[Show abstract][Hide abstract] ABSTRACT: Patients with lung tumors harboring activating mutations in the EGF receptor (EGFR) show good initial treatment responses to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. However, acquired resistance invariably develops. Applying a focused shRNA screening approach to identify genes whose knockdown can prevent and/or overcome acquired resistance to erlotinib in several EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, we identified casein kinase 1 alpha (CSNK1A1, CK1 alpha). We found that CK1 alpha suppression inhibits the NF-kappaB pro-survival signaling pathway. Furthermore, down-regulation of NF-kappaB signaling by approaches independent of CK1 alpha knockdown can also attenuate acquired erlotinib resistance, supporting a role for activated NF-kappaB signaling in conferring acquired drug resistance. Importantly, CK1 alpha suppression prevented erlotinib resistance in an HCC827 xenograft model in vivo. Our findings suggest that patients with EGFR-mutant NSCLC might benefit from a combination of EGFR TKIs and CK1 alpha inhibition to prevent acquired drug resistance and prolong disease-free survival.
[Show abstract][Hide abstract] ABSTRACT: The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for 'chromothripsis' in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.
[Show abstract][Hide abstract] ABSTRACT: Positioned nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we have generated the first genome-wide nucleosome positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome wide. Using this resource, we found surprising differences from the previously published nucleosome organization of the distantly related yeast Saccharomyces cerevisiae. DNA sequence guides nucleosome positioning differently: for example, poly(dA-dT) elements are not enriched in S. pombe nucleosome-depleted regions. Regular nucleosomal arrays emanate more asymmetrically-mainly codirectionally with transcription-from promoter nucleosome-depleted regions, but promoters harboring the histone variant H2A.Z also show regular arrays upstream of these regions. Regular nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs and requires a remodeler, Mit1, that is conserved in humans but is not found in S. cerevisiae. Nucleosome positioning mechanisms are evidently not universal but evolutionarily plastic.
[Show abstract][Hide abstract] ABSTRACT: The majority of nuclear eukaryotic DNA is packaged into nucleosome cores where DNA is wrapped tightly around histone protein octamers. Such histone bound nucleosomal DNA is less accessible than the short linker DNA between nucleosome cores or the DNA in extended nucleosome free regions. Therefore, the positions of nucleosomes relative to a DNA sequence feature, like a transactivator binding site, a transcriptional start site or an origin of replication, can have profound effects on nuclear processes like transcription, replication, recombination and repair. Now that many DNA related processes are studied in a genome-wide manner, it is increasingly important to map the basic organization of their chromosomal DNA substrate, i.e., the positions of nucleosomes, on a genome-wide scale as well. To this end, the protection of nucleosomal DNA from digestion with micrococcal nuclease (MNase) is used as an assay for the presence of a nucleosome. The MNase protected DNA fragments, so called mononucleosomal DNA, can be mapped genome-wide by hybridization to microarrays. This method has been established for Saccharomyces cerevisiae, and we present here the adaptation of the method for Schizosaccharomyces pombe. As an independent method to validate genome-wide data for individual loci, we also include a protocol for the determination of locus specific nucleosome positioning by indirect end labeling.