ABSTRACT
The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.
Subject(s)
Drosophila Proteins , Drosophila , Muscle, Skeletal , Nuclear Lamina , Animals , Lamin Type B/genetics , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/physiology , Nuclear Lamina/physiology , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Glycosylphosphatidylinositols/metabolismABSTRACT
Zygotic genome activation (ZGA) is a critical postfertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL (murine endogenous retrovirus-L) is transiently upregulated at the two-cell stage during ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we show that full-length MERVL transcripts, but not encoded retroviral proteins, are essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both knockdown and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcripts led to retention of an accessible chromatin state at, and aberrant expression of, a subset of two-cell-specific genes. Taken together, our results suggest a model in which an endogenous retrovirus plays a key role in regulating host cell fate potential.
Subject(s)
Gene Expression Regulation, Developmental , Retroelements , Mice , Animals , Retroelements/genetics , Gene Expression Regulation, Developmental/genetics , Embryonic Development/genetics , Chromatin/genetics , Chromatin/metabolism , Zygote/metabolismABSTRACT
Mounting evidence has established that subsets of transposable elements (TEs) function as gene regulatory elements in a cell type- and species-specific manner. Here we describe an in vitro system to ectopically activate TEs using CRISPR-mediated activation (CRISPRa) for functional studies in mouse embryonic stem cells (ESCs). We established a stable mouse CRISPRa ESC line, in which expression of guide RNA enables the activation of TE-derived enhancers and the expression of their adjacent genes. We show an example of ectopic activation of TE-derived enhancers that function in male meiosis, as well as the expression of adjacent germline genes in ESCs. This system can also be applied to functional studies of TEs that are not active in ESCs.
