Mutations of theDrosophilaZinc Finger-encoding GenevielfältigImpair Mitotic Cell Divisions and Cause Improper Chromosome Segregation

Abstract

We describe the molecular characterization and function of vielfä ltig (vfl ), a X-chromosomal gene that encodes a nuclear protein with six Krü ppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-offunction and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis. INTRODUCTIONZinc fingers constitute the most abundant structural motifs in the proteome predicted from the genome sequences of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans (Rubin et al., 2000) as well as the draft human genomic sequences (Lander et al., 2001). Zinc finger proteins are best known as transcriptional regulators that participate in a variety of cellular activities such as development, differentiation, and tumor suppression. The most common form of zinc finger domain is the so called C2H2 domain, first identified in the basal transcription factor TFIIIA in Xenopus laevis (Miller et al., 1985) and subsequently found also in DNA-binding transcription factors regulating polymerase II-dependent gene expression (Rosenberg et al., 1985). The three-dimensional structure of the basic C2H2 zinc finger is a small domain composed of a ␤-hairpin followed by an ␣-helix, a structure that is held in place by a zinc ion. DNA-binding C2H2 zinc fingers generally occur as tandem arrays with a minimum of two fingers needed to specify the DNA-binding site (Choo and Klug, 1995). In addition to DNA-binding, zinc finger proteins have been implicated in RNA-binding, protein-protein interactions, and lipid binding (Lorick et al., 1999;Bach, 2000;Tucker et al., 2001).DNA-binding C2H2 zinc finger proteins contain proteinbinding domains that provide the basis for the assembly of regulatory complexes involved in chromatin remodeling and transcriptional regulation (Aasland et al., 1995;David et al., 1998). They are frequently expressed in distinct spatial and temporal patterns, and their subcellular localization was found to be regulated in a cell cycle-dependent manner. The entry of mitosis is characterized by the transcriptional shutdown (Prescott and Bender, 1962) that involves inactivation of the general transcription machinery (Segil et al., 1996;Bellier et al., 1997;Akoulitchev and Reinberg, 1998;Long et al., 1998) as well as gene-specific transcription factors during the G 2 /M transition during mitosis. F...

Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape

“…For the C2H2 TF STZ, where mutants have enhanced tolerance to salt and abiotic stresses, we also saw enrichment in mitosis-related functions (Figure 4, box 4). As C2H2 family members from both plants and animals are known to regulate the cell cycle and DNA replication (Staudt et al, 2006;Welch et al, 2007), STZ enrichment for mitotic functions suggests that its stress response phenotype may involve direct regulation of cell division. Finally, bHLH122, known to be important for abiotic drought responses, targeted genes in immune processes (Figure 4, box 3), suggesting that it may also play a role in biotic defense.…”

The TAGteam motif facilitates binding of 21 sequence-specific transcription factors in the Drosophila embryo

“…Depletion of zygotically transcribed Vielfaltig results in chromosome segregation and DNA replication defects (Staudt et al 2006), leading us to hypothesize that TAGteam motifs might play a role in ORC positioning. Using published ORC ChIPseq data from mixed-stage embryos (Roy et al 2010), we confirmed previous reports (MacAlpine et al 2010) of a strong correlation between chromatin accessibility and ORC binding.…”

“…Vielfaltig has six C2H2 zinc fingers, two N-terminal and four C-terminal (Staudt et al 2006), of which the C-terminal zinc fingers are sufficient to recapitulate its known DNA-binding specificity (Liang et al 2008). Vielfaltig may help to maintain chromatin in an accessible state, participate in permissive protein-protein interactions, or have particular interaction partners.…”

“…Highly conserved sequence homologs of vielfaltig have been identified in other insects, but not in vertebrates (Staudt et al 2006). However, overlapping patterns of genome-wide binding of diverse transcription factors appears to be a common feature of undifferentiated animal cells.…”

Transcriptional Activation Is a Conserved Feature of the Early Embryonic Factor Zelda That Requires a Cluster of Four Zinc Fingers for DNA Binding and a Low-complexity Activation Domain

“…1A) (3,10). There are no other identifiable protein domains that might suggest an enzymatic activity.…”