Formation and maintenance of euchromatic domains of interphase chromosomes.

The organization of the interphase chromosome is inherited over cell generations as seen by the pattern of bands and interbands on polytene chromosomes, that reproducibly forms in different tissues and stages of development. The open chromatin of interbands is established by proteins with specific structural and enzymatic properties and is stabilized by elements insulating the spreading of nearby condensed chromatin. Starting from the molecularly characterized interband protein Z4, the aim of the project was to study the role and mechanistic interaction of interband specific proteins in establishing open chromatin structure. General mechanisms of boundary formation between chromosomal band/interband domains and in the formation of insulator elements were another focus of our project.
We first demonstrated that the interband zinc-finger protein Z4 protein plays an important role in these processes since its reduction by hypomorphic alleles results in a nearly complete loss of band/interband structure (Eggert et al. 2004). As shown by a series of mutations, the Z4 gene is essential and exhibits a haplo-Suppressor and triplo-Enhancer of position effect variegation phenotype like many proteins involved in the formation of chromatin substructure. Z4 protein, that by itself has no known enzymatic activity was found to interact with other chromatin proteins crucially required for the formation of structured interphase chromatin like the H3S10 kinase Jil-1 (Gan, 2009, Gan et al. in preparation) and the chromodomain protein Chriz (Gortchakov, 2005). Molecular mapping experiments suggest, that the 3 proteins together with other less well characterized proteins interact in an interband specific multisubunit complex.
Chriz is an essential chromodomain protein and like for Z4 band/interband structure is severely affected following RNAi knockdown. So far we only obtained loss of function alleles for the Chriz gene that are lethal at the embryo/larval transition and can not be studied on polytene tissues in 3rd instar larvae. The domains required for direct mutual interaction between Chriz and Z4 were mapped by CoIP, yeast two hybrid and GST-pulldown experiments in vitro and domains required for chromosomal and specific interband binding in vivo were mapped by a series of transgenes. Interestingly, the minimal aminoterminal fragment of Z4 required for interband binding is identical to the fragment required for its interaction with Chriz. RNAi induced knock down of Chriz in imaginal disc nuclei or salivary glands results in a gross reduction of Z4 binding as well. In contrast, reduction of Z4 by RNAi doesn’t significantly change Chriz binding (Gan, 2009, Gan et al. in preparation). Overexpression of a myc-tagged Z4 N-terminal Chriz interaction fragment competes and replaces endogeneous Z4 chromosomal protein. However, competition is seen only after an exposure of at least 24 hrs, indicating a low exchange rate within the complex (Möbus et al. in prep.). Currently we establish transgenic lines to directly measure chromosomal exchange rates by photodynamic studies.
We have generated and characterized transgenic strains containing random genomic insertion sites of attB sequences for site specific recombination using attP containing vectors and the PhiC31 recombinase. Insertion sites in condensed bands, either by P-element exchange or by construction of artificial bands are under construction (Thomas Zielke, Dandan Zhao unpublished). These strains can then be used to integrate 61C7-8 interband sequences, truncations and modifications to test their capacity for interband and boundary formation.
Keywords: Interphase, Chromosomal domains, Chromosomal borders, Interbands
Domain: chromosome research, development
Involved in the project: Dr. Harald Eggert, wiss. Mitarbeiter (Grundausstattung)
Dandan Zhao, stud. HK/ wiss. Mitarbeiterin (Diplom/Promotion)


> Eggert, H., Gortchakov, A, Saumweber H. (2004) Identification of the Drosophila interband-specific protein Z4 as a DNA binding zinc-finger protein determining chromosomal structure. J. Cell Sci. 117, 4253-4264.
> Gortchakov, A., Kaltenhäuser, J., Eggert, H. Saumweber, H. (2004) Construction of pMH, a convenient Escherichia coli protein expression vector. Appl. Mol. Biol. 38, 713-716.
> Gortchakov, A., Eggert, H., Gan, M., Mattow, J., Zhimulev, I.F., Saumweber, H. (2005) Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster. Chromosoma 114, 54-66.



Prof. Dr. Harald Saumweber, AG-Leiter
Institut für Biologie, Abt. Zytogenetik
Humboldt Universität zu Berlin
Chausseestr. 117, 10115 Berlin, Germany

Phone: +49 30 2093817 8
Fax: +49 30 2093817 7