RESUMEN
ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.
Asunto(s)
Replicación del ADN/genética , ADN/genética , G-Cuádruplex , Heterocromatina/genética , Proteína Nuclear Ligada al Cromosoma X/genética , Células Cultivadas , Secuenciación de Inmunoprecipitación de Cromatina/métodos , ADN/química , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Inestabilidad Genómica/genética , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Conformación de Ácido Nucleico , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
The heat shock protein GRP78 typically resides in the endoplasmic reticulum in normal tissues, but it has been shown to be expressed on the cell surface of several cancer cells, and some stem cells, where it can act as a signaling molecule by not-yet-fully defined mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that sGRP78 can function in a cell-context dependent manner, with a diverse array of reported binding partners, to regulate a variety of cellular responses. We had previously shown that sGRP78 was important in regulating pluripotent stem cell (PSC) functions, and hypothesized that embryonic-like mechanisms of GRP78 were critical to regulating aggressive breast cancer cell functions. Here, using proteomics we identify Dermcidin (DCD) as a novel sGRP78 binding partner common to both PSCs and breast cancer cells. We show that GRP78 and DCD cooperate to regulate stem cell and cancer cell migration that is dependent on the cell surface functions of these proteins. Finally, we identify Wnt/ß-catenin signaling, a critical pathway in stem cell and cancer cell biology, as an important downstream intermediate in regulating this migration phenotype.
Asunto(s)
Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico/metabolismo , Péptidos/metabolismo , Vía de Señalización Wnt , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Femenino , Humanos , Células Madre/metabolismoRESUMEN
Cancer-derived iPSCs have provided valuable insight into oncogenesis, but human cancer cells can often be difficult to reprogram, especially in cases of complex genetic abnormalities. Here we report, to our knowledge, the first successful generation of an iPSC line from a human immortalized acute myeloid leukemia (AML) cell line, the cell line HL-60. This iPSC line retains a majority of the leukemic genotype and displays defects in myeloid differentiation, thus providing a tool for modeling and studying AML.