Insights into the epigenetic regulation of Unstable factor for orange1 in maize
Open Access
- Author:
- Cui, Jin
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 27, 2019
- Committee Members:
- Surinder Chopra, Dissertation Advisor/Co-Advisor
Surinder Chopra, Committee Chair/Co-Chair
Majid R Foolad, Committee Member
Jesse R Lasky, Outside Member
David Robert Huff, Committee Member
Teh-Hui Kao, Program Head/Chair - Keywords:
- Unstable factor for orange1
Maize genetics
RNA-seq
small-RNA
DNA methylation
alternative splicing - Abstract:
- Transcriptional gene regulation is an important aspect of molecular biology, which involves many mechanisms. In eukaryotes, transcriptional factors can bind to specific DNA sequence motifs which activate or repress target gene transcription. On the other hand, since genomic DNA is packed by histones into chromatins, reversible changes of chromatin state can lead to decondensation of DNA, allowing for polymerase and their cofactors to facilitate transcription, and vice versa. Since these changes are heritable through mitosis and meiosis, and yet do not involve changes in DNA sequences, they are part of the epigenetic regulation. Several key factors also play a part in epigenetic mechanisms. DNA methylation is the addition of methyl group to cytosine and is considered an important epigenetic mark. Small RNAs are non-coding RNAs which are also highly involved in transcriptional regulation. The maize pericarp color1 (p1) is a well-established epigenetic marker which contains hundreds of easily distinguishable color patterns in the pericarp and cob glume tissues. p1 is an R2R3 MYB transcription factor which activates expression of several key enzymes in the flavonoid biosynthesis pathway, which leads to production of phlobaphene pigments in pericarps. Previous research has shown that DNA methylation at the cis-regulatory element of p1 can affect its transcript level. Two major epigenetic modifiers of p1 has been discovered and one of them is Unstable factor for orange1 (Ufo1). Ufo1 is a dominant natural mutation displaying ectopic accumulation of phlobaphenes in floral tissues as well as plant body, provided that a functional p1 allele is present in the background. The only allele of Ufo1, Ufo1-1, displays incomplete penetrance and low expressivity, which is associated with DNA methylation. Chapter 2 summarizes previous fine-mapping results which narrowed down Ufo1-1 mapping region to a ~40 Mb region adjacent to centromere in chromosome 10. An RNA-seq approach was used to identify a candidate gene for Ufo1-1 allele which was found to be 50~250 times up-regulated in various tissues of the mutant. The candidate gene for ufo1 contains a unique non-autonomous CACTA insertion in its first intron which plays key role in regulating gene expression and we discovered that Ufo1-1 expressivity is strongly correlated with the degree of DNA methylation at this element. We also explored Ufo1-1’s impact on gene expression and provide reasonable explanation on the molecular basis of pleiotropic defects in the presence of Ufo1-1. Subsequent functional validation of the cloned ufo1 candidate gene was performed by creating transgenic overexpression lines of maize which phenocopy the mutant phenotypes. In chapter 3, I explored various sequencing data related to the Ufo1-1 mutant and discovered interesting patterns. First, we discovered that Ufo1-1 has relatively small impact on overall small RNA accumulation as compared to another p1 epigenetic modifier, mediator of paramutation1 (mop1), which is known to be a major component of the RNA-directed DNA methylation (RdDM) in maize. However, locus-specific changes in small RNA was found to be highly correlated with changes in DNA methylation, especially CHH methylation. Furthermore, we discovered that the expressing Ufo1-1 mutant exhibits increased gene body methylation but decreased transposable element (TE) methylation. I show that although differential gene body methylation could be associated with small RNA accumulation in flanking regions and was largely uncorrelated with gene expression level. At last, we found that there is a significant overlap between differential gene body methylation and differential exon usage, and a subset of genes involved in nucleotide binding functions are particularly affected. RT-PCR and bisulfite sequencing validation were also performed. Finally, in chapter 4, I explored the inheritance of small RNAs (sRNAs) in Ufo1-1 hybrid (U/+) in comparison to parental lines. I summarize the results of de novo annotation of sRNAs from three maize tissues. We found widespread locus-specific changes in the presence of Ufo1-1 as well as non-additive inheritance pattern of sRNAs in Ufo1-1 hybrids. Homozygous Ufo1-1 (U/U) parent mainly targeted sRNAs from lincRNAs whereas U/+ hybrids mainly affected sRNAs from gene flanking regions and inverted repeat sequences. In this dissertation research, we first cloned the gene responsible for Ufo1-1 mutation and characterized potential function of the wild type ufo1 gene. We discovered that ufo1 gene expression is endosperm-specific and it may regulate or influence many genes and pathways in kernel development, sugar metabolism, hormone homeostasis and ribosomal biogenesis. Finally, genome-wide epigenetic studies have helped us advance our understanding of the sRNA inheritance pattern and the role of Ufo1-1 in gene body methylation and alternative splicing.