The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in threedimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin. INTRODUCTIONThe nuclear mitotic apparatus protein (NuMA) was first described in 1980 (Lydersen and Pettijohn, 1980) and observed to concentrate at the spindle poles during mitosis. Subsequently NuMA, variously named SPN antigen, p240 antigen, centrophilin, 1F1/1H1 antigen, SP-H antigen, and W1 antigen (Compton et al., 1991(Compton et al., , 1992Kallajoki et al., 1991Kallajoki et al., , 1993Maekawa et al., 1991;Tousson et al., 1991;Yang et al., 1992;Maekawa and Kuriyama, 1993;Tang et al., 1993), was shown to be a critical player in the formation and stabilization of the mitotic spindle, notably by associating with the minus end of spindle microtubules and interacting with dynein, dynactin, and LGN (Compton and Cleveland, 1993;Gaglio et al., 1995;Merdes et al., 2000;Du et al., 2001;Gehmlich et al., 2004). In addition to its location at the poles of the mitotic spindle, NuMA has been reported in the nucleus of both cycling and growth-arrested cells, as shown by immunostaining of cells in culture and tissue biopsy sections (Lelièvre et al., 1998;Merdes and Cleveland 1998;Gribbon et al., 2002;Taimen et al., 2004). However, a role for NuMA in interphase remains to be determined. The hypothesis that NuMA might organize nuclear structure (Compton and Cleveland, 1994) is supported by the existence of a long central coiled-coil region in the protein and the prevalence of NuMA in the nucleus upon detergent extraction. Furthermore, NuMA binds DNA matrix attachment regions (MARs) in vitro (Ludérus et al., 1994), and the cleavage of NuMA precedes DNA degradation during apoptosis (Weaver et al., 1996). The likelihood of a link...

The remodeling of nuclear organization during differentiation and the dramatic alteration of nuclear organization associated with cancer development are well documented. However, the importance of tissue architecture in the control of nuclear organization remains to be determined. Differentiation of mammary epithelial cells into functional tissue structures, in three-dimensional culture, is characterized by a specific tissue architecture (i.e. a basoapical polarity axis), cell cycle exit and maintenance of cell survival. Here we show that induction of partial differentiation (i.e. basal polarity only, cell cycle exit and cell survival) by epigenetic mechanisms in malignant breast cells is sufficient to restore features of differentiation-specific nuclear organization, including perinucleolar heterochromatin, large splicing factor speckles, and distinct nuclear mitotic apparatus protein (NuMA) foci. Upon alteration of nuclear organization using an antibody against NuMA, differentiated non-neoplastic cells undergo apoptosis, whereas partially differentiated malignant cells enter the cell cycle. Non-neoplastic cells cultured under conditions that prevent the establishment of apical polarity also enter the cell cycle upon NuMA antibody treatment. These findings demonstrate that the differentiation status rather than the non-neoplastic or neoplastic origin of cells controls nuclear organization and suggest a link between nuclear organization and epigenetic mechanisms dictated by tissue architecture for the control of cell behavior.

Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.

As part of the ongoing key comparison BIPM.QM-K1, a comparison has been performed between the ozone national standard of the Instituto de Salud Carlos III (ISCIII) and the common reference standard of the key comparison, maintained by the Bureau International des Poids et Mesures (BIPM). The instruments have been compared over a nominal ozone amount-of-substance fraction range of 0 nmol/mol to 500 nmol/mol. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).