Kevin G. Ford

Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1–0.8) and 2.1 (P<0.04, 95% CI, 1.1–4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients’ outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.

Specific targeting of cytosine methylation to DNA sequences in vivo

Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions.

Heritable Gene Repression through the Action of a Directed DNA Methyltransferase at a Chromosomal Locus

The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.

Use of altered-specificity binding Oct-4 suggests an absence of pluripotent cell-specific cofactor usage

Oct-4 is a POU domain transcription factor that is critical for maintaining pluripotency and for stem cell renewal. Previous studies suggest that transcription regulation by Oct-4 at particular enhancers requires the input of a postulated E1A-like cofactor that is specific to pluripotent cells. However, such studies have been limited to the use of enhancer elements that bind other POU-protein family members in addition to Oct-4, thus preventing a ‘clean’ assessment of any Oct-4:cofactor relationships. Other attempts to study Oct-4 functionality in a more ‘stand-alone’ situation target Oct-4 transactivation domains to DNA using heterologous binding domains, a methodology which is known to generate artificial data. To circumvent these issues, an altered-specificity binding Oct-4 (Oct-4RR) and accompanying binding site, which binds Oct-4RR only, were generated. This strategy has previously been shown to maintain Oct-1:cofactor interactions that are highly binding-site and protein/binding conformation specific. This system therefore allows a stand-alone study of Oct-4 function in pluripotent versus differentiated cells, without interference from endogenous POU factors and with minimal deviation from bound wild-type protein characteristics. Subsequently, it was demonstrated that Oct-4RR and the highly transactive regions of its N-terminus determined here, and its C-terminus, have the same transactivation profile in pluripotent and differentiated cells, thus providing strong evidence against the existence of such a pluripotent cell-specific Oct-4 cofactor.

Single zinc-finger extension: enhancing transcriptional activity and specificity of three-zinc-finger proteins.

Abstract In order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.