Possible control over cell cycle progression by TLK via chromatin. Altering TLK expression affects cell cycle progression most likely via Asf1. Altered Asf1 phosphorylation could affect chromatin assembly in S phase and/or during DNA repair. TLK may also act on chromatin through histone H3 (Li et al. 2001), and have additional functions via as yet unidentified substrates.

Apoptosis in tlk^Δ14 class I mutant embryos. DNA staining with SYTOX Green of preblastodermal wild-type (A,C) and tlk^Δ14 loss-of-function mutant embryos (B,D). Nuclei fail to replicate properly and undergo apoptosis. (E–G) DNA and lamin double staining shows DNA fragmentation and nuclear membrane breakdown in tlk^Δ14 mutant embryos, both characteristics of apoptotic death. The nuclear envelope is outlined with antibodies against lamin (LAM; white in F, red in G).

Asynchronous nuclear divisons in tlk^Δ14 class II mutant embryos. DNA is stained with SYTOX Green (DNA; green in A–D). The nuclear envelope is outlined with antibodies against lamin (LAM; inset, D). (A) Synchronous nuclear divisions (here: metaphase) in a preblastoderm wild-type embryo. (B) tlk^Δ14 mutant embryo of younger age showing that nuclear divisions are asynchronous. Note that nuclei at the left and right margins have already divided and are separated, whereas nuclei in the central region are still in anaphase and show a defective chromosome segregation pattern (long DNA bridges). (C,D) Nuclear cycle 12 wild-type (C) and corresponding tlk mutant embryo (D). Note the loss of mitotic synchrony and uneven spacing of mutant nuclei (inset, D). The nuclear envelope is labeled with antibodies against lamin (red). tlk embryos show heterogeneity in the size of the nuclei.

tlk^Δ14 mutants have reduced phosphorylated histone H3 levels. DNA is stained with SYTOX Green (DNA; white in A,D; green in C,F). Nuclei in M phase are visualized by staining with anti-phospho-histone H3 antibody (P-H3; white in B,E; red in C,F). (A–C) DNA and phosphorylated histone H3 patterns in stage 8 wild-type embryos. Note P-H3 antibody stained cells within the mitotic domains. (D–F) tlk^Δ14 mutant embryo stained with P-H3 antibody. Note few mitotic cells undergoing DNA fragmentation (large thin arrows). Most nuclei remain in interphase and accumulate big clumps of DNA (small fat arrows). (G) Western blots of SDS gels loaded with crude extracts from eggs that derived from either wild-type females (WT) or females containing homozygous tlk^Δ14 germ lines were probed with antibodies against phosphorylated histone H3 (anti-phospho H3). Controls for loading of protein extracts; antibody staining of nonphosphorylated histone H3 (anti-histone H3) and actin (anti-Actin); increasing amounts of the protein extracts are indicated by triangles. Note that the tlk^Δ14 mutants show reduced levels of phosphorylated histone H3.

Overexpression of the TLK–ASF1 pathway affects chromatin structure and endoreplication in salivary glands. (A,G) Salivary glands from larvae overexpressing egfp at 25°C under Heat-Shock-gal4 (HS-gal4) driver show normal cell number (128 per lobe) and morphology of polytene chromosomes. (B,H) Overexpression of asf1 results in smaller salivary glands with only half of the normal cell number (upper salivary gland). Occasionally, some of the salivary glands have normal size and almost normal cell number (lower salivary gland). Polytene chromosomes appear thinner, indicating that DNA is underreplicated. (C,I) Overexpression of tlk produces small salivary glands with nearly normal cell number, but with chromosomes that are extremely thin and unstructured, showing global changes in higher-order chromatin structure. (D,J) Coexpression of both tlk and asf1 gives rise to tiny salivary glands with few cells (∼1/10 of the normal) and tiny chromosomes. (E,K) Expression of the dominant negative tlk^dead allele almost completely abolishes cell proliferation in salivary glands, even at 18°C. In these glands, chromosomes appear to be underreplicated. Chromosomes from few preparations that could be obtained show a banded pattern (K, inset). (F,L) Coexpressing asf1 with tlk^dead partially rescues tlk^dead effects on cell cycle progression and DNA replication. Note that G–L (with the exception of the inset in K) are presented at the same magnification.

Distribution of tlk transcripts and protein. (A) In situ hybridization of whole-mount preparations from embryos (stages 3 and 9) as revealed by antisense RNA corresponding to LD15160. (B) Western blots of SDS gels loaded with crude extracts from eggs of either wild-type females (WT) or females containing a homozygous tlk^Δ14 germ line (tlk¹⁴) were probed with anti-TLK antibodies; anti-actin antibodies were used to monitor the protein loading. Asterisks indicate bands corresponding to predicted sizes of TLKs, dots indicate possible degradation products (for details, see text). (C,D) Confocal images of embryos showing DNA (green; SYTOX Green), lamin (red; antilamin antibodies), or TLK (blue; anti-TLK antibodies). (C) Stage 5 wild-type embryo with synchronized nuclei at interphase; TLK is concentrated in the nuclei (see enlarged area, right). (D) Stage 8 wild-type embryo with asynchronous cell divisions. TLK is enriched in interphase nuclei. During all other stages of the division cycle, TLK is distributed diffusely and not associated with condensed chromosomes (enlarged area, right). (E) Staining of whole-mount salivary glands for TLK (green) and DNA (blue). Note that TLK is concentrated in nuclei. (F) Magnification of nuclei showing that TLK is not associated with polytene chromosomes but concentrated around them.

TLK acts in the same genetic pathway as ASF1 and phosphorylates it in vitro. (A) tlk interacts with asf1 in vivo. In wild-type flies, synchronized cell divisions give rise to adult eyes with regularly distributed facets. In flies overexpressing either tlk kinase or asf1 in the eye under the control of eyeless-GAL4 driver, the facets distribution is slightly altered, giving rise to a rough eye phenotype. Coexpression of both tlk and asf1 in the eye using the eyeless-GAL4 driver leads to severe developmental defects, resulting in pin-head phenotype. Overexpression of tlk^dead alone in the eye with the same eyeless-GAL4 driver results in pin-head phenotype, whereas coexpression of both tlk^dead and asf1 rescues the tlk^dead pin-head phenotype. (B) GST pull-down assays with radiolabeled TLK and TLK^dead produced by coupled in vitro transcription/translation (IVT) in the presence of [³⁵S]methionine as detected by autoradiography. Both TLK and TLK^dead bind to GST–ASF1 fusion protein, but not to GST. A total of 10% of the input mixture was loaded as control (Input). A major fraction of TLK kinase (P) migrates slower on SDS-PAGE compared with TLK^dead due to autophosphorylation. (C) Stability of the interaction between TLK and ASF1 depends on TLKs autophosphorylation state. Radiolabeled TLK and TLK^dead were treated with either calf intestinal alkaline phosphatase (CIP) or with ATP. CIP treatment results in a fast migrating TLK form of same mobility as TLK^dead, which is pulled down by GST–ASF1. Addition of ATP results in a slower migrating form of TLK, which is presumably hyperphosphorylated (labeled PP) and does not bind GST–ASF1. Gel mobility of TLK^dead and binding to GST–ASF1 protein is unchanged after treatments either with CIP or ATP. (D) Nonradiolabeled, in vitro translated TLK phosphorylates GST–ASF1 protein and itself, but not GST alone, as detected by autoradiography with ³²P. TLK^dead lacks phosphorylation and autophosphorylation activities because of a mutation in the catalytic domain. TLK and TLK^dead proteins were not synthesized in sufficient amounts to be revealed by Coomassie staining; however, they were revealed by immunoblotting using anti-TLK antibodies (data not shown).

Characterization of the tlk gene, tlk mutations, and tlk protein. (A) Structure of the tlk gene and position of the P-element insertion l(1)G0054. cDNA was obtained from the LD15160 clone. AE refers to the contig number of the Drosophila genome project (Adams et al. 2000). Comparison of different tlk cDNA sequences and tlk genomic sequences (Berkeley Drosophila Genome Project, BDGP) showed that tlk encodes differentially spliced transcripts. The tlk LD15160 and GH07910 transcripts differ in their 3′ untranslated region (different polyadenylation sites), and they have an alternatively spliced first exon. The predicted protein sequences encoded by tlk LD15160 and tlk GH07910 (gene CG2829) share a common ORF of 1242 residues and differ in the first putative 13 (MPVVKQRKMSRFK) and 24 amino acids (MDQFQTSLNPRKLELLESRITGVR), respectively. Database searches revealed that cDNAs LD16185 and LD09833 also encode a protein with 12 alternative N-terminal residues (MCVQKDMRRLKK) and the cDNAs LD41810 and LD16882 lack the alternatively spliced first exon. The alleles tlk^Δ14 and tlk^Δ40 result from an imprecise excision in which 2357 bp have been deleted (tlk^Δ14) and 1143 bp have been replaced by a 22-bp sequence interval of the P-element (tlk^Δ40), respectively. Nontranslated regions and coding region of the gene are shown by open and black boxes, respectively; deleted genomic DNA of the mutants is shown by brackets. PK indicates the cDNA region that encodes the tlk kinase domain; ABP (green box) indicates the position of the peptide used for antibody production. (B) Alignment of the conserved C terminus containing the Tousled kinase domain of Drosophila tlk, Arabidopsis (Atha), and human homologs (Hsap TLK1 and Hsap TLK2) using the CLUSTALW program (Thompson et al. 1994). Identical residues or conservative replacements are shaded; dots denote gaps in the alignments. The amino acid position 217 is labeled with an asterisk; A indicates that the tlk^dead transgene carries a replacement of the aspartic acid residue by alanine. (C) Schematic representation of Drosophila, Arabidopsis, and human TLKs. The kinase domains (PK; yellow boxes), nuclear localization signals (red oval), leucine zipper motives (blue boxes), coiled-coil regions (green spirals), and glutamine-rich regions (green boxes) are shown. Numbers refer to positions of amino acids.