We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa ...MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G
2
/M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.
Specific knock-down of gene expression by RNA interference is the method of choice to study gene function in human cells. A uniquely detailed phenotypic readout of such hypomorphs is possible by live ...cell microscopy of appropriate fluorescent reporter proteins. Here, I will present a fully automated method for RNAi screens in cultured human cells, combining reverse transfection by siRNA cell arrays, automated time-lapse fluorescence microscopy and computational phenotype analysis by image processing. The strategy is illustrated using an automatically scored mitosis assay and provides an easily scalable platform that we currently use in genome-wide RNAi screens for several cellular functions
PDF
