The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa. Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens. We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells. Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins. Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins. Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes. In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase. Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.
Abstract:Cell migration through extracellular matrices requires nuclear deformation, which depends on nuclear stiffness. In turn, chromatin structure contributes to nuclear stiffness, but the mechanosensing pathways regulating chromatin during cell migration remain unclear. Here, we demonstrate that WD repeat domain 5 (WDR5), an essential component of H3K4 methyltransferase complexes, regulates cell polarity, nuclear deformability and migration of lymphocytes in vitro and in vivo, independent of transcriptional activity, suggesting non-genomic functions for WDR5. Similarly, depletion of RbBP5 (another H3K4 methyltransferase subunit) promotes similar defects. We reveal that a 3D environment increases the H3K4 methylation dependent on WDR5, and results in a globally less compacted chromatin conformation. Further, using atomic force microscopy, nuclear particle tracking and nuclear swelling experiments, we detect changes in nuclear mechanics that accompany the epigenetic changes induced in 3D conditions. Indeed, nuclei from cells in 3D environments were softer, and thereby more deformable, compared to cells in suspension or cultured in 2D conditions, again dependent on WDR5. Dissecting the underlying mechanism, we determined that actomyosin contractility, through the phosphorylation of myosin by MLCK (myosin light chain kinase), controls the interaction of WDR5 with other components of the methyltransferase complex, which in turn upregulates H3K4 methylation activation in 3D conditions. Taken together, our findings reveal a novel non-genomic function for WDR5 in regulating H3K4 methylation induced by 3D environments, physical properties of the nucleus, cell polarity and cell migratory capacity. 3 Significance:Cells require nuclear deformation to squeeze through tissue matrices. We have discovered that WDR5 (an epigenetic modulator of H3K4 methylation) is fundamental for cell polarity and migration in vitro and in vivo, independently of transcription. We have uncovered that the interactions between cells and the surrounding 3D confined conditions induce the upregulation of H3K4me3. Moreover, 3D environments control the deformability and the mechanical properties of the nucleus. We have identified that loss of WDR5 abrogates the H3K4 methylation and the nuclear changes induced by 3D conditions. Mechanistically, we found that MLCK and myosin function was required for WDR5-mediated H3K4 methylation in 3D matrices. Our findings uncover new functions of the epigenetic machinery when cells move through constricted conditions.
Nanoindentation enables the measurement of mechanical properties from single crystals with dimensions of a few micrometers. This experimental technique, however, has only recently been applied to molecular crystals. Key differences between the application of this technique to molecular crystals and metals and other inorganics are identified. From this, protocols for the measurement of hardness and elastic modulus of molecular crystals of pharmaceutical interest are proposed. Using form I aspirin as a model system, the impact of single crystal sample surface preparation (washing and cleaving) on the surface roughness is explored. We show the importance of using a calibration sample with hardness and stiffness close to that of molecular crystals for the acquisition of more accurate data. The issue of solvent occlusions formed during crystal growth is discussed as a source of material property variation as well as tip contamination. It is proposed that this in part explains the significantly larger variation of the measured mechanical properties among different single crystals compared to those performed on a unique sample. Because both the indentation modulus and the hardness can vary significantly for low depth indents, samples were tested over a wide range of depths, which revealed that a minimum depth of penetration is required for the acquisition of data. This experiment is crucial and needs to be carried out for every system under study since it allows for the determination of the minimum-working load. Post-indentation imaging combined with crystallographic analysis and molecular simulations allows for the characterization and rationalization of the material plastic deformation mechanisms.
The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4β1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4β1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4β1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.
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