Functional implications of IGF2-mediated post-translational modifications of REST

Repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) is a transcriptional silencer of neuronal gene expression. Experimental evidence supports its vital role in neuronal differentiation, repression of neuronal genes in non-neuronal cells and more recently, in the regulation of the cell cycle. Aberrant REST activity can lead to oncogenesis and indeed, REST can act as both a tumour suppressor and an oncogene, depending on the cellular and molecular context. Preliminary data, using microarray analysis on mouse embryos with differential IGF2 dose supply, identified REST as one of the key transcriptional hub genes that define the signalling output of IGF2. REST was stabilised within 6 hours of exogenous exposure to IGF2, in a proteasome and PI3K-dependent manner. IGF2 is a mitogenic peptide that is crucial for normal fetal growth. IGF2 dosage control has been shown to be essential in regulating the different neurological niches of the adult brain as well as in growth and development. The hypothesis tested in this thesis was whether IGF2 stimulation induces REST protein stabilisation by a post-translational modification preventing its ubiquitination and subsequent proteasomal degradation. Here, we evaluate the mechanism of this stabilisation.
Results showed that IGF2 stabilises REST by de-phosphorylation, preventing its ubiquitination and degradation by the proteasome, leading to an increase in global REST levels, which, in turn, drives a secondary increase in the nuclear and potentially functional form of REST. The IGF2-mediated REST stability was found to be dependent on the cell lines used, the timing of the IGF2 exposure and feedback signalling. I attempted to characterise the REST phosphosite that might be responsive to IGF2 by using an in silico approach followed by mass spectrometry of exogenously expressed tagged-REST. Multiple high molecular weight (MW) REST forms were identified by Western blotting, which initiated an attempt to characterise the different MW forms of REST systematically. The 220 kDa high MW REST form was found to be phosphorylated, O-glycosylated, nuclear in localisation, and hence potentially the functional REST form. A dimerisation mechanism, mediated by a covalent linkage, was hypothesised to explain the increase in MW. CRISPR/Cas9 technology was next utilised to achieve a REST knock out for functional studies. An efficiency of 38-54% in indel generation was achieved which resulted in heterozygous REST knock out cells and a complete failure to obtain REST null homozygotes, indicating knockoutrelated cell lethality or differentiation phenotype.
This work suggests a role for IGF2 in stabilising and maintaining levels of REST, possibly during development, when neuronal precursor cells undergo lineage commitment and terminal differentiation, as the brain and nervous system are forming. By stabilising REST and repressing neuronal differentiation, IGF2 might redirect cells towards proliferation. Hence, we have identified a key regulatory signalling pathway, which might regulate a balance between growth and neuronal differentiation.

Advisor: Hassan, B