Defining the role of DOT1L in chromocenter stabilization pre- and post-fertilization

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PROJECT SUMMARY Dramatic restructuring of chromatin is an essential event preceding and immediately following fertilization. The developing sperm nucleus undergoes extreme compaction, and this hypercompact genetic material is rapidly, aggressively repackaged into a chromatinized state soon after entering the oocyte. Failure to enact these changes leads to nuclear disorganization and produces inviable embryos. Chromocenters are large, dense nuclear structures that coordinate reprogramming of repressive heterochromatin during these transitions, and chromocenter integrity is important for fertility. Despite its importance, a major unsolved problem is what signal or signals trigger chromocenter disassembly in developing sperm heads and stimulate chromocenter reassembly in embryos. The overall objective of this project is to define the molecular pathways that govern chromocenter restructuring before and after the events of fertilization. The central hypothesis is that the same chromatin regulator, DOT1L, controls both chromocenter disassembly before fertilization and chromocenter reassembly after fertilization by stimulating transcription of pericentromeric major satellite repeats. This hypothesis is supported by preliminary data indicating that DOT1L has a specialized role in promoting expression of major satellite repeat elements and that this function is important for formation of chromocenters. Preliminary studies also demonstrated that DOT1L is required for male fertility, and that it is active in chromocenters and required for nuclear reprogramming and condensation in the late stages of sperm development, and that its activity is required for preimplantation embryogenesis. The hypothesis will be tested in two Specific Aims: the first Aim will test the contribution of DOT1L to chromocenter disassembly during sperm nuclear condensation, and the second Aim will determine how DOT1L regulates chromocenter assembly in early embryos. This project is innovative for its conceptual advance implicating a unifying regulatory factor in control of heterochromatin disassembly and reassembly before and after fertilization, its exploration of a novel mechanism for transmission of epigenetic information across generations, its establishment of a new molecular and biological function for DOT1L, and its use of precision heterochromatin visualization techniques in preimplantation embryos. This work is expected to reveal a fundamental missing link governing the dramatic heterochromatin reprogramming that occurs both before and after fertilization, advancing understanding of genome regulation during gamete and embryo development and revealing a new relationship between paternal chromatin and the epigenetic state of the embryo.