BIOLOGICAL OVERVIEW

It is suggested that Castor and Hunchback act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development. By silencing pdm expression in early and late NB sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the sequential expression of Hb, Pdm, and Cas. Embryos lacking Hb function suffer multiple defects. During CNS development, hb- embryos fail to develop labial and thoracic ganglia; gaps form between the subesophageal maxillary neuromeres and the abdominal ganglia. In addition, the seventh and eighth abdominal segments are fused due to the absence of parasegment 13. Missing are the highly ordered ventral cord axon scaffolds made up of longitudinal connective and commissural fascicles. Given the apparent transient overlap between Hb and Pdm-1 expression in the CNS and Hb's established role as a repressor of pdm expression in the cellular blastoderm, it is likely that Hb also silences pdm expression during early NB sublineage development. Pdm-1 expression patterns in hb- embryos confirm the hypothesis that Hb functions as a repressor during CNS development. In the absence of Hb, Pdm-1 is ectopically expressed in all CNS ganglia (Kambadur, 1998).

Likewise, Castor represses pdm. To determine if Cas is a pdm repressor, Pdm-1 and Pdm-2 expression were analyzed in cas null embryos. In stage 9 and in younger embryos, no differences were detected between the cas- and wild-type expression patterns of Pdm-1 or -2. However, starting at stage 10, NBs fail to terminate expression of both Pdms. Ectopic Pdm expression is observed in most, if not all, late developing sublineages in all CNS ganglia. The sustained Pdm expression is most likely due to transcriptional derepression. In a cas- background transgenes bearing a pdm-1 proximal promoter fragment are ectopically expressed in NBs during late sublineage development. This result demonstrates that the enhancer(s) within the 6.3 kb regulatory DNA are negatively regulated by Cas. Binding studies reveal that Cas can bind to the same DNA sites as Hb, raising the possibility that it modulates transcriptional activities of genes also regulated by Hb. DNA sequence analysis of Cas fragments reveals 32 potential DNA-binding sites, all sharing at least 8 out of the 10 bp with the Hb consensus sites. Cas is shown to be able to bind to these sites. These results suggest that Hb and Cas regulate pdm expression by interacting directly with their cis-regulators to deactivate controlling enhancer(s), with Hb repressing the pdm genes early in CNS development, while Cas silencing acts late in CNS development (Kambadur, 1998).

The tightly choreographed NB expressions of Hb, Pdm, and Cas suggest temporally integrated processes participate in their formation. Whereas Hb is expressed early in neuroblast lineages, Cas is expressed late (see Lateral views of Drosophila CNS). The fact that NBs are found co-expressing Hb/Pdm-1 or Pdm-1/Cas, but never Hb/Cas, further suggests that at least some early NBs make a Hb->Pdm->Cas transition. However, not all NBs undergo these transitions. This is particularly evident in NBs that enter the proliferative zone during later delamination waves. For example, the first ventral cord NBs to express Cas, the S3 NB6-1s, activate Cas shortly after delaminating from the ectoderm and do not express Hb. Late developing sublineages rely on cas to both insulate cell-fate programs and to secure the expression of factors that likely play key roles in their cell-identity decisions. Cas carries out the first of these regulatory roles by selectively silencing the expression of pdm genes. It is concluded that the Zn-finger proteins Hb and Cas act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development (Kambadur, 1998).

A fundamental question in developmental biology seeks to unravel the connection between cell cycle and differentiation. Do cells have to divide in order to turn on new genes? Do they have to undergo a regime of DNA synthesis? It has been found that neuroblasts require cell division to activate cas/ming expression, while single, identified neuroblasts require only cell cycle progression and not mitosis to activate even-skipped expression. Other genes, unplugged and achaete are expressed independently of cell cycle and cytokinesis (Cui, 1995). This observation also supports the late-in-development origin of cas/ming expressing cells: earlier neuroblasts would have had to go through a mitotic cycle before their neuroblast progeny could express cas/ming.

GENE STRUCTURE

Bases in 5' UTR - 688

Exons - four

Bases in 3' UTR - 591

PROTEIN STRUCTURE

Amino Acids - 799

Structural Domains

There are four consecutive zinc finger repeats. The motif is Cys2-His2-Cis2-His2. Multiple PEST sequences contributing to protein turnover are present. There is an N-terminal polyglutamine sequence, an acidic region, a serine proline rich region and a polylysine sequnce (Mellerick, 1992).

Castor functions as a DNA-binding transcription factor. It contains a centrally located Zn-finger domain made-up of four consecutive C2-H2C2-H2 repeats. The second C2-H2 of each repeat closely resembles fingers of the Xenopus TFIIIA C2-H2 class. Flanking this repeat are motifs that may constitute either transcription transactivation or repression domains. UV induced protein-DNA cross-linking in vivo studies reveal that Cas binds genomic DNA. To determine if Cas is a sequence-specific DNA-binding protein, the cyclic amplification of selected targets protocol was used. After six rounds of selection/amplification, sequencing of cloned fragments revealed that all had at least one sequence motif in common and some contained two core recognition sequences. DNA fragments containing one site homologous to the consensus site produce a single prominent Cas-DNA gel-shift; a fragment with two, generates two complexes. Addition of Cas-specific antisera causes a super-shift of the Cas-DNA complex. A search of known transcription factor DNA-binding sites shows that the Cas recognition sequence is almost identical to that of the Drosophila Zn-finger protein Hunchback. The Cas consensus matches 9 out of 10 bp for the reported Hb sites. To determine if Cas binds Hb sites, gel-shift experiments using DNA fragments were carried out with exact sequence matches to Hb targets. Cas does indeed bind to these sites. The sequence-specificity of Cas-DNA binding to Hb recognition sites was further tested by competition assays and base-pair substitutions. Taken together, these experiments demonstrate that Cas can bind to the same DNA sites as Hb, raising the possibility that it modulates transcriptional activities of genes also regulated by Hb. Secondary structure predictions of the Cas finger domain indicate that only the first and third of its TFIIIA-like fingers contain alpha-helices. Interestingly, optimal alignment of Cas and Hb fingers reveals that the first and third a-helices of Cas share the highest homology with the corresponding a-helices of Hb (33% identity for the first and 27% for the third). Although speculative, their shared DNA-binding preferences may be due in part to the shared residues found in these predicted reading heads. Outside of their Zn-fingers, Hb and Cas show no obvious sequence similarities (Kambadur, 1998).

date revised: 20 December 2006

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