The epigenetics and role of Dopa Decarboxylase in heart development
Genomic imprinting in mammals subjects a handful of genes to silencing on one allele depending on the parent-of-origin of that allele. The Ddc_exonla gene transcript is under the control of genomic imprinting in the developing and neonatal mouse heart with transcription occurring solely on the paternally inherited allele, with the maternally inherited allele epigenetically silenced. In all other tissues where Ddc_exonla is expressed transcription occurs from both parentally inherited alleles. CTCF plays a central role in controlling gene expression by regulating chromatin organisation, and has been shown to be fundamental for imprinting of the Igf2/H19 locus through its binding to the germline differentially methylated region (gDMR). This thesis explores the occurrence of CTCF binding both genome-wide and at imprinting gDMRs in mouse brain in order to assess its relevance to the control of transcription in vivo. 49,358 significant binding sites are detected across the genome and binding is enriched at gene coding regions but depleted at distal intergenic regions. 12/20 (60%) imprinted gDMRs are bound by CTCF, of these five bind in a parent-of-origin specific manner implicating CTCF in the control of a subset of imprinted genes including Ddc_exonla. Comparative analysis of CTCF binding in multiple tissues shows a high degree of overlap, and Motif analysis reveals CTCF binds the same canonical motif sequence in each tissue. CTCF binding in the absence of the canonical motif is more tissue-specific. Ddc_exonla expression is imprinted in the developing heart but is bi-allelic in brain, and the mechanism of imprinting is not known. To explore a model for imprinting control the epigenetic profile of the Ode imprinted locus was examined in detail. Methylation analysis reveals several regions that are differentially methylated between heart and brain. One region constitutes a CpG rich region at the promoter of GrblO, another imprinted gene located adjacent to Ddc_exonla. The second region is at the promoter of AK0066690, a non-coding antisense transcript which initiates in intron four of Ddc_exonla. CTCF binding at the Ddc/GrblO locus is assayed in heart and brain, and binding is invariant between tissue types. The AK0066690 transcript is expressed in the neonatal mouse heart but not in the neonatal brain, consistent with a model of silencing Ddc_exonla on the maternal allele via transcriptional interference. Ddc_exonla codes for the Dopa Decarboxylase (Ddc) protein, which is predominately expressed in the developing myocardium, this points to a role in fetal heart development. The role of Ddc in cardiogenesis is explored using knockout mice lacking Ddc_exonla expression in heart. Expression microarrays were used to detect changes in gene expression, and morphometric analysis using 3D imaging was performed to look for gross morphological changes. Results suggest that Ddc plays a role in regulating cellular proliferation and cardiogenesis of the developing myocardium as mice lacking Ddc show a significant thinning of the apical portion of the right ventricle, a region that shows abundant Ddc expression. In this thesis the significance of CTCF binding to imprinting control is examined and the observations applied in a locus specific manner to explore both the mechanistic control, and the functional role of Ddc in the developing mouse heart.
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