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To functionally characterize HP6, Greil et al


May 21, 2023

To functionally characterize HP6, Greil et al. Gal4-induced RNA interference system, we found that after depletion of Umbrea in salivary gland polytene chromosomes, they exhibit multiple telomeric fusions. Taken together, these results suggest JNJ-38877618 that Umbrea cooperates with Hip, HP1 and HOAP and plays a functional role in mediating normal telomere behaviour in does not require telomerase activity, and is JNJ-38877618 mainly provided by the attachment of terminal repeat retrotransposons, and and (HP1 protein contains an N-terminal chromo domain name. The chromo domain name mediates the association of HP1 and pericentric heterochromatin by binding the N-terminal tail of histone H3 methylated at lysine 9 (H3mLys9) (Bannister et al. 2001; Lachner et al. 2001). The C-terminal chromo shadow domain name forms a homodimer (Brasher et al. 2000) and interacts with some chromosomal proteins (Eissenberg and Elgin 2000; Li et al. 2002), including Hip (observe below and Schwendemann et al. 2008). JNJ-38877618 The hinge region links the chromo and the chromo shadow domains, is important for nuclear targeting (Smothers and Henikoff 2001), and affects HP1 protein interactions and chromosomal distributions (Badugu et al. 2005). Recently, we have recognized and characterized Hip, an HP1-interacting protein (Schwendemann et al. 2008). Hip and HP1 interact and haplo- and triplo-dosage mutations act as dominant suppressors of PEV. It has been shown that a multiprotein complex is required to establish a heterochromatin structure and that HP1, a key component of these complexes, interacts with a myriad of proteins (reviewed in Li et al. 2002). The results so far suggest that the HP1 protein functions as an adaptor bringing together different proteins in multiprotein complexes. Hence, in order to understand the mechanisms involved, identification of novel proteins that associate with heterochromatin would provide insight into the mechanisms of heterochromatin multiprotein function. Here, we characterize Umbrea, a Hip and HP1-interacting protein. The gene is identical to the recently described HP6 encoding gene (Greil et al. 2007). In this study, HP6 and three other proteins (HP3, HP4 and HP5) were characterized as four novel heterochromatin proteins. Greil et al. used the Interaction Database (Giot et al. 2003) to search for proteins that directly interact with HP1 and named these proteins HP3, HP4 (identical to Hip), and HP5. However, on the basis of information in the Interaction Database, Greil et al. propose that the fourth protein, HP6 (identical to Umbrea described here), does not directly bind HP1 but binds HP3, HP4, HP5 and HOAP. Although Greil et al. name the novel heterochromatin protein HP6, we refer to the official FlyBase name for the gene that we describe and characterize in this report. Umbrea contains only a chromo shadow domain. Hip, HP1 and Umbrea can be co-immunoprecipitated from nuclear extracts. Umbrea directly binds to HP1 and Hip cause telomeric associations of polytene chromosomes. This finding suggests a function of Umbrea in telomere protection in wild type. The mechanisms of telomere protection in are not well understood. The characterization of Umbrea will contribute to the understanding of telomeres. Materials and methods Cloning of (coding for Umbrea protein) was amplified by PCR using total genomic DNA from adult and primers umbrea-fwdfragment described above. The His-tagged Umbrea recombinant protein was expressed in BL21(DE3) cells JNJ-38877618 using construct pET21c-umbrea. The protein was purified using Ni-NTA columns (Novagen) and standard procedures. The eluate fraction was used as an immunogen in two guineapigs by Pineda Antikoerper Service (Berlin, Germany). The specificity of the anti-Umbrea antibodies was tested by immunoblots using salivary gland nuclear extract (for preparation see Lehmann and Korge 1995). Antibodies from these sera were used in chromosome staining and immunoprecipitation reactions. Whole-mount immunostaining of salivary glands was carried out essentially as described in Siegmund and Korge (2001). Polytene chromosomes were stained as described in Lehmann and Korge (1996) using monoclonal anti-HP1 antibody (a gift of S. C. R. Elgin, Washington University, St. Louis, MO, USA.), anti-Umbrea I antiserum described above, and anti-Hip II antiserum (Schwendemann et al. 2008). Cy3-conjugated JNJ-38877618 AffiniPure goat anti-guinea pig (Jackson ImmunoResearch, West Grove, PA, USA), Alexafluor 488 goat anti-rabbit IgG conjugate (Molecular Probes/Invitrogen, San Diego, CA, USA), and Cy3-conjugated goat anti-mouse (Jackson ImmunoResearch) were used as secondary antibodies. DNA was counterstained with the dye Hoechst33258. Preparations were examined with a Zeiss Axiophot fluorescence microscope and a Quantix (Photometrics) video camera. Images were processed with Photoshop 7.0. Immunoprecipitation and direct protein interaction assays Co-immunoprecipitation reactions Timp2 containing 90?l of salivary gland nuclear extract (preparation described in Lehmann and Korge 1995) and 50?l each of anti-Umbrea antisera I.