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First published online 3 August 2004
doi: 10.1242/jcs.01292

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Identification of the Drosophila interband-specific protein Z4 as a DNA-binding zinc-finger protein determining chromosomal structure

¹ Humboldt University Berlin, Institute of Biology, Department of Cytogenetics, Chausseestrasse 117, 10115 Berlin, Germany
² The Institute of Cytology and Genetics, Lavrentjeva 10, Novosibirsk, 630090, Russia

View larger version (21K): [in a new window]   Fig. 2. Generation and verification of Z4 mutations. The genomic region with the two alternatively spliced exons of the gene CG12974 (red bars) and the transcript of Z4 depicting its translated regions (blue bars) and untranslated regions (yellow bars) is shown. Arrows indicate the direction of transcription. The site of the EP-element (green triangle) insertion in line EP(3)0756 is shown, from which deletions were generated by imprecise excision to generate the homozygous lethal lines Z4-1.3 and Z4-7.1. The extent of the deletions is indicated by the broken lines. In line Z4-1.3 the deletion encompasses 800 bp of DNA including the 5'-transcription start of Z4 and the 5'-region of the gene CG12974 encoding two exons of the transcript CG12974-RA. In line Z4-7.1 1800 bp to the left of the EP-element are deleted, which removes part of the transcription units of gene CG12974, but leaves the coding region of Z4 unaffected. The bar below indicates the genomic region that complements the lethal mutation of lines Z4-1.3 and Z4-7.1. Within this bar, the vertical line shows the position of the myc-tag that was fused in frame to the 3'-end of the Z4 coding region. Chromosomes from larvae transgenic for the tagged genomic region were stained with DAPI (A) and a monoclonal anti-myc antibody (B). The composite image (C) reveals the localization of the tagged Z4 protein to the chromosomal interbands.

View larger version (75K): [in a new window]   Fig. 1. Z4 expression and localization to chromosomal interbands. (A) Polytene chromosomes of wild-type 3rd instar larvae were double labeled with DAPI (blue) and the Z4 antibody (red). Z4 stains the euchromatic arms of the chromosomes (a) and is absent from the heterochromatic chromocenter (b). Within euchromatin, Z4 stains exclusively the interbands (c). In heat-shocked wild-type larvae Z4 demarcates the distal edge of the 87A hsp70 heat-shock puff (d, marked by the white arrowhead) and the proximal edge of the hsp70 heat-shock puff in 87C (yellow arrowhead). (B) Early embryos from wild-type flies (a-d) and Kc cells (e,f) were double labeled with DAPI (a,c,e) and the Z4 antibody (b,d,f). In the early embryo Z4 is ubiquitously expressed. The staining of Kc cells shows that Z4 is chromosomally associated during interphase (e,f). By contrast, Z4 does not associate with mitotic chromosomes of early embryos but disperses within the cell (c,d). (C) Western blot of nuclear proteins from wild-type embryos (E) and Kc cells (Kc) with the Z4 antibody. In addition to antigens that are uniquely present in Kc cells or embryos, the antibody recognizes the 170 kDa Z4 protein, which is present in both fractions.

View larger version (47K): [in a new window]   Fig. 3. Dose-dependent effect of Z4 on wm4 position-effect variegation. (A) Representative eye phenotypes of males derived from a cross between homozygous wm4 females and males heterozygous for the Z4 mutants balanced over TM3, Sb. Compared with the males with the TM3 chromosomes, the eyes of sibling males mutant for Z4 have increased numbers of red ommatidia. (B) Quantitative measurement of eye pigments of the phenotypes shown in (A). Eye pigments were extracted from the heads of 20 representative individual males for each cross and their absorbance at 480 nm was determined. The columns show the ratios of absorbances between the Z4 mutants and their TM3 siblings derived from the identical cross. The control shows the ratios obtained from wm4/Y;e,st,spo/+ and wm4/Y;TM3,Sb/+ siblings. (C) Eye phenotypes of males from a cross between wm4/wm4 females and w1118/Y;P[Z4myc]/CyO males. Males with three copies of the Z4 gene have significantly reduced numbers of red ommatidia compared with their sibling males with two doses of Z4.

View larger version (121K): [in a new window]   Fig. 4. Morphology of chromosomes mutant for Z4. Tb+ homozygous mutant 3rd instar larvae were collected from the Z4-7.1/TM6,Tb stock and chromosomal squashes were stained with DAPI (A-C). Whole mount salivary glands from wild-type (D,G) and Z4 mutants transheterozygous for the alleles Z4-1.3/Z4-7.1 (E,H) were double stained with Sytox Green (D,E) and the Z4 antibody (G,H). Unfixed salivary glands dissected from larvae expressing the fusion construct BJ1-GFP (F) or from Z4-1.3/Z4-7.1 mutant larvae transgenic for BJ1-GFP (I) were analyzed by CLSM. In Z4 mutants a pertubation of chromosomes with the appearance of decondensed chromatin is evident (C,E,I).

View larger version (50K): [in a new window]   Fig. 5. Presence of Z4 in chromosomes of Z4 hypomorphic mutants. (A) Chromosomes were squashed from 3rd instar larvae homozygous mutant for Z4-7.1. The DNA of the squashed chromosomes was stained with Sytox Green (green) and with the Z4 antibody (red). The composite image shows the localization of Z4 to some telomeres in addition to a few internal chromosomal sites, which are still complementary to the DNA staining. (B,C) Chromosomes from homozygous mutant larvae of the line Z4-1.3 that were rescued by the expression of the Z4 cDNA were squashed and stained with DAPI (blue) and the Z4 antibody (red). Compared with its staining of the interbands, Z4 is concentrated on the telomere of the X chromosome (B) and on the telomere of the left arm of chromosome 2 (C).

View larger version (39K): [in a new window]   Fig. 6. Binding of Z4 to Notch sequences in vitro. (A) 5'-region of Notch. The black bar represents the transcribed region of Notch, and the gray bar indicates the region deleted in the faswb-allele. The position of the N1 and N2 fragments that were used for in vitro binding to Z4 are shown below. Both fragments were digested and endlabeled at the HinfI-sites indicated. (B) Electrophoretic mobility shift assay with 0.5 µg of purified Z4 (omitted in lanes 1 and 8), 5 ng of the end-labeled DNA fragments N1 (lanes 1-7) or N2 (lanes 8-14) and a 100-, 200-, 400- or 800-fold excess of poly(dI-dC) competitor DNA (lanes 3-6 and 10-13) as indicated on the top.

View larger version (63K): [in a new window]   Fig. 7. Identification of a chromodomain protein interacting with Z4. (A) Proteins were immunoprecipitated from nuclear extracts prepared from Kc cells with the anti-Z4 antibody, resolved by SDS-PAGE on a 8% gel and visualized by Coomassie staining. The major band at a relative molecular weight of 150-160 kDa was eluted from the gel and analyzed by MALDI mass spectrometry. The band labeled Chriz is the protein encoded by the gene CG10712. (B-D) Polytene chromosomes of a transgenic line that expressed a myc-tagged Chriz protein in salivary glands were stained with an antibody against the myc-tag. The DNA staining (blue) is complementary to the staining of myc-Chriz (red), showing its interband localization on all chromosomes (A). myc-Chriz is restricted to the euchromatic parts of the chromosomes and does not bind to the heterochromatic chromocentre (C). At higher resolution the localization of myc-Chriz to a high number of interbands is obvious from the optical section shown in (D). It is identical to the interband localization of Z4.

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