The control of transcriptional corepressors by synaptic activity

Title: novel routes to the activation of gene transcription by synaptic activity: Brain cells (neurons) communicate with each other by releasing chemical messengers (neurotransmitters) onto each other at structures called synapses, a process called 'synaptic activity'. These messengers are detected by special channels on the cell surface, which then open and allows calcium and sodium ions to flow into the cell. This triggers the release of neurotransmitter onto yet more neurons. This means of neuron-to-neuron communication is the way by which information flows round the brain. However, 'synaptic activity' also triggers changes inside neurons. The calcium ions which flow into the neuron activate signal pathways, which in turn activate the transcription of genes. Transcription is a crucial step in the process whereby genes (made of DNA and located in the nucleus) are read by the cell's machinery and decoded into new proteins. These new proteins are crucial for many fundamental processes in the neuron. For example, learning and memory involves changes in the way neurons communicate with each other, and this process relies on these new proteins made in response to 'synaptic activity'. These new proteins also control how neurons in the brain develop from the foetus, through infancy and on to adulthood. Equally importantly, these new proteins also make individual neurons healthier and more likely to survive for longer than neurons that don't experience synaptic activity. Therefore, an understanding of how synaptic activity activates gene transcription is an important problem for scientists studying the brain. Our proposed research will characterise a completely new way by which genes can be activated by synaptic activity. The transcription of many genes is suppressed by special molecules called corepressors. One particularly important one is called SMRT, which represses many different genes in the nucleus by blocking the action of the cell's transcription machinery. We have recently discovered that when calcium ions flow into neurons following synaptic activity, signals in the neuron are activated which cause SMRT to leave the nucleus and go into the cytoplasm. Once in the cytoplasm, SMRT is unable to suppress transcription because the genes and transcription machinery are all in the nucleus. Therefore these genes become much easier to activate. Our work will uncover the exact signalling events that take place that make SMRT stop repressing transcription in the nucleus, and go into the cytoplasm. In addition, we will identify exactly what type of genes are likely to be influenced by this 'export' of SMRT. We will also determine the effect that SMRT export has on the way in which a neuron develops, looking particularly at the way a neuron changes shape as it matures. Because SMRT is known to repress the transcription of so many types of gene, signals that stop SMRT from working have the potential to have a big effect on the neuron. As mentioned earlier, the activation of gene transcription by synaptic activity controls many very important processes. SMRT export triggered by synaptic activity is a previously undiscovered route by which transcription of many genes can be turned on. Therefore understanding the mechanism and consequences of this process is of utmost importance. While this work is centred on the study of neurons, SMRT represses genes in many cell types, so the relevance of this work is not restricted to neurons. Furthermore, calcium ions don't just have effects in neurons, they are able to activate signalling pathways in all types of cell, from white blood cells to egg cells. The gene transcription that calcium ions activate in these cells are important for other processes, such as for white blood cells to fight infection. therefore our discoveries regarding how calcium activates gene transcription in neurons will be of benefit to scientists researching a wide variety of problems.

Question J summarises the background to the project and the project objectives (1-4). There follows below a technical summary of the proposed research, outlining the experimental and methodological approaches that will be adopted to achieve each project objective: 1. Identify the direct activity-induced signalling events leading to SMRT export: To determine whether SMRT export relies on an intrinsic nuclear export signal (NES), we will mutate a conserved putative NES which we have found by an in silico search. A lack of effect would indicate either a highly non-consensual NES, or that export is promoted by other associated proteins. To determine the region of SMRT that confers activity-dependent export, we will exploit our finding that while SMRT undergoes activity-dependent nuclear export, its paralogue, N-CoR does not. We will analyse the effect of synaptic activity on the subcellular redistribution of GFP fusions of a series of SMRT/N-CoR chimeras, to identify which region of SMRT is necessary to confer activity-dependent export. We will then investigate whether this region confers export by being the target of activity-dependent phosphorylation events. Our recent work uncovered potential roles for nuclear CaM kinase IV and the Ras-MEK1-ERK1/2 pathway in triggering SMRT export. Therefore consensus sites for these kinases will be mutated (to alanine). If any of these affect activity dependent export, confirmation that these sites are phosphorylated in vivo will be determined by studying in vivo activity-dependent P-32 incorporation into SMRT. 2. Identify the contribution of activity-dependent class IIa HDAC export in promoting SMRT export: In the nuclei of electrically silent neurons we have shown that SMRT colocalises with the predominant neuronal nuclear class IIa HDAC, HDAC5. We will confirm this by co-immunoprecipitation, which we will also use to determine if any other class IIa HDACs associate with SMRT. To determine whether the prior export of HDAC5 causally influences the subsequent export of SMRT, we will express a constitutively nuclear, non-exportable mutant of HDAC5 (and look for impaired export of SMRT). Conversely we will perform siRNA mediated knock-down of HDAC5, and study whether SMRT's normal nuclear localisation is affected (even in the absence of synaptic activity). 3. Understand the activity-dependent disruption of corepressor complexes on endogenous SMRT-repressed promoters: We will study several endogenous promoters that are regulated by 4 different SMRT-repressed transcription factors (TR, CBF1, SRF, NF-kB). We will first confirm association of these transcription factors with the appropriate region of the promoter by chromatin-immunoprecipitation (ChIP). We will then analyse the association of SMRT at these promoters (also by ChIP) and study the activity-dependent dissociation of SMRT in each case. This will determine whether synaptic activity induces dissociation of SMRT irrespective of which transcription factor it is interacting with, or whether its effect is restricted to specific transcription factors. Furthermore, we will use ChIP to determine whether non-exportable mutants of SMRT (created in objective 1) or HDAC5 actually stabilise the corepressor complex at endogenous promoters, making it refractory to activity-induced signalling. 4. Analyse the role of activity-dependent SMRT export on a downstream physiological output: development of dendritic architecture Using a eGFP marker to track neurite outgrowth, we have preliminary evidence that CaM kinase IV potentiates Notch-dependent changes to dendritic outgrowth and branching. Using a similar approach, we will determine whether CaM kinase IV potentiates the known promoting effect of thyroid hormone on dendritic spine density (using confocal microscopy of eGFP-expressing neurons). We will also identify the effect of expressing non-exportable mutants of SMRT or HDAC5 on this CaM kinase IV- dependent potentiation.