Cell cycle

Summary

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that causes it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division. In cells with nuclei (eukaryotes, i.e., animal, plant, fungal, and protist cells), the cell cycle is divided into two main stages: interphase and the mitotic (M) phase (including mitosis and cytokinesis). During interphase, the cell grows, accumulating nutrients needed for mitosis, and replicates its DNA and some of its organelles. During the mitotic phase, the replicated chromosomes, organelles, and cytoplasm separate into two new daughter cells. To ensure the proper replication of cellular components and division, there are control mechanisms known as cell cycle checkpoints after each of the key steps of the cycle that determine if the cell can progress to the next phase. In cells without nuclei (prokaryotes, i.e., bacteria and archaea), the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells. In single-celled organisms, a single cell-division cycle is how the organism replicates itself. In multicellular organisms such as plants and animals, a series of cell-division cycles is how the organism develops from a single-celled fertilized egg into a mature organism, and is also the process by which hair, skin, blood cells, and some internal organs are regenerated and healed (with possible exception of nerves; see nerve damage). After cell division, each of the daughter cells begin the interphase of a new cell cycle.

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown.DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization(FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress.One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination.CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling.We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

Class I histone deacetylases (HDAC1-3) are histone lysine delactylases

Carlos Moreno Yruela, Alexander Lund Nielsen, Di Zhang

Lysine L-lactylation [K(L-la)] is a newly discovered histone mark stimulated under conditions of high glycolysis, such as the Warburg effect. K(L-la) is associated with functions that are different from the widely studied histone acetylation. While K(L-la) can be introduced by the acetyltransferase p300, histone delactylases enzymes remained unknown. Here, we report the systematic evaluation of zinc- and nicotinamide adenine dinucleotide-dependent histone deacetylases (HDACs) for their ability to cleave epsilon-N-L-lactyllysine marks. Our screens identified HDAC1-3 and SIRT1-3 as delactylases in vitro. HDAC1-3 show robust activity toward not only K(L-la) but also K(D-la) and diverse short-chain acyl modifications. We further confirmed the de-L-lactylase activity of HDACs 1 and 3 in cells. Together, these data suggest that histone lactylation is installed and removed by regulatory enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway's regulatory elements.

2022

Recognition of pathogen-derived molecules through germline-encoded receptors is a fundamental principle of innate immunity. Pattern recognition receptors detect specific intracellular danger signals to trigger potent immune responses. The DNA sensor cyclic GMP-AMP synthase (cGAS) detects double-stranded DNA derived from viruses and microbes in the cytoplasm. However, due to its ability to recognize DNA independent of sequence, aberrant detection of self-DNA in various settings can lead to autoinflammatory diseases. Under physiological conditions, genomic DNA is sequestered in the nucleus and shielded from cytosolic molecules by the nuclear envelope (NE). Nevertheless, cGAS gains access to the nuclear compartment in some instances, and the mechanisms controlling its activity on genomic DNA are not fully known.In this study, we investigate the regulation of nuclear cGAS localization and activity in different contexts. We first describe how cGAS activity against nuclear DNA is restricted in instances of spontaneous loss of nucleo-cytoplasmic compartmentalization. Upon NE rupture, cGAS enters the nucleus, but is prevented from activation by Barrier-to-autointegration factor 1 (BAF). BAF dynamically competes with cGAS for DNA binding, thereby preventing the formation of stable cGAS-DNA complexes and cGAS enzymatic activity. We thus identify a cellular safeguard mechanism, beyond physical separation, that restricts cGAS activity against genomic self-DNA.In addition, we investigate cGAS activation in conditions associated with a disrupted NE. Instability of the NE is a hallmark of laminopathies, diseases of the nuclear lamina that can manifest in premature aging phenotypes. While we find no contribution of basal cGAS signaling to the pathogenesis of two different progeria syndromes, we detect a decreased capacity to respond to DNA challenge on the cellular level, suggesting that cGAS may be sequestered inside the nucleus in these cells.Lastly, because cGAS is increasingly recognized to reside inside the nucleus at steady state, we delineate cGAS nuclear localization in time and space. In cycling cells, nuclear cGAS levels oscillate with the cell cycle and are highest after mitosis followed by a sharp decrease in G1 phase. We report that cGAS is not exported during G1, but rather subjected to degradation inside the nucleus by the nuclear proteasome. Moreover, we establish a technique to study potential interaction partners of nuclear cGAS, which opens up intriguing possibilities for further investigation.Together, our findings contribute to a better understanding of the regulatory processes that govern nuclear cGAS localization and activity, and will help to uncover potential disease-causing aberrations in the future.

EPFL2022