Biology subjects

King, M. C.

Publications and source records attributed to King, M. C..

23 records · Page 2Linked to original sources

bioRxiv2020

Nucleosome-constrained loop extrusion model for the origin of topologically associating domains

The chromosomes - DNA polymers and their binding proteins - are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse- model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number loops or residence times of loop extrusion factors influences the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taken together we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

cell biology↗

bioRxiv2019

Ablation of SUN2-containing LINC complexes drives cardiac hypertrophy without interstitial fibrosis

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear-cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but an full understanding of disease etiology in these patients is lacking. Here we show SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from pro-fibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGF{beta} signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGF{beta} signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of pro-fibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.

cell biology↗

bioRxiv2018

Loss of Rap1 supports recombination-based telomere maintenance independent of RNA-DNA hybrids in fission yeast

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant \"alternative lengthening of telomeres\" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 orthologue, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that up-regulation of telomere-engaged TERRA or altered recruitment of shelterin components can support telomerase-independent telomere maintenance.

genetics↗

bioRxiv2018

LEM domain proteins control the efficiency of adaptation through copy number variation

While loss of genome integrity is at the basis of numerous pathologies, including cancer, genome plasticity is necessary to adapt to a changing environment and thus is essential for long-term organismal success. Here we present data supporting a targeted mechanism that promotes adaptation to environmental stress by driving site-specific genome instability tied to transcriptional induction and the formation of RNA-DNA hybrids. Using an in vitro evolution assay we observe that the inner nuclear membrane LEM domain proteins Heh1 and Heh2 play antagonistic roles in inhibiting or promoting adaptation through copy number expansion, respectively, which is also reflected in their genetic interaction networks with genes responsible for transcription-dependent genome instability. Taken together, our data suggest the existence of a LEM domain protein-mediated mechanism by which an immediate transcriptional response to a changing environment drives targeted genome instability to promote increased variation on which selection can act to support long-term adaptation.

genetics↗

bioRxiv2017

Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5 ends of a DSB. For example, loss of 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that \"rewiring\" of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

genetics↗