Frank Hänel

LINC, a human complex that is related to pRB-containing complexes in invertebrates regulates the expression of G2/M genes.

Here we report the identification of the LIN complex (LINC), a human multiprotein complex that is required for transcriptional activation of G2/sub>/M genes. LINC is related to the recently identified dREAM and DRM complexes of Drosophila and C.elegans that contain homologues of the mammalian retinoblastoma tumor suppressor protein. The LINC core complex consists of at least five subunits including the chromatin-associated LIN-9 and RbAp48 proteins. LINC dynamically associates with pocket proteins, E2F and B-MYB during the cell cycle. In quiescent cells, LINC binds to p130 and E2F4. During cell cycle entry, E2F4 and p130 dissociate and LINC switches to B-MYB and p107. Chromatin Immunoprecipitation experiments demonstrate that LINC associates with a large number of E2F-regulated promoters in quiescent cells. However, RNAi experiments reveal that LINC is not required for repression. In S phase, LINC selectively binds to the promoters of G2/sub>/M genes whose products are required for mitosis and plays an important role in their cell cycle dependent activation.

Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development

ABSTRACT The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1s function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

Characterization of the interaction between the human DNA topoisomerase IIβ-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein

TopBP1 (topoisomerase IIbeta-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G(1)/S boundary of cell cycle; only weak interaction could be found at the G(2)/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.

Chlamydia psittaci: New insights into genomic diversity, clinical pathology, host–pathogen interaction and anti-bacterial immunity

The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen.

The DNA topoisomerase IIβ binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)†

We investigated the physical association of the DNA topoisomerase IIβ binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP‐ribose) polymerase‐1 (PARP‐1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C‐terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP‐1 in vitro containing the DNA‐binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP‐1 proteins could be shown in HeLa‐S3 cells by co‐immunoprecipitation. TopBP1 and PARP‐1 are localized within overlapping regions in the nucleus of HeLa‐S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP‐ribose) (PAR) synthesis takes place and is ADP‐ribosylated by PARP‐1. Finally, cellular (ADP‐ribosyl)ating activity impairs binding of TopBP1 to Myc‐interacting zinc finger protein‐1 (Miz‐1). The results indicate an influence of post‐translational modifications of TopBP1 on its function during DNA repair. J. Cell. Biochem. 102: 171–182, 2007.

Chlamydial protease CT441 interacts with SRAP1 co-activator of estrogen receptor α and partially alleviates its co-activation activity

Chlamydiae are obligate intracellular pathogens which secrete host-interactive proteins capable of directly modulating eukaryotic pathways. Using the PDZ domain of the protease CT441 of Chlamydia trachomatis as a bait in a yeast two-hybrid screen, we identified the SRAP1 co-activator of estrogen receptor alpha (ERalpha) as an interacting protein. SRAP1 is a unique modulator of steroid receptor activity, as it is able to mediate its co-regulatory effects both as a RNA and a protein. GST pull-down experiments confirmed the interaction of CT441 and SRAP1 in vitro. Furthermore, it was shown that the CT441-PDZ domain fused to a nuclear localization signal was able to bind and to target SRAP1 to the nucleus in mammalian cells. CT441 did not cleave SRAP1, but retained the protein in the cytoplasm and thereby partially alleviated its co-activation of ERalpha in a heterologous yeast system and in mammalian cells. Possible implications of chlamydial regulation of host metabolism by targeting ERalpha activity are discussed. Moreover, the property of CT441-PDZ domain to specifically sequester SRA1 protein but not SRA1 RNA may be used to distinguish between the cellular functions of the SRA1 RNA and protein. This has clinical relevance as it has been proposed that disturbance of the balance between SRAP1-coding and non-coding SRA1 RNAs in breast tumor tissues might be involved in breast tumorigenesis.

Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation.

Proteomic identification of PSF and p54(nrb) as topBP1-interacting proteins

TopBP1 is a BRCT domain‐rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1‐interacting proteins by co‐immunoprecipitation assays and LC‐ESI‐MS‐analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull‐down assays, co‐immunoprecipitations and by yeast two‐hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double‐strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 200 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair. J. Cell. Biochem. 113: 1744–1753, 2012.

Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIβ binding protein 1

Topoisomerase IIβ binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy‐terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP‐ribose) polymerase 1 (PARP‐1), where the latter interaction is responsible for the poly(ADP‐ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 Å. The crystal structure reveals a degenerate phospho‐peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho‐peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho‐peptide binding domain. We further provide insight into poly(ADP‐ribose) binding and sites of potential modification by PARP‐1.

Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Chlamydia (C.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. During a unique developmental cycle of this obligate intracellular pathogen, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. C. psittaci uses dynein motor proteins for optimal early development. Chlamydial proteins that mediate this process are unknown. Two-hybrid screening with the C. psittaci inclusion protein IncB as bait against a HeLa Yeast Two-hybrid (YTH) library revealed that the host protein Snapin interacts with IncB. Snapin is a cytoplasmic protein that plays a multivalent role in intracellular trafficking. Confocal fluorescence microscopy using an IncB-specific antibody demonstrated that IncB, Snapin, and dynein were co-localized near the inclusion of C. psittaci-infected HEp-2 cells. This co-localization was lost when Snapin was depleted by RNAi. The interaction of Snapin with both IncB and dynein has been shown in vitro and in vivo. We propose that Snapin connects chlamydial inclusions with the microtubule network by interacting with both IncB and dynein.