Understanding gene regulation by HDAC1 complexes in development and cancer

Packaging DNA into nucleosomes helps protect the long fragile genomes of eukaryotic species. However, in doing so it becomes an ever-present physical barrier to the machinery required for its replication, repair and transcription. Wrapped up tightly in its histone overcoat, how do cells gain access to the underlying DNA? Universally, in species as diverse as brewers’ yeast, fruit flies, worms and man, the answer is to post-translationally modify (PTM) the histones to either help open it up, or compress the chromatin still further. Lysine-acetylation (Lys-Ac) is one of the most common histone PTMs and occurs on the unstructured and Lys-rich N-terminal tails of the core histones (H2A, H2B, H3 and H4). Given the density of Lys residues within histone tails, neutralization of their positive charge by acetylation reduces the overall affinity of histones for the negatively charged DNA backbone, opening chromatin and making it more transcriptionally permissive. Histone acetylation is highly dynamic with its level regulated by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). There are 18 HDAC enzymes in mammalian cells that can be sub-divided according to the presence of Zn2+-dependent (Class I, II and IV) or NAD+-dependent (Class III/Sirtuins) catalytic domains. As master regulators of chromatin accessibility, HDACs have been implicated in almost all nuclear functions, including DNA repair, DNA replication, chromosome segregation and gene expression. There is a compelling motivation to understand the fundamental biology of HDAC enzymes and to exploit this knowledge to improve their use as drug targets.

Because of their role in cell cycle progression, HDAC inhibitors (HDACi), such as SAHA, have been utilised as anticancer agents. HDACi are also established treatments for epilepsy and bi-polar disorder, and have shown promise as therapeutics for neurodegenerative disorders, such as Huntingdon’s disease. However, the use of pan-HDACi in patients is associated with multiple debilitating side-effects. Even specific HDACi, such as Entinostat (MS-275) which target predominantly HDAC1/2, results in many of the same problems, presumably because it still targets all four HDAC1/2 containing complexes non-specifically. Given the positive therapeutic value of HDAC inhibition in numerous disease states, and the detrimental side-effects of generic HDAC inhibition, there is a strong imperative to design novel HDAC inhibitors (HDACi) with improved specificity and alternative modes of action. Or to phrase the question in another way, can we drug individual HDAC1/2 complexes? And if so, what are the consequences?

Projects in the Cowley lab:

1) The role of HDAC1/2 complexes in development

HDAC1/2 complexes are essential for embryonic development. We have therefore used gene editing methods, such as CRISPR, to generate embryonic stem (ES) cell lines in which we can specifically ‘switch off’ HDAC1 and HDAC2, or components of specific complexes e.g. LSD1 and Sin3A, to examine their critical roles in development using a number of ES cell differentiation systems. Currently, we are performing transcriptomic experiments in ‘gastruloids’, an organoid system formed from aggregating ES cells that closely mimics the gastrulating embryo.

2) Targeting HDAC1/2 for degradation in cancer cells

In collaboration with Dr James Hodgkinson (Dept. of Chemistry) at the University of Leicester, have been developing novel PROTACs directed towards HDAC1/2. Proteolysis Targeting Chimaeras (PROTAC) are hetero-bifunctional molecules which incorporate a known binding moiety to the protein of interest (POI, e.g. an inhibitor), coupled to a ligand for an E3 ubiquitin ligase complex. Direct recruitment of the E3 ligase to the POI via the PROTAC, targets it for ubiquitination and ultimately degradation. We recently published the first PROTACs to target HDAC1/2 specifically in cancer cells and are currently optimising additional molecules that target specific complexes.

Keywords:

Biochemistry, Cancer, CRISPR, Gene regulation, Molecular Biology, Epigenetics and Chromatin

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