Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave ...concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components—the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33—unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.
Mastermind (Mam) is one of the elements of Notch signaling, an ancient system that plays a pivotal role in metazoan development. Genetic analyses in Drosophila and Caenorhabditis elegans have shown ...Mam to be an essential positive regulator of this signaling pathway in these species. Mam proteins bind to and stabilize the DNA-binding complex of the intracellular domains of Notch and CBF-1, Su(H), Lag-1 (CSL) DNA-binding proteins in the nucleus. Mammals have three Mam proteins, which show remarkable similarities in their functions while having an unusual structural diversity. There have also been recent indications that Mam-1 functionally interacts with other transcription factors including p53 tumor suppressor. We herein describe that Mam-1 deficiency in mice abolishes the development of splenic marginal zone B cells, a subset strictly dependent on Notch2, a CSL protein and Delta1 ligand. Mam-1 deficiency also causes a partially impaired development of early thymocytes, while not affecting the generation of definitive hematopoiesis, processes that are dependent on Notch1. We also demonstrate the transcriptional activation of a target promoter by constitutively active forms of Notch to decrease severalfold in cultured Mam-1-deficient cells. These results indicate that Mam-1 is thus required to some extent for Notch-dependent stages in lymphopoiesis, thus supporting the notion that Mam is an essential component of the canonical Notch pathway in mammals.
Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. ...This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known
Drosophila
insulators. To further study functioning of the Su(Hw)-dependent complexes, we used the previously described
su(Hw)
E8
mutation with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)
E8
protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)
E8
protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein–protein interactions that are stabilized by Mod(mdg4)-67.2.
Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact ...that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.
Chromatin architecture is critical for the temporal and tissue-specific activation of genes that determine eukaryotic development. The functional interaction between enhancers and promoters is ...controlled by insulators and tethering elements that support specific long-distance interactions. However, the mechanisms of the formation and maintenance of long-range interactions between genome regulatory elements remain poorly understood, primarily due to the lack of convenient model systems.
became the first model organism in which architectural proteins that determine the activity of insulators were described. In
, one of the best-studied DNA-binding architectural proteins, Su(Hw), forms a complex with Mod(mdg4)-67.2 and CP190 proteins. Using a combination of CRISPR/Cas9 genome editing and
-dependent integration technologies, we created a model system in which the promoters and enhancers of two reporter genes are separated by 28 kb. In this case, enhancers effectively stimulate reporter gene promoters in
and
only in the presence of artificial Su(Hw) binding sites (SBS), in both constructs. The expression of the mutant Su(Hw) protein, which cannot interact with CP190, and the mutation inactivating Mod(mdg4)-67.2, lead to the complete loss or significant weakening of enhancer-promoter interactions, respectively. The results indicate that the new model system effectively identifies the role of individual subunits of architectural protein complexes in forming and maintaining specific long-distance interactions in the
model.
To better understand the molecular basis for the cytotoxic effects of suramin, we have developed suramin-resistant DC-3F/SU 1000 cells by continuous exposure of fibrosarcoma cells to increasing ...concentrations of suramin. The suramin resistance (approximately 10-fold) is not associated with changes in uptake or intracellular distribution of the drug. The sensitivity to actinomycin D, cytarabine, aphidicolin, hydroxyurea, vincristine, and 5-fluorouracil is unaltered. In contrast, DC-3F/SU 1000 cells are about 2-fold resistant to classical DNA topoisomerase II inhibitors such as doxorubicin, amsacrine, and etoposide, whereas the cells are 1.5-fold more sensitive to the topoisomerase I inhibitor camptothecin. The cross-resistance to topoisomerase II inhibitors occurred earlier than the collateral sensitivity to camptothecin. Amsacrine- and etoposide-induced DNA-protein complex formation is reduced about 2-fold in DC-3F/SU 1000 cells, compared with DC-3F cells, whereas camptothecin-induced DNA-protein complex formation is increased 1.5-fold. Western blot analysis of cellular lysates from the two cell lines shows no significant differences in the level of topoisomerase II, whereas the level of topoisomerase I is increased 2.5-fold in DC-3F/SU 1000 cells. The catalytic activities of topoisomerases I and II in nuclear extracts from DC-3F/SU 1000 cells are both about 2-fold higher than those in extracts from DC-3F cells, whereas amsacrine- and etoposide-induced DNA-protein complex formation is comparable between the two cell lines. Taken together, our results support the involvement of DNA topoisomerases in the cytotoxic activity of suramin. We further believe that the DC-3F/SU 1000 cells may be a useful model for the elucidation of factors that lead to low, clinically relevant, levels of resistance to topoisomerase II inhibitors.
DREF was first characterized for its role in the regulation of transcription of genes encoding proteins involved in DNA replication and found to interact with sequences similar to the DNA recognition ...motif of the BEAF-32 insulator protein. Insulators are DNA-protein complexes that mediate intra- and inter-chromosome interactions. Several DNA-binding insulator proteins have been described in Drosophila, including BEAF-32, dCTCF and Su(Hw). Here we find that DREF and BEAF-32 co-localize at the same genomic sites, but their enrichment shows an inverse correlation. Furthermore, DREF co-localizes in the genome with other insulator proteins, suggesting that the function of this protein may require components of Drosophila insulators. This is supported by the finding that mutations in insulator proteins modulate DREF-induced cell proliferation. DREF persists bound to chromatin during mitosis at a subset of sites where it also co-localizes with dCTCF, BEAF-32 and CP190. These sites are highly enriched for sites where Orc2 and Mcm2 are present during interphase and at the borders of topological domains of chromosomes defined by Hi-C. The results suggest that DREF and insulator proteins may help maintain chromosome organization during the cell cycle and mark a subset of genomic sites for the assembly of pre-replication complexes and gene bookmarking during the M/G
1
transition.
Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA ...physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.
Insulators are DNA‐protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific ...subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190‐binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190‐interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190‐binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA‐binding proteins that form hetero‐oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer‐blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.
Synopsis
CP190 is a common component of various Drosophila insulator complexes, but its role at genomic binding sites not shared with other known insulator factors is unclear. This study now identifies two new proteins that interact and cooperate with CP190 in insulator function.
Characterization of CP190 interactors identifies Ibf1 and Ibf2 as novel insulator binding proteins.
Ibf1 and Ibf2 are necessary for enhancer‐blocking function of Drosophila insulators.
Chromatin binding of the Ibf1/Ibf2 complex involves recognition of a novel DNA motif.
Recruitment of the CP190 insulator protein to a subset of its chromatin binding sites depends on Ibf1/Ibf2.
Two new factors join the ranks of fly insulator proteins that cooperate with CP190, helping to understand CP190 action at genomic binding sites not shared with other known insulator proteins.
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