ABSTRACT
Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.
Subject(s)
DNA Helicases/genetics , Dyskeratosis Congenita/genetics , Fetal Growth Retardation/genetics , Intellectual Disability/genetics , Microcephaly/genetics , Mutation , Telomere/genetics , Animals , Base Sequence , Blotting, Western , Cell Proliferation , Cells, Cultured , DNA Helicases/metabolism , Dyskeratosis Congenita/metabolism , Dyskeratosis Congenita/pathology , Family Health , Female , Fetal Growth Retardation/metabolism , Fetal Growth Retardation/pathology , Gene Expression , Genomic Instability/genetics , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Intellectual Disability/metabolism , Intellectual Disability/pathology , Male , Mice , Microcephaly/metabolism , Microcephaly/pathology , Pedigree , Reverse Transcriptase Polymerase Chain Reaction , Telomere Shortening/genetics , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/metabolismABSTRACT
Telomeres play crucial roles in the maintenance of genome integrity and control of cellular senescence. Most eukaryotic telomeres can be transcribed to generate a telomeric repeat-containing RNA (TERRA) that persists as a heterogeneous nuclear RNA and can be developmentally regulated. However, the precise function and regulation of TERRA in normal and cancer cell development remains poorly understood. Here, we show that TERRA accumulates in highly proliferating normal and cancer cells, and forms large nuclear foci, which are distinct from previously characterized markers of DNA damage or replication stress. Using a mouse model for medulloblastoma driven by chronic Sonic hedgehog (SHH) signaling, TERRA RNA was detected in tumor, but not adjacent normal cells using both RNA fluorescence in situ hybridization (FISH) and northern blotting. RNA FISH revealed the formation of TERRA foci (TERFs) in the nuclear regions of rapidly proliferating tumor cells. In the normal developing cerebellum, TERRA aggregates could also be detected in highly proliferating zones of progenitor neurons. SHH could enhance TERRA expression in purified granule progenitor cells in vitro, suggesting that proliferation signals contribute to TERRA expression in responsive tissue. TERRA foci did not colocalize with γH2AX foci, promyelocytic leukemia (PML) or Cajal bodies in mouse tumor tissue. We also provide evidence that TERRA is elevated in a variety of human cancers. These findings suggest that elevated TERRA levels reflect a novel early form of telomere regulation during replication stress and cancer cell evolution, and the TERRA RNA aggregates may form a novel nuclear body in highly proliferating mammalian cells.
Subject(s)
Cerebellar Neoplasms/pathology , Medulloblastoma/genetics , Medulloblastoma/pathology , Neural Stem Cells/metabolism , RNA/genetics , Repetitive Sequences, Nucleic Acid/genetics , Telomere/genetics , Animals , Brain/embryology , Brain/pathology , Cell Proliferation , Cerebellar Neoplasms/genetics , Coiled Bodies/metabolism , DNA Damage , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Hedgehog Proteins/pharmacology , Histones/metabolism , Humans , In Situ Hybridization, Fluorescence , Interphase , Mice , Models, Biological , Neural Stem Cells/pathologyABSTRACT
Cerebellum development depends on the correct differentiation of progenitors into neurons, a process controlled by a transcriptional program that remains poorly understood. Here we show that neural-specific deletion of the BTB/POZ zinc-finger transcription factor-encoding gene Rp58 (Znf238, Zfp238) causes severe cerebellar hypoplasia and developmental failure of Purkinje neurons, Bergmann glia and granule neurons. Deletion of Rp58 in mouse embryonic Atoh1(+) progenitors leads to strong defects in growth and foliation owing to its crucial role in the differentiation of granule neurons. Analysis of the Rp58 mutant at E14.5 demonstrates that Rp58 is required for the development of both glutamatergic and GABAergic neurons. Rp58 mutants show decreased proliferation of glutamatergic progenitors at E14.5. In addition, Rp58 ablation results in a reduced number of GABAergic Pax2(+) neurons at E16.5 together with defects in the transcriptional program of ventricular zone progenitors. Our results indicate that Rp58 is essential for the growth and organization of the cerebellum and regulates the development of both GABAergic and glutamatergic neurons.
