Periodic Reporting for period 1 - NGECA (REGULATION OF NEURONAL GENE EXPRESSION THROUGH CHROMOSOME ARCHITECTURE)
Periodo di rendicontazione: 2016-11-01 al 2018-10-31
Development of the brain is a complex and highly ordered process, requiring activation and silencing of specific repertoires of genes. There are many levels of regulation to ensure these changes occur in a coordinated and timely manner; disruption of appropriate gene expression can lead to neurological disorders. One aspect of gene regulation that has not been fully explored in the brain is the impact of gene location within the 3D nucleus. It is known that the nuclear periphery is generally repressive, and that some genes move to the interior during activation. Moreover, separated genomic regions can come together in 3D space through looping, enabling co-regulation. This can be of different genes in the same biological pathway, or of genes with enhancers that increase their transcription. The objectives of my project were to explore the role of genome looping and gene movement within the nucleus on neuronal gene expression and function.
I began with a panel of genes that are important for brain development and assessed the genomic regions they interact with. This led me to identify the first enhancer for Bdnf, which encodes a neurotrophin that is critical for neuronal differentiation and synaptic plasticity. The interacting region bears many hallmarks of enhancers, including chromatin accessibility, histone modifications and transcription factor binding. Importantly, I have demonstrated that the putative enhancer is transcribed, and that inhibition of the region prevents Bdnf functioning in dendritic growth. Furthermore, I have found that Bdnf moves away from the nuclear periphery during its developmental activation, and that the chromosome region in which it resides undergoes topological restructuring. Mis-regulation of Bdnf expression is implicated in a variety of neurological disorders, including those arising during development, and in neurodegeneration. Therefore, understanding its many-faceted regulation is important for understanding and treating disease.
I began with a panel of genes that are important for brain development and assessed the genomic regions they interact with. This led me to identify the first enhancer for Bdnf, which encodes a neurotrophin that is critical for neuronal differentiation and synaptic plasticity. The interacting region bears many hallmarks of enhancers, including chromatin accessibility, histone modifications and transcription factor binding. Importantly, I have demonstrated that the putative enhancer is transcribed, and that inhibition of the region prevents Bdnf functioning in dendritic growth. Furthermore, I have found that Bdnf moves away from the nuclear periphery during its developmental activation, and that the chromosome region in which it resides undergoes topological restructuring. Mis-regulation of Bdnf expression is implicated in a variety of neurological disorders, including those arising during development, and in neurodegeneration. Therefore, understanding its many-faceted regulation is important for understanding and treating disease.
Bdnf encodes a neurotrophin with critical roles in brain development, neuronal regeneration and synaptic plasticity. I performed 4C-seq to understand which genomic regions interact with Bdnf in cortical neurons, as enhancers or co-regulated genes often loop to the genes they regulate. Excitingly, I identified a loop from Bdnf to an intergenic site 45 kb downstream, which we postulate is a novel enhancer. Experiments using a reciprocal viewpoint at the intergenic site confirm the interaction. Analysis of published data suggests that in neurons the region has peaks of enhancer-associated histone modifications, and displays DNase hypersensitivity. It is bound by coactivators and transcription factors, which are associated with enhancer activation, supporting our hypothesis that the region could act as an enhancer.
I wanted to investigate whether the putative enhancer plays a role during developmental Bdnf activation. I used a primary neuron differentiation system to isolate neural progenitor cells (NPCs) and postmitotic neurons (PMNs), and found significant upregulation of all Bdnf isoforms from NPCs to PMNs. Preliminary 4C-seq data suggests that the loop to the intergenic region is present in PMNs but not NPCs. Preliminary double DNA-FISH experiments labelling Bdnf and the putative enhancer suggest that the inter-probe distance decreases from NPCs to PMNs, in keeping with the population level looping data. I have also established that during neuronal development the Bdnf locus moves away from the repressive nuclear periphery, and towards RNA polymerase II foci.
Transcription from active enhancers is thought to be vital for their function. I mapped published data and found nascent transcription at the putative Bdnf enhancer in neurons. qRT-PCR confirms expression in the region, which increases from NPC to PMN concomitantly with Bdnf mRNA, and is sensitive to transcriptional inhibition. These data show that the intergenic region we have identified as a site of interaction with Bdnf bears many characteristics of a neuronal enhancer.
To functionally dissect how the putative enhancer influences Bdnf expression levels in vivo, I established lentiviral infection of CRISPR guide RNA (gRNA)-directed dCas9-KRAB. This transcriptional repression system attains >65% infected PMNs, which are notoriously hard to target. I am currently using this scheme to repress the enhancer and assess Bdnf transcription during development. Bdnf is an essential factor in neuronal growth, including axonal branching and dendrite arborization. I have analysed activity-induced dendritic growth and found that repression of the enhancer abrogates Bdnf-driven dendritic arborization. I am now performing rescue experiments to demonstrate that the effect of enhancer inhibition is mediated through failure to upregulate Bdnf. We further plan to assess the role of the enhancer on brain development in vivo using in utero electroporation.
We hope to finish this work and submit for open access publication by the end of 2020. During this Fellowship I published a review article on the regulation of neuronal gene expression through genome architecture (Current Opinion in Neurobiology, 2019). I presented the project in a selected short talk at the Academy of Medical Sciences ‘The Developing Brain in Heath and Disease’ conference (March 2019) as well as in internal seminars and lab meetings. No website has been developed for this project.
I wanted to investigate whether the putative enhancer plays a role during developmental Bdnf activation. I used a primary neuron differentiation system to isolate neural progenitor cells (NPCs) and postmitotic neurons (PMNs), and found significant upregulation of all Bdnf isoforms from NPCs to PMNs. Preliminary 4C-seq data suggests that the loop to the intergenic region is present in PMNs but not NPCs. Preliminary double DNA-FISH experiments labelling Bdnf and the putative enhancer suggest that the inter-probe distance decreases from NPCs to PMNs, in keeping with the population level looping data. I have also established that during neuronal development the Bdnf locus moves away from the repressive nuclear periphery, and towards RNA polymerase II foci.
Transcription from active enhancers is thought to be vital for their function. I mapped published data and found nascent transcription at the putative Bdnf enhancer in neurons. qRT-PCR confirms expression in the region, which increases from NPC to PMN concomitantly with Bdnf mRNA, and is sensitive to transcriptional inhibition. These data show that the intergenic region we have identified as a site of interaction with Bdnf bears many characteristics of a neuronal enhancer.
To functionally dissect how the putative enhancer influences Bdnf expression levels in vivo, I established lentiviral infection of CRISPR guide RNA (gRNA)-directed dCas9-KRAB. This transcriptional repression system attains >65% infected PMNs, which are notoriously hard to target. I am currently using this scheme to repress the enhancer and assess Bdnf transcription during development. Bdnf is an essential factor in neuronal growth, including axonal branching and dendrite arborization. I have analysed activity-induced dendritic growth and found that repression of the enhancer abrogates Bdnf-driven dendritic arborization. I am now performing rescue experiments to demonstrate that the effect of enhancer inhibition is mediated through failure to upregulate Bdnf. We further plan to assess the role of the enhancer on brain development in vivo using in utero electroporation.
We hope to finish this work and submit for open access publication by the end of 2020. During this Fellowship I published a review article on the regulation of neuronal gene expression through genome architecture (Current Opinion in Neurobiology, 2019). I presented the project in a selected short talk at the Academy of Medical Sciences ‘The Developing Brain in Heath and Disease’ conference (March 2019) as well as in internal seminars and lab meetings. No website has been developed for this project.
The Bdnf gene encodes a neurotrophin which exerts positive effects on neuronal survival and differentiation, and on synaptic plasticity. It is therefore important in brain development, learning and memory, and neuronal regeneration. Aberrant Bdnf expression is implicated in neurological diseases, including schizophrenia, depression, Alzheimer’s disease, Rett syndrome and ADHD. Conversely, enhanced Bdnf expression is linked to the neuroprotective effects of environmental enrichment, exercise and anti-depressants. Accordingly, there is great interest in Bdnf as a biomarker and therapeutic target. By identifying the first enhancer for Bdnf, we are contributing significantly to our understanding of the transcriptional regulation of this critical neurotrophin.
High-throughput screens have identified putative enhancers genome-wide using hallmarks such as histone modifications or DNA accessibility. However, relatively few have been mechanistically tested during neuronal development. My use of CRISPR-Cas9 technologies to assess enhancer influence on biological function is a key facet of this project’s impact.
The critical effect of 3D location and genome topology on gene regulation is a fascinating and growing field. My project contains detailed analysis of multiple aspects of genome architecture during the developmental activation of an important neuronal gene: movement with respect to the nuclear periphery, changing of topological boundaries, and enhancer-promoter looping. Understanding the interplay of different nuclear organization levels is an innovative approach that will help us fully understand transcriptional regulation.
High-throughput screens have identified putative enhancers genome-wide using hallmarks such as histone modifications or DNA accessibility. However, relatively few have been mechanistically tested during neuronal development. My use of CRISPR-Cas9 technologies to assess enhancer influence on biological function is a key facet of this project’s impact.
The critical effect of 3D location and genome topology on gene regulation is a fascinating and growing field. My project contains detailed analysis of multiple aspects of genome architecture during the developmental activation of an important neuronal gene: movement with respect to the nuclear periphery, changing of topological boundaries, and enhancer-promoter looping. Understanding the interplay of different nuclear organization levels is an innovative approach that will help us fully understand transcriptional regulation.