HDAC1,2 inhibition impairs EZH2- and BBAP-mediated DNA repair to overcome chemoresistance in EZH2 gain-of-function mutant diffuse large B-cell lymphoma.

Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor - B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells.

Dioxin exposure blocks lactation through a direct effect on mammary epithelial cells mediated by the aryl hydrocarbon receptor repressor.

In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene ß-casein. As TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition.

Phosphorylation of Kaposi's sarcoma-associated herpesvirus processivity factor ORF59 by a viral kinase modulates its ability to associate with RTA and oriLyt.

ORF59 of Kaposi's sarcoma-associated herpesvirus (KSHV) plays an essential role in viral lytic replication by providing DNA processivity activity to the viral DNA polymerase (ORF9). ORF59 forms a homodimer in the cytoplasm and binds and translocates ORF9 into the nucleus, where it secures ORF9 to the origin of lytic DNA replication (oriLyt) in order to synthesize long DNA fragments during replication. ORF59 binds to oriLyt through an immediate early protein, replication and transcription activator (RTA). Here, we show that viral kinase (ORF36) phosphorylates serines between amino acids 376 and 379 of ORF59 and replacement of the Ser378 residue with alanine significantly impairs phosphorylation. Although mutating these serine residues had no effect on binding between ORF59 and ORF9, viral polymerase, or ORF36, the viral kinase, it significantly reduced the ability of ORF59 to bind to RTA. The results for the mutant in which Ser376 to Ser379 were replaced by alanine showed that both Ser378 and Ser379 contribute to binding to RTA. Additionally, the Ser376, Ser378, and Ser379 residues were found to be critical for binding of ORF59 to oriLyt and its processivity function. Ablation of these phosphorylation sites reduced the production of virion particles, suggesting that phosphorylation is critical for ORF59 activity and viral DNA synthesis.

Kaposi's sarcoma-associated herpesvirus-encoded LANA recruits topoisomerase IIß for latent DNA replication of the terminal repeats.

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays a major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. TR elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent genes. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified topoisomerase IIß (TopoIIß) as a LANA-interacting protein. Here, we show that TopoIIß forms complexes with LANA that colocalize as punctuate bodies in the nucleus of KSHV-infected cells. The specific TopoIIß binding region of LANA has been identified to its N terminus and the first 32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TopoIIß with LANA. TopoIIß plays an important role in LANA-dependent latent DNA replication, as addition of ellipticine, a selective inhibitor of TopoII, negatively regulated replication mediated by the TR. DNA break labeling and chromatin immunoprecipitation assay using biotin-16-dUTP and terminal deoxynucleotide transferase showed that TopoIIß mediates a transient DNA break on viral DNA. These studies confirm that LANA recruits TopoIIß at the origins of latent replication to unwind the DNA for replication.

Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication.

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences.