Investigating the role of Smchd1 in control of clustered protocadherin expression

Hu, Jiang (2016) Investigating the role of Smchd1 in control of clustered protocadherin expression. PhD thesis, Queensland University of Technology.


Smchd1 (Structural Maintenance of Chromosomes Hinge Domain containing 1), a previously uncharacterised gene, was identified during an N-ethyl-nitrosourea (ENU) mutagenesis screen in mice, which was designed to identify modifiers of epigenetic reprogramming. The mutant allele of Smchd1 is named MommeD1 (Modifier of murine metastable epialleles, D1) and results in significantly reduced Smchd1 transcript levels, due to nonsense mediated mRNA decay. Accumulating studies have revealed that Smchd1 plays a critical role in X inactivation, as well as regulating a subset of clustered autosomal genes which are subject to monoallelic expression (e.g. imprinted genes and the clustered protocadherin genes). Loss of SMCHD1 function is also implicated in Facioscapulohumeral Muscular Dystrophy Type 2 (FSHD2) in humans.

The clustered protocadherin (Pcdh) genes are expressed in a random combinatorial monoallelic manner and encode cell surface adhesion proteins. The enormous diversity of the protocadherins extracellular domain, which is displayed on the surface of neurons is sufficient to confer each with an individual identity. It has been proposed that this individual identity is critical for forming the cellular connections and interactions necessary to develop complex neuronal networks. Several studies have suggested that the clustered Pcdh genes may play a critical role in autism and schizophrenia.

In this project, neural stem cells (NSCs) were used to perform qRT-PCR in bulk cells and at the single cell level to analyse expression of the clustered Pcdh genes, as well as conducting Transcriptome-seq and MBD-seq (methyl-CpG binding domain (MBD)). Results showed that the expression of most clustered Pcdha and Pcdhb isoforms were up-regulated in Smchd1MommeD1/MommeD1 NSCs, and at the individual cell level, more Smchd1MommeD1/MommeD1 NSCs expressed more individual Pcdha and b isoforms per cell than was found in Smchd1+/+ NSCs.

Attempts were made to isolate single Purkinje cells from the cerebella of Smchd1+/+ and Smchd1MommeD1/MommeD1 male mice, and analyse the expression of clustered Pcdh genes by single cell qRT-PCR. Unfortunately, the methods used to isolate single Purkinje cells required further optimisation and no definitive results were obtained from this analysis to date. However, the analysis of dendritic morphology of Purkinje cells was successful, which showed a "loss of self-avoidance" in the dendrites of Smchd1MommeD1/MommeD1 compared to Smchd1+/+ Purkinje cells.

The restricted expression pattern of PCDHA isoforms was not altered to any great extent after knockdown or knockout of SMCHD1 (in combination with the loss of some DNA methylation) in human neuroblastoma cell lines SK-N-SH and SH-SH5Y. This finding indicates that once the pattern of PCDHA isoform expression has been chosen and epigenetically stabilised in the presence of SMCHD1, the subsequent loss of SMCHD1 is not sufficient to totally disrupt the pattern, even in combination with the loss of DNA methylation.

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ID Code: 96573
Item Type: QUT Thesis (PhD)
Supervisor: Hafner, Gregory
Keywords: Clustered protocadherin, DNA methylation, Epigenetics, Monoallelic Expression, Smchd1
Divisions: Current > Institutes > Institute of Health and Biomedical Innovation
Institution: Queensland University of Technology
Deposited On: 06 Jul 2016 05:10
Last Modified: 06 Jul 2016 05:10

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