Examining the chromatin profile at transposon-gene boundaries and developing a genetic map of the B centromere in maize
Ellis, Nathanael Andrew
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The primary constriction site on a chromosome is called a centromere and is necessary for the faithful segregation of DNA during cell division. In maize, centromeres are primarily made up of the class I transposable elements (TEs), CRM2 and tandem repeat, CentC. These repetitive sequences interact with the centromere defining histone variant, CENH3. In this study, transposon display (TD) was carried out to amplify CRM2 junction sites, creating 40 unique markers that are specific to the B centromere. CRM2-TD markers were genetically mapped to the B centromere by assaying a series of lines with different centromere breakpoints, and the markers were joined to make a ~10kb pseudocontig or B minimal map. Chromatin immunoprecipitation for CENH3 associated DNA was carried out in B73 lines with and without B chromosome. Centromere specific reads were mapped to the B minimal map and B73 genome to identify CRM2-TD marker sequences associated with the active centromere and 31 markers were found that span the centromere cores, necessary for centromere formation. Lack of these markers were associated with ectopic neocentromere formation. The genetic map and minimal map of the B centromere will be essential for further analyzing of centromere deletions lines and formation of a physical map spanning the entirety of the B centromere. TEs play numerous important roles for genome evolution and centromere sequence is an example for class I elements. Non-autonomous derivatives of class II DNA transposons are called Miniature Inverted-Repeat Transposable Elements (MITE) and contribute to genetic diversity in maize. In the grasses, MITEs are abundant in the 3’ and 5’ regions of genes and associated with high gene expression. In this study, superfamilies of MITEs were analyzed by whether or not they act as boundary elements between genes and a group of class I TEs shown to spread heterochromatin into nearby low-copy regions. We found that when a MITE is present between a gene and spreading TE, gene expression levels are higher and DNA methylation levels lower than when a MITE is absent. Methylation levels drastically reduce over a subset of MITE superfamilies, and these MITEs have a unique chromatin profile and sequence content.