Repair of DNA lesions must occur within the chromatin landscape and is associated with alterations in histone modifications and nucleosome rearrangement. To directly associate these chromatin features with DNA damage and repair, it is necessary to be able to map DNA adducts. We have developed a cyclobutane pyrimidine dimer (CPD)-specific immunoprecipitation method and mapped ultraviolet damage hotspots across human chromosomes 1 and 6. CPD hotspots occur almost equally in genic and intergenic regions. However, these hotspots are significantly more prevalent adjacent to repeat elements, especially Alu repeats. Nucleosome mapping studies indicate that nucleosomes are consistently positioned at Alu elements where CPD hotspots form, but by 2 h post-irradiation, these same regions are significantly depleted of nucleosomes. These results indicate that nucleosomes associated with hotspots of CPD formation are readily rearranged, potentially making them accessible to DNA repair machinery. Our results represent the first chromosome scale map of ultraviolet-induced DNA lesions in the human genome, and reveal the sequence features and dynamic chromatin changes associated with CPD hotspots.

Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection IMPORTANCEOur previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.

Mechanical signals regulate adipogenic differentiation of mesenchymal stem cells (MSCs). Critical to the mechano-regulation of MSCs, Linker of the Nucleoskeleton and Cytoskeleton (LINC) complexes are integral to both nucleo-cytoskeletal signal transduction and structural integrity of the nucleus. The LINC complex is made of Nesprin proteins that associate with the cytoskeleton on the outer nuclear membrane (ONM) and Sun proteins that bound to nuclear lamina and chromatin at the inner nuclear membrane (INM). In addition to their role in the LINC complex function, depletion of Sun1/2 effects chromosomal tethering to the nuclear envelope, nuclear morphology, and chromatin organization. Suggesting that Sun1/2 proteins may regulate chromatin organization and adipogenic differentiation independent of the LINC complex mediated nucleo-cytoskeletal connectivity. To test this hypothesis Sun1/2 depletion was compared to expression of a dominant-negative KASH (dnKASH) domain to decouple nucleus from cytoskeleton by inhibiting Nesprin-SUN association. Sun1/2 depletion inhibited fat droplet formation and production of adipogenic proteins such as Adipoq, which were supported by RNA-seq showing decreased adipogensis. In contrast dnKASH responded oppositely, increasing fat droplet formation, Adipoq and adipogenic gene expression. At the chromatin level, Sun1/2 depletion increased H3K9me3 levels, increased H3K9me3 foci count, and enrichment on Adipoq. No increase of H3K9me3 levels, foci count, or increased H3K9me3 enrichment on Adipoq was found during dnKASH expression. We conclude that physically decoupling of the LINC complex via dnKASH accelerates adipogenesis and that depletion of Sun1/2 increases heterochromatin accrual and inhibits adipogenesis independent of the LINC complex function.

This report describes the development of a novel dual color Southern protocol to visualize two distinct genomes or genic regions simultaneously on a single Southern blot. The blot is developed with IRDye-conjugated antibody (Ab) and streptavidin that recognize Digoxigenin- (Dig) or biotin-labeled probes, respectively and visualized on an infrared imager. This protocol was validated by visualizing viral and host genomes of human cytomegalovirus (HCMV)-infected human fibroblasts. This technique utilizes extremely sensitive fluorescent imaging, allowing the detection of nanogram quantities of DNA, as opposed to microgram quantities needed in Southerns using radioactively labeled probes, and eliminates the inherent loss in signal after stripping and reprobing a Southern blot. The probes are labeled with non-radioactive Dig and biotin and can be stored for extended periods of time. This protocol will aid in studies of any system with two genomes, such as cells infected with numerous types of microorganisms (virus/parasites/bacteria), or studies of mitochondrial and nuclear DNA within the same cells.

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM ؊ lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM ؊ lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM ؊ line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM ؊ cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. T he human cytomegalovirus (HCMV) life cycle involves a complex interplay between the virus and the host, with the virus exploiting the host cellular machinery for many of its own functions and, ultimately, releasing fully infectious virions. During a permissive HCMV infection, after virions have entered the cell, the tegument proteins and virus genome are independently trafficked to the nucleus. In fibroblasts, large bipolar viral replication centers (RCs) are formed within 48 h postinfection (hpi) and certain host cellular proteins become strongly associated with these RCs (1; reviewed in reference 2). These proteins include the regulatory protein p53 (3), as well as numerous components of the host cellular DNA damage response (DDR) and repair pathways (4-8).Many virus infections affect the DDR. The interactions span a range of up-and downregulations and include a complex dynamic between the virus and its host's damage response (as reviewed in references 6 and 9). Some viruses appear to require DDR proteins for efficient replication (10, 11), while for other viruses an efficient DDR can be detrimental to their DNA replication (12-21). Studies from several labs, including our own, have shown that HCMV infection initiates the ataxia telangiectasia mutated (ATM)-dependent double-strand break (DSB) ...

Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, [alpha]UVssDNA-1 (mAb C3B6), recognizing the thymidine(6-4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure-function studies and application to human investigations.

We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV.