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Stem Cell Hub, California Institute for Regenerative Medicine CESCG at UC Santa Cruz

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Fan lab genomic analysis of stem cell differentiation in human overgrowth syndrome and ADCA-DN syndrome

Lab information [ Lab website | CIRM grants ]

Experimental design

Flowchart

Summary

Overgrowth Syndrome

In humans, mutations in any of the three DNA methyltransferases cause diverse diseases. Mutations in DNMT3A have been shown to cause acute myeloid leukemia (AML). More recently, a study identified non-overlapping DNMT3A mutations that causes overgrowth syndrome with intellectual disability. However, how specific DNMT3A mutations contribute to the molecular pathogenesis of overgrowth syndrome and intellectual disability is unclear. Patients carrying de novo DNMT3A mutations exhibit abnormally tall stature, craniofacial anomalies, and mild to moderate intellectual disability. These findings raise underappreciated and fundamental questions surrounding the epigenetics of overgrowth syndrome. For example, how do mutations in DNMT3A alter the epigenome and transcriptome, and subsequently contribute to overgrowth syndrome and intellectual disability? Precisely when during development would overgrowth phenotype begin to manifest?

In this project, the Fan Lab plans to generate isogenic human ESC lines carrying DNMT3A overgrowth disease point mutations using CRISPR. Then, they will differentiate hESCs to determine effects of DNMT3A mutation on stage-specific neural crest cells, neural precursor cells, and mesenchymal stem cells. The Fan Lab will characterize transcriptome and epigenome (DNA methylome, Chromatin configuration and Histone modification) in mutant and control NPCs, MSCs, neurons, and osteoblasts, to understand epigenetic mechanism of the disease.

ADCA-DN Syndrome

In mammalian cells, DNA methylation relies on 3 major DNA methyltransferases (DNMTs) called Dnmt1, Dnmt3a and Dnmt3b. Dnmt3a and Dnmt3b function in de novo methylation, while Dnmt1 maintains DNA methylation status during DNA replication or repair. Complete deletion of Dnmt1 in mouse is embryonic lethal, but mutation leads to phenotype resemble human diseases, such as HSAN1E (hereditary sensory and autonomic neuropathy type 1E) and ADCA-DN (autosomal dominant cerebellar ataxia-deafness and narcolepsy). Previous research identified three point mutations in DNMT1 RFT domain is the cause of ADCA-DN, a neuronal degeneration disease that onset at patients' middle age. Since the mutations on DNMT1 is the cause of the disease, the Fan Lab hypothesizes that DNA methylation play a role in the pathogenesis. In this project, they use CRISPR to introduce nucleotide mutation into mouse embryo stem cell (J1 cell line) and differentiate the ESC to neuron progenitors and then neurons in vitro. They will also construct mouse model as a in vivo differentiation system. The Fan Lab will characterize transcriptome and DNA methylome, in mutant and control ESC, neuron progenitors and neurons, to look for clues about how mutations causes the disease.

Publications

King, A.D., Huang, K., Rubbi, L., Liu, S., Wang, C.Y., Wang, Y., Pellegrini, M. and Fan, G., 2016. Reversible regulation of promoter and enhancer histone landscape by DNA methylation in mouse embryonic stem cells. Cell reports, 17(1), pp.289-302.

Primary files

Lab analysis

Biomarkers, protocols, clustering or other supplementary files supplied by the lab

Secondary analysis

Expression Matrix (lab-generated) | Expression matrix (UCSC) | QC Metrics

CESCG Center Standard Analysis

FastQC | Picard | RSEM | STAR | bigWig

Tertiary analysis

Cell Browser

Sample Psychic

SCIMITAR

RIGGLE

SurfacePlots

JCVI BioMarkers


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