence and localisation of KDAC8 and KDAC1 were assessed using Western blot and immunofluorescence microscopy. Cells were treated for 16 hours with 0.5µM TSA or vehicle controls. RNA was extracted and gene expression assessed on Illumina HT-12 chips (n=3 per group). Analysis was performed with Perseus software, comparisons made by 2 sample t-test and bioinformatic pathway analysis performed in STRING (v10). KDAC8 was expressed predominantly in non-nuclear regions, whereas KDAC1 was mostly in the nucleus, suggesting similar localisations to SM cells. KDAC8 and KDAC1 proteins were absent in their respective KOs. TSA treatment increased acetylation of α-tubulin and histone H3, indicating TSA action on non-nuclear- and nuclear-resident KDACs. TSA affected the expression of 3693 genes in WT, 2955 genes in KDAC8 KO and 2770 genes in KDAC1 KO cells (FDR 0.01,>2 fold change). Of note, TSA altered expression of 1624 genes (>2-fold) common to WT, KDAC1 KO and KDAC8 KO. In addition, direct comparison of KDAC1 KO and KDAC8 KO clones showed no differences in basal or, surprisingly, TSA-induced gene expression profiles. Pathway analysis of the 400 most downregulated genes by TSA in each group indicated prominent alteration of transcriptional machinery components, including genes encoding KDAC7, KDAC9 and many zinc finger proteins. Genes upregulated by TSA (top 400 in each group) included many encoding proteins involved in membrane transport. The absence of non-nuclear KDAC8 protein, or nuclear KDAC1 protein, has little effect on basal or TSA regulated gene transcription, suggesting a robust and modular control of gene expression by the KDAC superfamily. The aforementioned TSA-mediated alteration of expression of genes encoding transcriptional proteins may be influential in this regulation.

Where applicable, experiments conform with Society ethical requirements