RESUMO
ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.
Assuntos
DNA de Neoplasias/metabolismo , DNA Ribossômico/metabolismo , Dosagem de Genes , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteína Nuclear Ligada ao X/metabolismo , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , DNA Ribossômico/genética , Instabilidade Genômica , Humanos , Naftiridinas/farmacologia , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteína Nuclear Ligada ao X/genéticaRESUMO
Human ALT cancers show high mutation rates in ATRX and DAXX. Although it is well known that the absence of ATRX/DAXX disrupts H3.3 deposition at heterochromatin, its impact on H3.3 deposition and post-translational modification in the global genome remains unclear. Here, we explore the dynamics of phosphorylated H3.3 serine 31 (H3.3S31ph) in human ALT cancer cells. While H3.3S31ph is found only at pericentric satellite DNA repeats during mitosis in most somatic human cells, a high level of H3.3S31ph is detected on the entire chromosome in ALT cells, attributable to an elevated CHK1 activity in these cells. Drug inhibition of CHK1 activity during mitosis and expression of mutant H3.3S31A in these ALT cells result in a decrease in H3.3S31ph levels accompanied with increased levels of phosphorylated H2AX serine 139 on chromosome arms and at the telomeres. Furthermore, the inhibition of CHK1 activity in these cells also reduces cell viability. Our findings suggest a novel role of CHK1 as an H3.3S31 kinase, and that CHK1-mediated H3.3S31ph plays an important role in the maintenance of chromatin integrity and cell survival in ALT cancer cells.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Proteínas Quinases/metabolismo , Western Blotting , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quinase 1 do Ponto de Checagem , Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Células HT29 , Células HeLa , Histonas/genética , Humanos , Microscopia de Fluorescência , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/genética , Serina/metabolismo , Telômero/genética , Telômero/metabolismo , Proteína Nuclear Ligada ao XRESUMO
We have previously shown that α-thalassemia mental retardation X-linked (ATRX) and histone H3.3 are key regulators of telomeric chromatin in mouse embryonic stem cells. The function of ATRX and H3.3 in the maintenance of telomere chromatin integrity is further demonstrated by recent studies that show the strong association of ATRX/H3.3 mutations with alternative lengthening of telomeres in telomerase-negative human cancer cells. Here, we demonstrate that ATRX and H3.3 co-localize with the telomeric DNA and associated proteins within the promyelocytic leukemia (PML) bodies in mouse ES cells. The assembly of these telomere-associated PML bodies is most prominent at S phase. RNA interference (RNAi)-mediated knockdown of PML expression induces the disassembly of these nuclear bodies and a telomere dysfunction phenotype in mouse ES cells. Loss of function of PML bodies in mouse ES cells also disrupts binding of ATRX/H3.3 and proper establishment of histone methylation pattern at the telomere. Our study demonstrates that PML bodies act as epigenetic regulators by serving as platforms for the assembly of the telomeric chromatin to ensure a faithful inheritance of epigenetic information at the telomere.