Biology subjects

Kafer, G. R.

Publications and source records attributed to Kafer, G. R..

3 recordsLinked to original sources

bioRxiv2022

Replication stress tolerance and management differs between naive and primed pluripotent cells

Replication stress is an endemic threat to genome stability. For reasons unknown, replication stress response factors become essential during peri-implantation development. This coincides with a stem cell potency switch from the naive to the primed state. Using genetically matched, chimera-derived mouse naive embryonic (mESC) and primed epiblast stem cells (mEpiSC) we found that replication stress management differs between potency states. Primed mEpiSCs rely on Atr activity to prevent replication catastrophe, minimize genomic damage, avoid apoptosis, and re-enter the cell cycle. Conversely, under replications stress, mESCs readily activate Atm regardless of Atr activity, undergo replication catastrophe, and induce apoptosis. Primed pluripotent cells therefore engage Atr to counteract replication difficulties and maintain viability, whereas cells in the naive state are more readily cleared under the same conditions. We anticipate these divergent strategies enable pluripotent cells of different potency states to meet associated proliferative or developmental demands during early development.

developmental biology↗

bioRxiv2020

Sequential enrichment at the nuclear periphery of H2A.Zac and H3K9me2 accompanies pluripotency loss in human embryonicstem cells

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure to effect the required changes to the transcriptional program. This involves covalent modification of histone tails, replacement of histone variants, and alteration in the subnuclear position of genes, including associations with the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone variants and covalent modifications for changes in nuclear periphery association during differentiation of human embryonic stem cells to a trophoblast-like lineage. This differentiation process is rapid and homogeneous, facilitating the use of a relatively fine timecourse (12h, 24h, and 48h post-initiation) to enable detection of transient changes. With this scheme, we detected two modifications with significant changes in enrichment at the nuclear periphery: acetylation of histone variant H2A.Z, and dimethylation of histone H3 at lysine 9. We show that these chromatin marks increase specifically at the nuclear periphery in a sequential, complementary manner, with a H2A.Z acetylation preceding H3K9 dimethylation. The increase of H3K9 dimethylation occurred coincidentally with but independently of accumulation of Lamin A, since Lamin A-/- hES cells showed no changes in the localization pattern of H3K9 dimethylation. Inhibition of histone deacetylases led to persistent and increased H2A.Z acetylation at the periphery, and failure to differentiate. Our results show that a concerted dynamic change in the nature of peripheral chromatin is required for differentiation into the trophoblast state.

cell biology↗

bioRxiv2018

Telomere-loop dynamics in chromosome end protection

We used super-resolution microscopy to investigate the role of macromolecular telomere structure in chromosome end protection. In murine and human cells with reduced TRF2, we find that ATM-activation at chromosome ends occurs with a structural change from t-loops to linearized chromosome ends through t-loop unfolding. Comparably, we find Aurora B kinase regulates telomere linearity concurrent with ATM activation at telomeres during mitotic arrest. Using a separation of function allele, we find that the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that telomere linearity and ATM activation occur separately from telomere fusion via non-homologous end-joining (NHEJ). Further, we show that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1-arrest when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that regulate ATM activation at chromosome ends independent of mechanisms to suppress chromosome end fusion.

molecular biology↗