Epigenetics and Epigenomics

Chemical modifications to DNA and histone proteins form a complex regulatory network that modulates chromatin structure and genome function. The epigenome refers to the complete description of these potentially heritable changes across the genome. The composition of the epigenome within a given cell is a function of genetic determinants, lineage, and environment.

With the sequencing of the human genome completed, investigators now seek a comprehensive view of the epigenetic changes that determine how genetic information is made manifest across an incredibly varied background of developmental stages, tissue types, and disease states. Selected areas of our work in genome regulation are:

DNA Methylation

DNA methylation is essential for mammalian development and is required in most somatic cells. It is established and maintained by three catalytically active enzymes: DNA methyltransferase (Dnmt) 1, Dnmt3a and Dnmt3b. Two additional, homologous enzymes, Dnmt2 and Dnmt3l, are expressed in several cell types, including ES cells. Deletion of Dnmt2 has no apparent phenotype in vitro or in vivo. The ability of Dnmt2 to methylate a specific cytosine in the anticodon loop structure of tRNAs suggests that it might not function as a DNA methyltransferase. Loss of Dnmt1 results in embryonic lethality around embryonic day (E) 8.5–9, and Dnmt1 mutant embryos retain only one-third of the normal amount of DNA methylation. Dnmt1-deficient embryos show rudiments of the major organs, but they are smaller than normal and appear to be developmentally delayed. Dnmt3b mutant embryos appear to develop normally before E9.5, but show multiple developmental defects later and do not develop to term. Conditional deletion of Dnmt3b in mouse embryonic fibroblasts (MEFs) results in partial loss of methylation, indicating the importance of this enzyme, together with Dnmt1, for maintaining epigenomic patterns in proliferating cells. Unlike mice lacking Dnmt1 or Dnmt3b, homozygous Dnmt3a knockout mice can develop to term, but become runted and die ~1 month after birth. Conditional deletion of Dnmt3a results in imprinting defects in the germline. Homozygous Dnmt3l mice are viable, but male mice are sterile and heterozygous offspring of homozygous females die owing to imprinting defects. This phenotype is similar to that of Dnmt3adeficient mice and suggests that both enzymes might be involved in establishing correct imprinting patterns. Dnmt3l is a close homolog of Dnmt3a and Dnmt3b that lacks the catalytic domain but is highly expressed in the early embryo, ES cells and germ cells. It has been suggested to function as a co-regulator of both Dnmt3a and Dnmt3b and has recently been shown to interact with the N-terminal tail of histone H3 when it lacks methylation at lysine 4. Importantly, the genome is transiently hypomethylated during two phases of normal development without adverse effects. As described above, the first phase is preimplantation development. The totipotent zygote and blastomeres, the pluripotent blastomeres, the pluripotent ICM cells and trophectoderm cells do not require substantial DNA methylation. A second wave of demethylation commences after the specification of primordial germ cells (PGCs) around day E7.25. Genome-wide bisulfite sequencing was used to show that E13.5 PGCs have only 5–20% of genomic DNA methylation left, confirming that PGCs show transient reduction of DNA methylation without adverse effects on viability.

Histone Modifications

Histone modifications provide an additional and complex layer of the epigenetic code. Many of the enzymes that regulate these modifications have been studied extensively, including histone acetyltransferases, deacetylases, methyltransferases and histone demethylases. Among the best-characterized mediators are protein complexes of the polycomb (PcG) and trithorax (trxG) groups. PcG proteins catalyze two distinct histone modifications: tri-methylation of lysine 27 of histone 3 (H3K27me3) by polycomb repressive complex (PRC) 2 and mono-ubiquitination of lysine 119 H2A (H2AK119ub1) by PRC1. H3K27 is tri-methylated by the enhancer of zeste (Ezh2 or KMT6), which contains a SET (su[var]3–9, enhancer of zeste, Trx) domain and, with Eed (embryonic ectoderm development) and Suz12 (suppressor of zeste 12), are components of PRC2. Loss of any one of the PRC2 subunits results in severe gastrulation defects, highlighting its essential role in normal development. Ezh2 knockout embryos are underdeveloped and die around E8.5. Ezh2 is upregulated upon fertilization and its expression remains high during pre-implantation development. Its close homolog, Ezh1, is expressed in the fertilized oocyte but is barely detectable at the blastocyst stage. However, it is expressed in ES cells and found later in the adult. Eed does not appear until day E5.5, suggesting that Ezh2 and maybe Ezh1 also have roles in preimplantation development that are independent of PRC2 and Eed. Eed-deficient embryos show gastrulation defects and do not maintain X inactivation in extraembryonic cells. Like mice deficient in the other PRC2 components, Suz12 homozygous mice die during the early postimplantation stages (before day E10.5). Similar to loss of PRC2, loss of PRC1 components, such as Ring1B, results in an early embryonic lethal phenotype. Bmi-1–null mice show several hematopoietic and neurological abnormalities51, and loss of the H3K9 methyltransferases Eset (SetDB1) or G9a causes peri- and postimplantation lethality. Finally, although not discussed here, mutants for most of the chromatin remodeling and histone chaperones also show early embryonic lethality. Together, the knockout studies have clearly established that DNA methylation and histone modification are essential for normal development. But many questions remain regarding the specific contributions of these epigenetic marks to the regulation of gene expression throughout development. The genomic distribution and global patterns of these marks have not been studied in detail. Mice with mutations in most of these genes die early, probably owing to failure to establish early epigenetic patterns that are presumed to dictate later developmental decisions. It is less clear what the effect of such mutations would be after initial specification has taken place. Strategies for exploring these questions in future research include conditional deletions in mouse somatic lineages and cell types and genome-wide mapping of epigenetic modifications in early development and single cell approaches.

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