Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.
The tumor suppressor protein p16 INK4a (p16) is a well‐established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy–lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress‐response proteins and has also been shown to attenuate age‐related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time‐lapse fluorescence microscopy using an endogenous p16‐mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3‐II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age‐related diseases.
Genome-wide association studies (GWAS) have identified over 100 loci associated with osteoarthritis (OA) risk, but the majority of OA risk variants are non-coding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multi-omic approach and genome editing to identify and functionally characterize potential OA risk genes. Computational analysis of GWAS and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for OA risk variants. We constructed a chondrocyte specific regulatory network by mapping 3D chromatin structure and active enhancers in human chondrocytes. We then intersected these data with our previously collected RNA-seq dataset of chondrocytes responding to fibronectin fragment (FN-f), a known OA trigger. Integration of the three genomic datasets with recently reported OA GWAS variants revealed a refined set of putative causal OA variants and their potential target genes. One of the putative target genes identified was SOCS2, which was connected to a putative causal variant by a 170 Kb loop and is differentially regulated in response to FN-f. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from three independent donors led to heightened expression of inflammatory markers after FN-f treatment. These data suggest that SOCS2 plays a role in resolving inflammation in response to cartilage matrix damage and provides a possible mechanistic explanation for its influence on OA risk. In total, we identified 56 unique putative OA risk genes for further research and potential therapeutic development.
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