Objetivo

4.2.1 Summary of Findings

The genetic strategies that I developed to both separate and profile nuclear gene expression in cortical excitatory and inhibitory neurons allowed me to 1) identify genes whose expression is enriched in a particular cell type over the other, and 2) determine how neuronal genes, and particularly excitatory- and inhibitory-enriched genes, are typically expressed within an unperturbed physiological context. Previous studies have demonstrated that neuronally- expressed genes tend to be longer in gene length relative to genes expressed in other somatic tissues and cell types (Gabel et al., 2015; King et al., 2013; Zylka et al., 2015). I confirmed as

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such among genes expressed in excitatory and inhibitory cortical neurons of adult mice, which were significantly longer than genomic distributions of gene length. The same was true for excitatory- and inhibitory-enriched genes, which tended to be cumulatively longer and shorter in gene lengths, respectively.

However, I observed additional cell-specific behaviors among active expressed genes in each cell type that have not been previously characterized. Excitatory and inhibitory neurons largely express the same group of genes. These include genes whose expression is lowly or moderately enriched (log2 FC < 3) in one cell type as opposed to the other. The manner in which

these genes are expressed, however, is dramatically different. Short inhibitory-enriched genes, which are associated with cellular respiration and energy metabolism, are expressed in a length- dependent manner in both excitatory and inhibitory neurons, which would be expected of metabolically active neurons regardless of subtype. It is likely that these genes are more highly expressed in inhibitory neurons because, as mitochondria-rich cell types, they have perpetual role in dynamically suppressing the activity of excitatory neurons to maintain overall network

homeostasis in the brain (Kann et al., 2014; Maffei and Fontanini, 2009). In contrast, long excitatory-enriched genes are positively correlated with the level of gene expression exclusively within excitatory neurons; this relationship is completely absent in inhibitory neurons. Excitatory- enriched genes encode proteins that promote axogenesis (signaling molecules, actin-associated proteins), are localized to the plasma membrane (synaptic proteins, ion channels,

transmembrane receptors) and extracellular matrix (secreted and cell adhesion molecules, integrins), and participate in intracellular signaling cascades (protein kinases and phosphatases). Thus these genes are consistent with the role of cortical pyramidal neurons that undergo

extensive changes in synaptic plasticity at the plasma membrane in response to neuronal circuit activity (Beaulieu, 1993; Emes et al., 2008; Holtmaat and Svoboda, 2009).

4.2.2 Broader Significance

Because excitatory- and inhibitory-enriched genes and their associated functions are consistent with the known roles of these two broad cortical cell types, I posit that excitatory

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neurons exhibit a strong positive association between gene length and expression level in order to carry out an assortment of cell-specific functions required for proper neuronal activity in the brain. I would thus expect cortical pyramidal neurons to be particularly sensitive to factors that perturb this length-dependent transcriptional regulation, which is supported by the effects of DNA supercoiling and topoisomerase inhibition on mammalian cells (King et al., 2013; Kouzine et al., 2013; Mabb et al., 2014; Madabhushi et al., 2015; Naughton et al., 2013; Puc et al., 2015).

Topoisomerases are enzymes that relieve the torsional stress associated with DNA supercoiling, which occur as a consequence of both cell division and gene transcription (Chen et al., 2013; Tsao et al., 1989; Wang, 2002). Cultured cortical neurons treated with 300µm

topotecan, a reversible TOP1 inhibitor, display impairments in excitatory synapse formation and function and deficits in inhibitory synapse function, leading to overall reduced spontaneous network activity without compromising cellular health. These effects are caused by an inability to resolve DNA supercoiling at particularly long genes, leading to impaired transcriptional elongation of RNA polymerase II and length-dependent downregulation of neuronal genes important for synaptic function, many of which include genes associated with autism. In addition to length- dependent transcription, topoisomerase 1 has also been shown to be required for ligand-

dependent androgen receptor-mediated enhancer activation in prostate cancer cells by relieving torsional strain required for productive elongation of eRNAs (Puc et al., 2015). Immediate early gene activation and expression in depolarized primary cortical neurons also requires the

production of double-stranded breaks by topoisomerase IIβ to relieve torsional strain and mediate synaptic changes (Madabhushi et al., 2015). Beyond the role of topoisomerases, these studies support the notion that high efficiency of transcriptional elongation may promote the expression of neuronal genes.

Notably, transcriptional elongation of genes can be impeded by well-ordered

nucleosomes along the gene body, which increases the frequency of productively elongating Pol II pausing across the gene body in eukaryotic yeast cells (Churchman and Weissman, 2011). Increases in torsional stress can also destabilize well-order nucleosomes in Drosophila S2 cells

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and lead to increased nucleosome turnover, which may impact overall chromatin structure at certain genes (Teves and Henikoff, 2014). Alternatively, high efficiency of transcriptional elongation may be promoted by participating in long-range chromatin interactions within transcriptionally active topologically associating domains (TADs) or other active sites of

transcription in the nucleus (Dixon et al., 2012; Osborne et al., 2004). Transcriptional activity and elongation can also effect changes in large-scale chromatin structure by promoting large regions of positive or negative supercoiling of DNA that help to establish broad topological domains of condensed or decondensed chromatin, respectively (Naughton et al., 2013).

The complex interplay being chromatin structure and the transcriptional elongation of long genes in pyramidal neurons is particularly interesting in light of studies that detect significant genetic risk associations among chromatin modifiers, transcriptional regulators, and synaptic genes in autism, schizophrenia, and intellectual disability (Cotney et al., 2015; De Rubeis et al., 2014; Iossifov et al., 2014). I conclude that excitatory neurons promote the length-dependent expression of long genes required for overall synaptic function at the plasma membrane and may be particularly sensitive to chromatin-mediated barriers of efficient transcriptional elongation.

4.3 DOWNREGULATED GENES MODULATE RETT PHENOTYPES