The current study aimed to determine the selective advantage of lateral amygdala (LA) neurons overexpressing the transcription factor CREB that enables their preferential incorporation into the fear memory trace. I hypothesized that overexpression of CREB drives the formation of dendritic spines, potentially providing these neurons with greater connectivity to sensory inputs at the time of learning. Using viral-mediated gene transfer, CREB tagged with GFP, or GFP as a control, was overexpressed in the LA of wild-type mice. Spine number and morphology were compared in homecage mice at the time when mice are normally trained in fear conditioning. Spine density was increased in neurons with CREB vector compared to neurons with GFP vector whereas spine head diameter and length was not different. Therefore, LA neurons overexpressing CREB have increased spine number at the time of learning, potentially providing these neurons with a selective advantage for incorporation into the fear memory trace.

<p>Abstract</p><p>Transcription factors control numerous vital processes in the cell through their ability to control gene expression. Dysfunctional regulation by transcription factors lead to disorders and disease. Transcription factors regulate gene expression by binding to DNA sequences (motifs) on the genome and altering chromatin. DNase-seq footprinting is a well-established assay for identification of DNA sequences that bind to transcription factors. We developed computational techniques to analyze footprints and predict transcription factor binding. These transcription factor specific predictive models are able to correct for DNase sequence bias and characterize variation in DNA binding sequence. We found that DNase-seq footprints are able to identify cell-type or condition specific transcription factor activity and may offer information about the type of the interaction between DNA and transcription factor. Our DNase-seq footprint model is able to accurately discover high confidence transcription factor binding sites and discover alternative interactions between transcription factors and DNA. DNase-seq footprints can be used with ChIP-seq data to discover true binding sites and better understand transcription regulation.</p> / Dissertation

The mammalian cerebral cortex is a remarkable product of brain evolution, and is the structure that most distinctively delineates the human species from others (Northcutt and Kaas, 1995; Rakic, 1988). Neurons in the adult brain are organised into cytoarchitectonic areas, defined by distinct biochemical, morphological and physiological characteristics (Rakic 1988). Remarkably, this complex structure is generated from a simple neuroepithelium. (For complete abstract open document)

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

This thesis was presented by Aaron Webber on the 4th December 2013 for the degree of Doctor of Philosophy from the University of Manchester. The title of this thesis is ‘Transcriptional co-regulation of microRNAs and protein-coding genes’. The thesis relates to gene expression regulation within humans and closely related primate species. We have investigated the binding site distributions from publically available ChIP-seq data of 117 transcription regulatory factors (TRFs) within the human genome. These were mapped to cis-regulatory regions of two major classes of genes,  20,000 genes encoding proteins and  1500 genes encoding microRNAs. MicroRNAs are short 20 - 24 nt noncoding RNAs which bind complementary regions within target mRNAs to repress translation. The complete collection of ChIP-seq binding site data is related to genomic associations between protein-coding and microRNA genes, and to the expression patterns and functions of both gene types across human tissues. We show that microRNA genes are associated with highly regulated protein-coding gene regions, and show rigorously that transcriptional regulation is greater than expected, given properties of these protein-coding genes. We find enrichment in developmental proteins among protein-coding genes hosting microRNA sequences. Novel subclasses of microRNAs are identified that lie outside of protein-coding genes yet may still be expressed from a shared promoter region with their protein-coding neighbours. We show that such microRNAs are more likely to form regulatory feedback loops with the transcriptional regulators lying in the upstream protein-coding promoter region. We show that when a microRNA and a TRF regulate one another, the TRF is more likely to sometimes function as a repressor. As in many studies, the data show that microRNAs lying downstream of particular TRFs target significantly many genes in common with these TRFs. We then demonstrate that the prevalence of such TRF/microRNA regulatory partnerships relates directly to the variation in mRNA expression across human tissues, with the least variable mRNAs having the most significant enrichment in such partnerships. This result is connected to theory describing the buffering of gene expression variation by microRNAs. Taken together, our study has demonstrated significant novel linkages between the transcriptional TRF and post-transcriptional microRNA-mediated regulatory layers. We finally consider transcriptional regulators alone, by mapping these to genes clustered on the basis of their expression patterns through time, within the context of CD4+ T cells from African green monkeys and Rhesus macaques infected with Simian immunodeficiency virus (SIV). African green monkeys maintain a functioning immune system despite never clearing the virus, while in rhesus macaques, the immune system becomes chronically stimulated leading to pathogenesis. Gene expression clusters were identified characterizing the natural and pathogenic host systems. We map transcriptional regulators to these expression clusters and demonstrate significant yet unexpected co-binding by two heterodimers (STAT1:STAT2 and BATF:IRF4) over key viral response genes. From 34 structural families of TRFs, we demonstrate that bZIPs, STATs and IRFs are the most frequently perturbed upon SIV infection. Our work therefore contributes to the characterization of both natural and pathogenic SIV infections, with longer term implications for HIV therapeutics.

Genetic dissection of loci important in the control of neurogenesis has improved our understanding of both the evolutionarily conserved and divergent processes in neurodevelopment. These loci include not only protein coding genes [1, 2], but also noncoding RNAs [3-5]. One important family of non-coding RNAs is miRNAs, which control gene expression fundamental in developmental regulation and mature cell maintenance [3, 5-9]. Here, we will first focus our efforts by surveying miRNA regulation in the developing brain. We hypothesize a strong regulatory role of miRNAs during proliferation, cell death, migration and differentiation in the developing mammalian forebrain that has yet to be adequately described in the literature. Second, we will assess miRNA's role in the evolutionary divergence of brain-related gene expression. We hypothesize that a human specific single nucleotide change(s) in the miRNA recognition element of transcription factors 3' untranslated regions contributes to species-specific differences in transcription factor expression and ultimately alters regulatory function.

Having evolved longer than any other group of organisms, bacteria have been perfecting mechanisms of sensing their environments for billions of years. Recent advances in the field of biosensing have enabled miniaturization of existing biosensors, but the number of characterized biosensor elements remains limited. Sensing parts derived from bacteria make promising targets for integration into biosensor devices and could expand the repertoire of easily detectable compounds. Here, RNA sequencing screening was used to identify a novel TetR family 3-ketosteroid inducible transcription factor called SRTF1 (Steroid Responsive Transcription Factor 1) from the Gram-positive soil bacterium Pimelobacter simplex. This is the first transcription factor confirmed to be inducible by these steroids in-vitro. A potential regulon was identified using in-vitro chromatin immunoprecipitation sequencing, revealing a conserved 20 base pair long palindrome within several promoters in a region of the P. simplex genome highly differentially expressed on exposure to steroids. Biolayer interferometry and intrinsic tryptophan fluorescence were used to quantitatively characterize the transcription factor’s DNA binding strength and hormone induction specificity. Circular dichroism study of SRTF1 revealed it is primarily alpha-helical like almost all other TetR family transcription factors. Bases in a core GCCG repeat within the palindrome were identified as important for SRTF1 binding and a disrupted palindrome was generated that greatly increased a quantum-dot based SRTF1 biosensor’s sensitivity for progesterone. As the transcription factor displays cross reactivity to cortisol and aldosterone that is undesirable in a diagnostic device, a fluorescent reporter assay based on the transcription factor was constructed. This reporter assay showed a similar steroid induction profiles as purified SRTF1, and was used to select for mutant SRTF1 variants generated using error-prone polymerase chain reaction with reduced inducibility by these 11-hydroxy steroids. This pipeline for identifying novel transcription factors, characterizing their DNA and ligand binding profiles, and altering them through mutation of DNA and protein sequences could allow for an expanded number of biosensor parts.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19274号 / 医博第4038号 / 新制||医||1011(附属図書館) / 32276 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 吉村 長久, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13157号 / 論医博第2144号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 影山 龍一郎, 教授 別所 和久, 教授 辻川 明孝 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

GABAergic inhibitory neurons are known to play a critical regulatory role in memory formation and learning. During motor learning, pyramidal neurons (PNs) of the primary motor cortex (M1) undergo spine reorganization and firing pattern refinement. Cortical PNs are directly inhibited and regulated by two inhibitory neuronal subtypes: somatostatin-expressing interneurons (SOM-INs) and parvalbumin-expressing interneurons (PV-INs). Interestingly, SOM-mediated inhibition has been shown to regulate the observed dynamics of PNs during motor learning. Despite our expanded understanding, the molecular mechanisms that underlie these processes remain unclear. Here, I identified that the immediate-early gene transcription factor, NPAS4, is selectively expressed in a subset of SOM-INs, but not in PV-INs or PNs, during the head-fixed pellet reaching motor learning task. Furthermore, I characterized its expression pattern within the SOM-INs of M1 and found that there was no change at early phases; but as training progressed, there was a gradual increase and plateau in the number of NPAS4-expressing SOM-INs. In collaboration with other lab members, we showed that Npas4 region- and cell-type specific deletion within SOM-INs of M1, impaired motor skill acquisition and disrupted the motor learning-induced spine reorganization. In addition, I validated and employed the novel NRAM system to examine if NPAS4 is continually expressed within the same subset of SOM-INs and found that an ensemble of SOM-INs repetitively express NPAS4 at various phases of learning. Lastly, chronic in vivo two-photon Ca²⁺ imaging during training showed that the ensemble of NPAS4-expressing SOM-INs had reduced activity during task-related movements compared to other SOM-INs. Together, our results reveal an important instructive role of NPAS4 within the microcircuits of M1, in which it modulates the inhibition of a distinct subset of SOM-INs during motor learning to promote spine stabilization of downstream task-related PNs that are important for motor skill acquisition.