Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.
Rabindranath De La Fuente, Claudia Baumann and Maria M Viveiros
Xiaotian Wang, Claudia Baumann, Rabindranath De La Fuente and Maria M Viveiros
Acentriolar microtubule-organizing centers (aMTOCs) play a critical role in stable meiotic spindle assembly in oocytes, necessary for accurate chromosome segregation. Yet, there is a limited understanding of the essential regulatory components of these unique MTOCs. In somatic cells, CEP215 (Centrosomal Protein 215) serves as an important regulator of centrosome maturation and spindle organization. Here, we assessed whether it has a similar function in mouse oocytes. CEP215 was detected in oocyte lysates and specifically localized to aMTOCs throughout the progression of meiosis in a pericentrin-dependent manner. Super-resolution microscopy revealed CEP215 co-localization with pericentrin and a unique pore/ring-like structural organization of aMTOCs. Interestingly, inhibition of Aurora Kinase A in either MI or MII-stage oocytes resulted in a striking loss of the ring-like aMTOC organization and pronounced CEP215 clustering at spindle poles, as well as shorter spindles with highly focused poles. In vitro siRNA-mediated transcript knockdown effectively reduced CEP215 in approximately 85% of the oocytes. Maturation rates to MII were similar in the Cep215 siRNA and injected controls; however, a high percentage (~40%) of the Cep215-knockdown oocytes showed notable variations in spindle pole focusing. Surprisingly, pericentrin and γ-tubulin localization and fluorescence intensity at aMTOCs were unaltered in knockdown oocytes, contrasting with mitotic cells where CEP215 depletion reduced γ-tubulin at centrosomes. Our results demonstrate that CEP215 is a functional component of oocyte aMTOCs and participates in the regulation of meiotic spindle pole focusing. Moreover, these studies reveal a vital role for Aurora Kinase A activity in the maintenance of aMTOC organization in oocytes.
Luhan Yang, Claudia Baumann, Rabindranth De La Fuente and Maria M Viveiros
Accurate chromosome segregation relies on correct chromosome-microtubule interactions within a stable bipolar spindle apparatus. Thus, exposure to spindle disrupting compounds can impair meiotic division and genomic stability in oocytes. The endocrine disrupting activity of bisphenols such as bisphenol A (BPA) is well recognized, yet their damaging effects on spindle microtubules (MTs) is poorly understood. Here, we tested the effect(s) of acute exposure to BPA and bisphenol F (BPF) on assembled spindle stability in ovulated oocytes. Brief (4 h) exposure to increasing concentrations (5, 25, and 50 µg/mL) of BPA or BPF disrupted spindle organization in a dose-dependent manner, resulting in significantly shorter spindles with highly unfocused poles and fragmented pericentrin. The chromosomes remained congressed in an abnormally elongated metaphase-like configuration, yet normal end-on chromosome-MT attachments were reduced in BPF-treated oocytes. Live-cell imaging revealed a rapid onset of bisphenol-mediated spindle MT disruption that was reversed upon compound removal. Moreover, MT stability and regrowth were impaired in BPA-exposed oocytes, with few cold-stable MTs and formation of multipolar spindles upon MT regrowth. MT-associated kinesin-14 motor protein (HSET/KIFC1) labeling along the spindle was also lower in BPA-treated oocytes. Conversely, cold stable MTs and HSET labeling persisted after BPF exposure. Notably, inhibition of Aurora Kinase A limited bisphenol-mediated spindle pole widening, revealing a potential interaction. These results demonstrate rapid MT disrupting activity by bisphenols, which is highly detrimental to meiotic spindle stability and organization. Moreover, we identify an important link between these defects and altered distribution of key spindle associated factors as well as Aurora Kinase A activity.