The Role and Mechanisms of Action of PML II on Regulating Gene Expression

Promyelocytic leukaemia protein (PML), initially identified in acute promyelocytic leukaemia (APL) cancer patients, is a pleiotropic protein implicated in a range of cellular functions such as regulating transcription, defence against viral infection and control of apoptosis and cell growth. The PML gene consists of nine exons giving rise to several different splice-variant transcripts. The isoforms are divided into seven groups, PML I - VII, and all contain an identical N-terminal region encoded by exons 1 to 6 including a RING-finger, two B-box and a Coiled-Coil motif. The differences in isoform structure leads to variation in isoform activity. PML nuclear bodies (NBs) are organised by the PML protein and recruit numerous partner proteins to regulate cellular activities.

PML-II has been shown to be required for efficient interferon response gene expression, and transcription and modification of chromatin architecture are two functions controlled by PML NBs. This project aims to understand how PML-II functions in the IFN response, focusing on events at the IFNBeta promoter. In uninfected cells, the IFNBeta gene is constitutively repressed, however, upon viral infection the gene is transiently activated. ChIP assays have shown there is a direct correlation between acetylation at histone H4 and the transcriptional activity of the IFNBeta promoter region. While repressed, the IFNBeta promoter region was hypoacetylated, silencing the IFNBeta genes. Upon inhibition of histone deacetylase, hyperacetylation of histone H4 occurs at the IFNBeta promoter leading to increased IFN levels, conferring the antiviral state. Other modifications found in the IFNBeta promoter site, such as methylations on a subset of lysine residues on histones H3 and H4 occur during activation of gene transcription. I will investigate these modifications at the IFNBeta gene to look for markers of transcription activation, such as histone 3 lysine 4 trimethylation (H3K4me3) and modifications for repression of transcription such as histone 3 lysine 27 trimethylation (H3K27me3). The balance between H3K4me3 verses H3K27me3 may indicate the transcriptional activity at the genes of interest. By looking at differences that arise when PMLII has been reduced, we will identify functions associated with histone modifications that involve PML.

Class switching of whole histones also occurs during activation of gene transcription. Eukaryotic DNA is packed into nucleosomes composed of 4 different histone proteins: H2A, H2B, H3 and H4. There are 5 variants of H3 histone protein, the main two being H3.1 and H3.3. H3.1 is enriched in regions of transcriptional repression and coincides with H3K27me3 sites. However, at regions with high transcriptional activity, H3.3 is more abundant. There is a suggested role for the PML protein in class switching from H3.1 to H3.3 via its interactions with death-associated protein 6 (DAXX) at SUMOylated sites on PML in PML NBs. PML opposes H3.3 histone deposition by DAXX and loss of PML alters chromatin composition, shifting histone modifications to repressive H3K27me3 markers and increased H3.3 deposition. The exchange of H3.1 to H3.3 at the IFN-Beta promoter is something I will investigate in wildtype and knockdown models of PMLII.