OUR lab has three main focuses, including epigenetic regulation of plants under biotic and abiotic stresses, epigenetic dynamics between introduced alien chromosomes and wheat chromosomes, as well as cis-regulatory elements and their regulation in gene expression.
UNDERSTANDING EPIGENETIC REGULATION OF PLANT UNDER MULTIPLE STRESSE
Transcription is orchastrated by the interaction between cis- and trans-regulatory elements in a spatiotemporal manner. The regulation of gene transcription in response to developmental and multiple environmental cues is a crucial mechanism for all plants species. We are interested in the question how the transcription is regulated at different developmental stages as well as under multiple stresses including cold and drought. We mainly focus on how genomic regulatory elements (promoters and enhancers) as well as epigenetic modifications play a role in the mediation of gene expression. We employ an interdisciplinary approach using genome-wide high throughput functional chromatin assays and bioinformatics. We aim to understand the regulation of transcription and improve the adaptivity of crops for the incoming environmental challenges.
TECHNIQUES APPLIED IN OUR RESEARCH
ATAC-Seq: Assay for Transposase-Accessible Chromatin using Sequencing
We exploit ATAC-seq to identify cis-regulatory elements by measuring the accessibility of the chromatin on a genome-wide scale. ATAC-seq was first introduced in 2013 and it becomes more popular in both animal and plant researches. ATAC-seq utilizes the transposase Tn5, which allows for highly efficient cutting of ‘naked DNA’ (open chromatin), whereas it’s insensitive to protein protected chromatin. Genomic regions associated with cis-regulatory elements are usually depleted of nucleosomes or under dynamic nucleosome displacement. Therefore, ATAC-seq enables identification of cis-regulatory elements for genes in their regulatory vicinity and quantifies the accessibility (openness) of the chromatin under stresses.
DNase-Seq: DNase I Hypersensitive Site Sequencing
DNase-Seq has been recognized as a reliable approach and widely used in cis-regulatory element study. Genomic regions that associated with cis-regulatory elements have a characteristic of pronounced sensitivity to DNase I digestion. Thus, DNase I digestion followed by Illumina sequencing enables to identify cis-regulatory elements not only proximal but also distal to their target genes.
TSS-Seq: Transciption Start Site Sequencing
Alternative transcription start site (ATS) leads to the production of mRNA isoforms from the same locus and further results in protein isoforms. Alternative transcription regulated by alternative cis-regulatory elements is involved in various biological processes, such as light and stress responses. TSS-seq employs a pyrophosphatase, which hydrolyzes inorganic pyrophosphate from the m7GpppG “cap” of mature mRNA to orthophosphate, to specifically target transcription start sites of mature mRNA (5′ ends). We use TSS-seq to understand how gene expression is regulated under various stresses.
ChIP-Seq: Chromatin Immunoprecipitation Sequencing using 10,000 nuclei
ChIP-seq has been extensively used for investigating DNA sequences bound by histones with particular modifications or transcription factors (TFs). Various histone modification marks are associated with active gene expression (such as H3K4me3 and H3K36me3) and gene repression (H3K27me3 and H3K9me2). We developed and benchmarked ChIP-seq using input material as low as 10,000 nuclei, which enables us to observe histone modifications in relation to gene expression at different developmental stages and under various stresses.
Live imagining and GUS validation
We employ a chemiluminescence imaging system to characterize functional cis-regulatory elements in a live plant using transiently transformed vector containing candidate cis-regulatory elements along with LUC or GFP. The system provides fast screening of cis-regulatory element candidates, even for some stress-responsive candidates. We also utilize classic GUS reporter system to validate tissue-specific, developmental stage-specific and stress-responsive cis-regulatory element candidates. Permanent transformation of candidate DNA fragments with GUS reporter gene provide accurate and visible information of the specificity for a cis-regulatory element.