CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

MMS22L-TONSL functions in sister chromatid cohesion in a pathway parallel to DSCC1-RFC.

The leading strand-oriented alternative PCNA clamp loader DSCC1-RFC functions in DNA replication, repair, and sister chromatid cohesion (SCC), but how it facilitates these processes is incompletely understood. Here, we confirm that loss of human DSCC1 results in reduced fork speed, increased DNA damage, and defective SCC. Genome-wide CRISPR screens in DSCC1-KO cells reveal multiple synthetically lethal interactions, enriched for DNA replication and cell cycle regulation. We show that DSCC1-KO cells require POLE3 for survival. Co-depletion of DSCC1 and POLE3, which both interact with the catalytic polymerase ε subunit, additively impair DNA replication, suggesting that these factors contribute to leading-strand DNA replication in parallel ways. An additional hit is MMS22L, which in humans forms a heterodimer with TONSL. Synthetic lethality of DSCC1 and MMS22L-TONSL likely results from detrimental SCC loss. We show that MMS22L-TONSL, like DDX11, functions in a SCC establishment pathway parallel to DSCC1-RFC. Because both DSCC1-RFC and MMS22L facilitate ESCO2 recruitment to replication forks, we suggest that distinct ESCO2 recruitment pathways promote SCC establishment following either cohesin conversion or de novo cohesin loading.

TSC loss distorts DNA replication programme and sensitises cells to genotoxic stress.

Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood. We report here the occurrence of futile checkpoint responses and an unusual type of replicative stress (RS) in TSC1-/- fibroblasts exposed to low-dose genotoxins. This RS is characterized by elevated nucleotide incorporation rates despite only modest origin over-firing. Strikingly, an increased propensity for asymmetric fork progression and profuse chromosomal aberrations upon mild DNA damage confirmed that TSC loss indeed proved detrimental to stress adaptation. We conclude that low stress tolerance of TSC-/- cells manifests at the level of DNA replication control, imposing strong negative selection on genomic instability that could in turn detain TSC-mutant tumours benign.

The residue at position 5 of the N-terminal region of Src and Fyn modulates their myristoylation, palmitoylation, and membrane interactions.

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.

Chromatin condensation dynamics and implications of induced premature chromosome condensation.

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing 'prophasing' of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.