Periodic Reporting for period 1 - ROQ-WACh (Identification and Characterization of Cis-Regulatory ModuleDysfunction in Rett Syndrome with ROQ-WACh)
Período documentado: 2016-03-01 hasta 2018-02-28
Proper gene expression is essential for the execution of cellular function. Disruption of this process results in pathologies such as autism and cancer. These diseases have an enormous impact on society both economically as well as for the quality of life of its members. As such this proposal aimed to address an important issue that will impact a large portion of society.
The regulation of gene expression is achieved on the molecular level by the coordinated action of genomic elements termed cis-regulatory modules (CRMs). Recent genomics studies suggest each gene has ~ 20 CRMs (e.g. promoters and enhancers) to establish precise transcription patterns. CRMs recruit transcription factors and RNA polymerase to a gene’s transcription start site1 in a sequence specific manner and may be sensitive to epigenetic modifications such as DNA methylation.
DNA methylation is an important epigenetic modification whose dysregulation plays an important role in the onset of diseases such as cancer and autism. Specifically, for example, mutations in the MeCP2 gene, which encodes protein that binds methylated DNA and regulates gene expression in neurons, results an Autism Spectrum Disorder termed Rett Syndrome (RTT). While thought to modulate CRM activity as a repressor, MeCP2’s function remains ambiguous and would benefit from a functional genomics characterization.
The purpose of this Marie Sklodowska-Curie (MSC) action proposal was three fold. My prior studies led to the development of a novel method to identify active CRMS called FIREWACh (functional identification of regulatory elements within accessible chromatin) and the first goal of this study was to utilize FIRE-WACh to identify active CRMS in neuronal cells. The second aim of this study was to further develop the methodology to adapt FIREWACh into a quantitative approach termed ROQ-WACh(regulatory output quantification within accessible chromatin) so as to detect changes in the level of activity of CRMs. Lastly, I hoped to develop an in-vitro model of RTT using stem cell derived neurons to probe changes in CRM activity with ROQ-WACh. As with all studies, serious challenges arose in the execution of these aims, but those obstacles were informative and have led to a better understanding of both cis-regulatory modules and the etiology of RTT.
The work performed under this action resulted in the discovery of neuronal specific CRMs as well as the development of an in vitro model system of RTT. Importantly this action allowed the transfer of knowledge both to the host lab as well as to the public throught the creation of a science outreach program.
The regulation of gene expression is achieved on the molecular level by the coordinated action of genomic elements termed cis-regulatory modules (CRMs). Recent genomics studies suggest each gene has ~ 20 CRMs (e.g. promoters and enhancers) to establish precise transcription patterns. CRMs recruit transcription factors and RNA polymerase to a gene’s transcription start site1 in a sequence specific manner and may be sensitive to epigenetic modifications such as DNA methylation.
DNA methylation is an important epigenetic modification whose dysregulation plays an important role in the onset of diseases such as cancer and autism. Specifically, for example, mutations in the MeCP2 gene, which encodes protein that binds methylated DNA and regulates gene expression in neurons, results an Autism Spectrum Disorder termed Rett Syndrome (RTT). While thought to modulate CRM activity as a repressor, MeCP2’s function remains ambiguous and would benefit from a functional genomics characterization.
The purpose of this Marie Sklodowska-Curie (MSC) action proposal was three fold. My prior studies led to the development of a novel method to identify active CRMS called FIREWACh (functional identification of regulatory elements within accessible chromatin) and the first goal of this study was to utilize FIRE-WACh to identify active CRMS in neuronal cells. The second aim of this study was to further develop the methodology to adapt FIREWACh into a quantitative approach termed ROQ-WACh(regulatory output quantification within accessible chromatin) so as to detect changes in the level of activity of CRMs. Lastly, I hoped to develop an in-vitro model of RTT using stem cell derived neurons to probe changes in CRM activity with ROQ-WACh. As with all studies, serious challenges arose in the execution of these aims, but those obstacles were informative and have led to a better understanding of both cis-regulatory modules and the etiology of RTT.
The work performed under this action resulted in the discovery of neuronal specific CRMs as well as the development of an in vitro model system of RTT. Importantly this action allowed the transfer of knowledge both to the host lab as well as to the public throught the creation of a science outreach program.
Using mouse embryonic stem cells and established protocols I was able to derive a near pure population (>~90%) of somatic spinal motor neurons. These were then subjected to the FIREWACh procedure to identify more than a thousand regulatory elements active within the neuronal population. Gene ontology analysis of these elements found that they resided near genes important to neuronal development. This is consistent with the notion that active regulatory elements govern gene expression important to cell type specific functions, for example in this case synapse formation, rather than processes important to all cells such as RNA synthesis pathways. It further provides evidence that FIREWACh is applicable to a variety of cell types. A list of the genomic location of these elements will be uploaded to the Gene Expression Omnibus and made publically available.
The second aim of this proposal was to adapt FIRE-WACh into an approach that could be used to quantify the activity of CRMs. The strategy I adopted involved two major modifications: firstly, rather than use lentiviral vectors to deliver the reporter construct as used in FIRE-WACh I aimed to use a transgenic approach. This new approach took advantage of the rapid advances in CRISPR technology to enable homologous recombination to specifically target CRM reporters to a specific location within the genome. Here, as with any new technology, there were significant challenges. Firstly, the efficiency of homologous recombination I achieved was
considerably less than that that had been reported in the literature. However using several different approaches I was able to optimize first the design of the guide RNA which directs the CRISPR molecular machinery to the correct genomic location. Secondly I optimized the conditions that allow homologous recombination by using small synthetic molecules that inhibit non-homologous end repair and promote homologous recombination.
Unfortunately,even under optimized conditions the efficiency of the method is insufficient to provide the genome wide coverage initially hoped for. Luckily rapid advances in the field of functional genomics have provided alternate approaches such as STARR-seq that can be used in lieu of the adapted FIREWACh approach. These experiments therefore have revealed new challenges in the development of functional genomic approaches that require future study.
Lastly, the development of the stem cell based model of RTT was largely successful. Here, I utilized the ability of embryonic stem cells to develop in functional neurons. First, using CRISPR again, I generated mouse embryonic stem cells that carry a deletion of the MeCP2 allele. These cells were then differentiated to neurons that phenocopy neurons of RTT patients. That is, the neurons generated from the stem cells lacked extensive dendritic arborization, had smaller cell bodies (somas), and had lower electrical activity. Three key characteristics found in the neurons of RTT patients. However, due to the limitations of my ROQ-WACh approach I was unable to quantify the impact of MeCP2 mutations on the activity of CRMs in these cells.
The second aim of this proposal was to adapt FIRE-WACh into an approach that could be used to quantify the activity of CRMs. The strategy I adopted involved two major modifications: firstly, rather than use lentiviral vectors to deliver the reporter construct as used in FIRE-WACh I aimed to use a transgenic approach. This new approach took advantage of the rapid advances in CRISPR technology to enable homologous recombination to specifically target CRM reporters to a specific location within the genome. Here, as with any new technology, there were significant challenges. Firstly, the efficiency of homologous recombination I achieved was
considerably less than that that had been reported in the literature. However using several different approaches I was able to optimize first the design of the guide RNA which directs the CRISPR molecular machinery to the correct genomic location. Secondly I optimized the conditions that allow homologous recombination by using small synthetic molecules that inhibit non-homologous end repair and promote homologous recombination.
Unfortunately,even under optimized conditions the efficiency of the method is insufficient to provide the genome wide coverage initially hoped for. Luckily rapid advances in the field of functional genomics have provided alternate approaches such as STARR-seq that can be used in lieu of the adapted FIREWACh approach. These experiments therefore have revealed new challenges in the development of functional genomic approaches that require future study.
Lastly, the development of the stem cell based model of RTT was largely successful. Here, I utilized the ability of embryonic stem cells to develop in functional neurons. First, using CRISPR again, I generated mouse embryonic stem cells that carry a deletion of the MeCP2 allele. These cells were then differentiated to neurons that phenocopy neurons of RTT patients. That is, the neurons generated from the stem cells lacked extensive dendritic arborization, had smaller cell bodies (somas), and had lower electrical activity. Three key characteristics found in the neurons of RTT patients. However, due to the limitations of my ROQ-WACh approach I was unable to quantify the impact of MeCP2 mutations on the activity of CRMs in these cells.
The funding provided by this Marie Sklodowska-Curie Action has allowed me to further develop key functional genomic approaches and their application to diseases such as Autism Spectrum Disorders. Importantly this action has led to the dissemination and transfer of a considerable amount of knowledge. I was able to advance the capabilities in functional genomics of my host lab through seminars, collaboration, and the mentorship of trainees. All three of my undergraduate trainees are on their way to matriculating into competitive PhD programs. Further, through the host lab I was able to embark in collaborations that led to publication.