Noncanonical functions of Ku may underlie essentiality in human cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

The multifaceted role of GCM1 during trophoblast differentiation in the human placenta.

Remodeling of the uterine vasculature by invasive extravillous trophoblasts (EVTs) is a critical aspect of human placentation. Insufficient EVT invasion can lead to severe obstetrical complications like preeclampsia, intrauterine growth restriction, and preterm birth. Glial cells missing-1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is highly expressed in human syncytiotrophoblast (ST) and EVTs. GCM1 is classically considered a master regulator of ST formation, but little is known about its contribution to the development and function of EVTs. Therefore, in this study we test the hypothesis that GCM1 is a critical regulator of both EVT and ST development and function. We show that GCM1 is highly expressed in human trophoblast stem (TS) cells differentiated into either ST or EVTs. Knockdown of GCM1 in TS cells hindered differentiation into both ST and EVT pathways. When placed in ST media, GCM1-knockdown cells formed small, unstable clusters; when placed in EVT media, cells had altered morphology and transcript profiles resembling cells trapped in an intermediate state between CT and EVT, and invasive capacity through matrix was reduced. RNA sequencing analysis of GCM1-deficient TS cells revealed downregulation of EVT-associated genes and enrichment in transcripts related to WNT signaling, which was linked to decreased expression of the EVT master regulator ASCL2 and the WNT antagonist NOTUM. Our findings reveal an essential role of GCM1 during ST and EVT development, and suggest that GCM1 regulates differentiation of human TS cells into EVTs by inducing expression of ASCL2 and NOTUM.

The Ku complex: recent advances and emerging roles outside of non-homologous end-joining.

Since its discovery in 1981, the Ku complex has been extensively studied under multiple cellular contexts, with most work focusing on Ku in terms of its essential role in non-homologous end-joining (NHEJ). In this process, Ku is well-known as the DNA-binding subunit for DNA-PK, which is central to the NHEJ repair process. However, in addition to the extensive study of Ku's role in DNA repair, Ku has also been implicated in various other cellular processes including transcription, the DNA damage response, DNA replication, telomere maintenance, and has since been studied in multiple contexts, growing into a multidisciplinary point of research across various fields. Some advances have been driven by clarification of Ku's structure, including the original Ku crystal structure and the more recent Ku-DNA-PKcs crystallography, cryogenic electron microscopy (cryoEM) studies, and the identification of various post-translational modifications. Here, we focus on the advances made in understanding the Ku heterodimer outside of non-homologous end-joining, and across a variety of model organisms. We explore unique structural and functional aspects, detail Ku expression, conservation, and essentiality in different species, discuss the evidence for its involvement in a diverse range of cellular functions, highlight Ku protein interactions and recent work concerning Ku-binding motifs, and finally, we summarize the clinical Ku-related research to date.