Periodic Reporting for period 3 - TotipotentZygotChrom (Mechanisms of chromatin organization and reprogramming in totipotent mammalian zygotes)
Período documentado: 2021-11-01 hasta 2023-04-30
Our research addresses the question of how life starts. Fertilization of egg by sperm generates a one-cell embryo that has the potential to generate an entire organism. Initially, the one-cell embryo is not competent to express its genome. Instead, this large cell is stockpiled with maternal RNA and protein that control diverse biological processes. A key transition is the handover of control from the egg genome to the embryo genome. This genome awakening is essential for life to proceed and is controlled by largely unidentified maternal factors in mammals. We focus on identifying these activators of genome awakening and to study their mechanism in vivo and in vitro. Our work also investigates how the embryonic genome is folded in three-dimensional space. Elucidating the mechanisms of genome awakening and folding are important because they are fundamental to life. Since the factors are efficient at reprogramming and activating the embryonic genome, their manipulation in other cell types has potential implications for regenerative medicine.
Our key finding is that genome folding is affected by proteins that are essential for DNA replication. Prior to this work, it was known that DNA is folded into loops by a process of loop extrusion that is mediated by cohesin proteins in interphase cells. We have also shown that cohesin is required for loop formation in early embryos. The progressive growth of loops stops when the extrusion machinery encounters a transcription factor called CTCF. This was the only known barrier to loop extrusion in vertebrates. Our work revealed that the replicative helicase MCM complex forms a randomly placed extrusion barrier that hinders loop extrusion and affects gene expression. These findings open up the possibility to investigate the molecular properties of MCM proteins that make them into barriers and to determine which other barriers exist in eukaryotic cells.
Our work on identifying the key regulators of embryonic genome awakening is ongoing. We expect to have identified some of these before the end of the project. In preparation for studying their mechanisms in vivo and in vitro, we are optimizing ultra-low input approaches and have gained the first insights into where transcription factors bind to chromatin in the early embryo. This method will enable a mechanistic study of the reprogramming factors. To complement genomics-based approaches, we also developed an imaging-based approach to study accessible chromatin that we term Chromatin Accessibility Revealed by Microscopy (ChARM). These approaches combined with genetic perturbations are expected to reveal the mechanism of reprogramming factors including transcription factors that initiate life in the early embryo.