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dc.contributor.advisorElliot J. Androphy, M.D.
dc.contributor.authorEvans, Matthew C.
dc.date2022-08-11T08:08:43.000
dc.date.accessioned2022-08-23T16:05:37Z
dc.date.available2022-08-23T16:05:37Z
dc.date.issued2011-06-20
dc.date.submitted2011-12-29
dc.identifier.doi10.13028/r8sr-5t09
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31908
dc.description.abstractSpinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. In this study we attempt to characterize novel chemical compounds identified as potential activators of the SMN2 gene. Additionally, we sought to determine the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 gene. We used quantitative PCR to determine the effects of novel compounds and shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that the compounds identified in multiple reporter high throughput screens increased SMN protein levels via transcriptional activation of the SMN2 gene. Other compounds identified in the same screen functioned post-transcriptionally, possibly stabilizing the SMN protein itself by decreasing degradation. Furthermore, we determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This collective work defines a quantitative RNA based protocol to determine mechanism of SMN reporter increase in response to any chosen treatment method. Additionally, this work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectMuscular Atrophy
dc.subjectSpinal
dc.subjectSurvival of Motor Neuron 2 Protein
dc.subjectHistone Deacetylases
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectEnzymes and Coenzymes
dc.subjectNervous System Diseases
dc.subjectNeuroscience and Neurobiology
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.subjectPathological Conditions, Signs and Symptoms
dc.titleQuantitative Analysis of Novel Chemical and shRNA Based Methods to Increase Survival of Motor Neuron Protein Levels
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1568&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/566
dc.legacy.embargo2011-08-31T00:00:00-07:00
dc.identifier.contextkey2425595
refterms.dateFOA2022-08-25T05:13:07Z
html.description.abstract<p>Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (<em>SMN1</em>). The <em>SMN2</em> gene is nearly identical to <em>SMN1</em>, however is alternatively spliced. The close relationship to <em>SMN1</em> results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. In this study we attempt to characterize novel chemical compounds identified as potential activators of the <em>SMN2</em> gene. Additionally, we sought to determine the regulatory role individual HDAC proteins use to control expression of full length protein from the <em>SMN2</em> gene.</p> <p>We used quantitative PCR to determine the effects of novel compounds and shRNA silencing of individual HDACs on the steady state levels of a <em>SMN2</em>-luciferase reporter transcripts. We determined that the compounds identified in multiple reporter high throughput screens increased SMN protein levels via transcriptional activation of the <em>SMN2</em> gene. Other compounds identified in the same screen functioned post-transcriptionally, possibly stabilizing the SMN protein itself by decreasing degradation. Furthermore, we determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription.</p> <p>Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This collective work defines a quantitative RNA based protocol to determine mechanism of SMN reporter increase in response to any chosen treatment method. Additionally, this work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.</p>
dc.identifier.submissionpathgsbs_diss/566
dc.contributor.departmentMedicine
dc.description.thesisprogramInterdisciplinary Graduate Program


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