Graduation Date
Fall 2022
Document Type
Thesis
Program
Master of Science degree with a major in Biology
Committee Chair Name
Amy Sprowles
Committee Chair Affiliation
HSU Faculty or Staff
Second Committee Member Name
Kyle Fink
Second Committee Member Affiliation
Community Member or Outside Professional
Third Committee Member Name
Brigitte Blackman
Third Committee Member Affiliation
HSU Faculty or Staff
Fourth Committee Member Name
Catalina Cuellar Gempeler
Fourth Committee Member Affiliation
HSU Faculty or Staff
Keywords
Epigenetics, X chromosome, Bioinformatics, Stem cells
Subject Categories
Biology
Abstract
Females with heterozygous X-linked mutations are prone to pseudo-haploinsufficiency because random X chromosome inactivation (XCI) silences one of their two X chromosomes. A prior study explored the theory that reactivating the silenced healthy allele could be a treatment for pseudo-haploinsufficient females. The next step was to evaluate this approach in a clinically relevant stem cell model of the rare neurological disease CDKL5 Deficiency Disorder (CDD). It was necessary to validate X-inactivation state with respect to CDKL5 allele expression in one of these models. We explored CDKL5 allele expression in two populations of CDD female patient-derived induced pluripotent stem cells (iPSCs) by determining whether expression of the CDKL5 allele from the inactive X chromosome was the result of a mixed population or incomplete XCI. To examine incomplete XCI due to naïve cell state, the iPSCs were differentiated into neuronal stem cells (NSCs), but sequencing revealed that NSCs maintained inactive allele expression. By contrast, sequencing of individual clones selected from both iPSC populations revealed clonal expression of CDKL5 alleles. This result indicated that a mixed cell population was the likely cause of "inactive" allele expression. This supports the use of these particular iPSCs as reactivation models. To develop a pipeline to observe epigenetic states on the inactive X, standard bioinformatic analysis of ChIP-seq data was combined with AlleleSeq to assess allele specific activity. This pipeline was used on publicly available ChIP-seq data from GM12878 cells. The results reveal bottlenecks for allele specific epigenetic research. Collectively, this work contains both methods and considerations for researchers studying inactivation of specific X-linked genes in iPSC models and epigenetic features in the female X chromosome.
Citation Style
CSE Name Year
Recommended Citation
Mackie, Ariah Dawn, "Determining haplotype specific X chromosome inactivation state and related epigenetic features in female X-linked genes" (2022). Cal Poly Humboldt theses and projects. 605.
https://digitalcommons.humboldt.edu/etd/605