Christian Ibarra

Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.

The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.

MOTIVATION: MethylCoder is a software program that generates per-base methylation data given a set of bisulfite-treated reads. It provides the option to use either of two existing short-read aligners, each with different strengths. It accounts for soft-masked alignments and overlapping paired-end reads. MethylCoder outputs data in text and binary formats in addition to the final alignment in SAM format, so that common high-throughput sequencing tools can be used on the resulting output. It is more flexible than existing software and competitive in terms of speed and memory use. AVAILABILITY: MethylCoder requires only a python interpreter and a C compiler to run. Extensive documentation and the full source code are available under the MIT license at: https://github.com/brentp/methylcode. CONTACT: bpederse@gmail.com.

Brucellosis is an infectious disease of worldwide distribution, which has a great economic impact due to the productive and reproductive losses that it causes, in addition to the serious public health problem. The aim of this study is to estimate the economic losses, through financial analysis, caused by bovine brucellosis in the province of Carchi, over a one-year period. A random sampling was used to determine the prevalence in the study area, where 2976 animals were considered, and the Rose Bengal (RB) test was used as a screening test and the Fluorescence Polarized Assay (FPA) as a confirmatory test, obtaining a prevalence of 8.2% (244/2976). In addition, parameters associated to the losses caused by brucellosis in cattle were determined by literature review. To estimate costs, field information was collected through a survey of a total of 100 randomly selected farmers. The loss estimated due to calves lost as a result of abortions and neonatal death was USD. 79170.00. The loss due to death of 4 cows as a result of metritis was estimated at USD. 5000.00. The cost of examination and treatment of aborted cows was USD. 20100.00. The losses due to reduction in milk production from aborted and non-aborted seropositive cows were estimated at USD. 158114.21. The financial losses due to brucellosis in province of Carchi were estimated at USD. 262384.21.

DNA methylation is one of the most well studied repressive epigenetic marks in eukaryotes. In plants, DNA methylation silences genes and transposons, and establishes genomic imprinting. Genomic imprinting is the mono-allelic expression of a gene occurring in a parent-of-origin specific manner. Imprinted genes tend to be expressed in nutritive tissues and structures that function to support the growing embryo. In mammals, this is the placenta, and in plants this is the endosperm. In Arabidopsis, the female gametophyte contains the egg and central cell, while the male gametophyte contains the vegetative cell and two sperm cells. Both the central cell and vegetative cells are companion cells to the egg and sperm cells respectively. When pollen lands on specialized cells at the tip of the gynoecium, the vegetative cell forms a pollen tube that transports the two sperm cells to the ovule. Within the ovule, one sperm fertilizes the egg cell to form the embryo, whereas a second sperm fertilizes the central cell to form the endosperm, which supports embryo development by providing nutrients. In Arabidopsis, the DME DNA glycosylase carries out active DNA demethylation in the central cell of the female gametophyte, which has been shown to establish genomic imprinting in Arabidopsis. Recently, DME-mediated DNA demethylation has also been shown to occur in the vegetative cell of the male gametophyte, as well. The aim of the work carried out in my thesis has been to investigate the role of DNA methylation and DME-mediated DNA demethylation in the central cell and vegetative cell. In particular, I was interested in identifying and understanding the regions in the genome where DME-mediated DNA demethylation occurs. My overall approach was to perform high-throughput bisulfite sequencing using the illumina<sup>TM</sup> platform on the Arabidopsis endosperm, embryo, and dme-mutant endosperm, and to analyze their DNA methylation profiles (methylomes). In my initial study (Chapter II), I measured non-allele-specific methylomes in which endosperm and embryo dissected from selfed-plants were used. In my subsequent study (Chapter III), I determined allele-specific (maternal versus paternal) methylomes by analyzing endosperm and embryo derived from seeds generated by crossing different Arabidopsis ecotypes. Because there are no current methods for determining the Arabidopsis central cell methylome, I sought to use the maternal endosperm genome as a proxy for the maternal central cell genome. Through collaboration, I also had the opportunity to investigate and explore the methylomes of wild-type and dme-mutant vegetative cell and sperm cells, which enabled me to compare the maternal and paternal reproductive methylomes of the plant. To the best of my knowledge, this is the first time a high-throughput DNA methylation study was done on the endosperm of a flowering plant. With my collaborators, we discovered that the Arabidopsis endosperm exhibits a genome-wide reduction in DNA methylation compared to the embryo. We also found that the DME DNA glycosylase regulates non-CG methylation in the endosperm, particularly siRNA-mediated CHH methylation. Moreover, our analysis of DME-dependent DNA demethylation in companion cells suggested a mechanism for reinforcing silencing of transposons in the plant male and female gamete cells. We found that DME demethylated many of the same loci in both the maternal endosperm and vegetative cell genomes, suggesting that DME has a much more robust and primitive role in Arabidopsis reproduction than previously thought. Interestingly, many of these DME-dependent sites were heavily enriched in small transposons and repetitive elements. Moreover, these small transposons tended to reside adjacent to genes, enabling us to propose a new model for the observed phenomenon of gene-adjacent DNA demethylation in Arabidopsis. That is, regulation of gene imprinting may not be the basal function of DME demethylation. These studies suggest that the primary function of demethylation of transposons in companion cells may be to reinforce transposon silencing in plant gametes.