Biology subjects

Greally, J.

Publications and source records attributed to Greally, J..

3 recordsLinked to original sources

bioRxiv2018

High efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells.

While lymphoblastoid cell lines (LCLs) represent a valuable resource for population genetic studies, they are usually regarded as difficult for CRISPR-mediated genomic editing. It would be valuable to be able to take the results of their functional variant studies and test them in the same LCLs. We describe a protocol using a single-stranded donor oligonucleotide (ssODN) strategy for scarless editing in LCLs. The protocol involves optimized transfection, flow cytometric sorting of transfected cells to single cells in multi-well plates and growth in conditioned, serum-rich medium, followed by characterization of the clones. Amplicon sequencing reveals the relative proportions of alleles with different editing events, with sequencing of DNA from clones showing the frequencies of events in individual cells. We find 12/60 (20%) of clones selected in this manner to have the desired ssODN-mediated recombination event. Long-range PCR of DNA at the edited locus and of RT-PCR products for the gene traversing the edited locus reveals 3/6 characterized clones (50%) to have large structural mutations of the region that are missed by sequencing just the edited site. The protocol does not require the use of lentiviruses or stable transfection, and makes LCLs a realistic cell type for consideration for CRISPR-mediated genomic targeting.

genomics↗

bioRxiv2018

Re-targeting of macroH2A following mitosis to cytogenetic-scale heterochromatic domains

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly-incorporated macroH2A re-targets the same, large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell.

biochemistry↗

bioRxiv2018

A novel approach to modeling transcriptional heterogeneity identifies the oncogene candidate CBX2 in invasive breast carcinoma

Oncogenes promote the development of and serve as therapeutic targets against subsets of cancers. Here, a new statistical approach that captures transcriptional heterogeneity in tumor and adjacent normal (i.e. tumor-free) mRNA expression profiles was developed to identify oncogene candidates that were overexpressed in a subset of breast tumors. Intronic DNA methylation was strongly associated with the overexpression of chromobox 2 (CBX2), an oncogene candidate that was identified using our method but not through prior analytical approaches. CBX2 overexpression in breast tumors was associated with the upregulation of genes involved in cell cycle progression and is associated with poorer 5-year survival. The predicted function of CBX2 was confirmed in vitro providing the first experimental evidence that CBX2 promotes breast cancer cell growth. Modeling mRNA expression heterogeneity in tumors is a novel powerful approach with the potential to uncover therapeutic targets that benefit subsets of cancer patients.

systems biology↗