The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing

DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing. © 2023 The Author(s). Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics

Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture

Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and Is Important for the Proper Aggregation of Developing Cells

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development

Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability

The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatin-binding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosome-nucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregatio