DNA methylation is an epigenetic mechanism critical for tissue development, cell specification and cellular function. Mammalian brains consist of millions to billions of neurons and glial cells that can be subdivided into many distinct types of cells. We hypothesize that brain methylomes are heterogeneously methylated across different types of cells and the transcription factors play key roles in brain methylome programming. To dissect brain methylome heterogeneity, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) loci which demonstrate bipolar DNA methylation pattern, i.e., hypermethylated in one cell subset but hypomethylated in others. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Importantly, we found that putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the important role of putative CSM loci during brain development, implying that dramatic changes in functions and complexities of the brain may be companied by a rapid change in epigenetic heterogeneity. To explore epigenetic regulatory mechanisms during brain development, as described in Chapter 3, we adopted unbiased data-driven approaches to re-analyze methylomes for human and mouse frontal cortices at different developmental stages. We predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated that thousands of EGR1 binding sites are with cell-type specific methylation patterns established during postnatal frontal cortex development. More specifically, the CpG dinucleotides within these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 programs the brain methylome together with TET1 during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome. / Ph. D. / DNA methylation is a widespread epigenetic mark on DNA, serving as a “switch” to turn on or off gene expression. It plays essential roles in cellular functions, tissue development. Mammalian brains contain millions to billions of neurons and glial cells, which can be further divided into many different types of cells. We hypothesize that brain cells have different methylation profiles across the genome, and transcriptional factors play important roles in programming methylation in the mammalian brain genome. To study the diversity of methylation profiles across the genomes of different brain cells, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) genomic regions which show bipolar DNA methylation pattern, i.e., hypermethylated in one type of cell but hypomethylated in others. By applying a technique called the genome-scale hairpin bisulfite sequencing to mouse frontal cortices, we demonstrated that the majority of CSM genomic regions predicted likely resulted from the methylation differences among brain cells, rather than from methylation differences between DNA double strands. Surprisingly, we found that these predicted CSM genomic regions increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the importance of predicted CSM genomic regions, implying that dramatic changes in brain function and structure may be companied by a rapid change in DNA methylation diversity during brain development. To explore underlying epigenetic mechanisms during brain development, as described in Chapter 3, we re-analyzed methylomes for human and mouse frontal cortices at different developmental stages, and predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain methylome programming. We found thousands of EGR1 binding sites showed cell-type specific methylation patterns, and were established during postnatal frontal cortex development. More specifically, the methylation level of these EGR1 binding sites was low in mature neurons but pretty high in glial cells. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the Egr1 knockout or Tet1 knockout mice show strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 works together with TET1 to program the brain methylome during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome.

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