Lynn M. Powell

Context dependence of proneural bHLH proteins

A key point in neural development is the commitment of progenitor cells to a specific neural fate. In all animals studied, proneural proteins - transcription factors of the basic helix-loop-helix (bHLH) family - are central to this process. The function of these factors is strongly influenced by the spatial and temporal context in which they are expressed. It is important to understand the molecular mechanisms by which developmental context interacts with and modifies the intrinsic functions and properties of the proneural proteins. Recent insights have been obtained in Drosophila and vertebrates from analysis of how bHLH proteins interact with other transcription factors to regulate target genes.

The proneural proteins Atonal and Scute regulate neural target genes through different E-box binding sites.

ABSTRACT For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. We investigated the contribution of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). We show that the proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, we define an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus, thereby defining distinct EAto and ESc sites. These E-box variants are crucial for function. First, tandem repeats of 20-bp sequences containing EAto and ESc sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo. Second, interchanging EAto and ESc sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity.

Specificity of Atonal and Scute bHLH factors: analysis of cognate E box binding sites and the influence of Senseless

The question of how proneural bHLH transcription factors recognize and regulate their target genes is still relatively poorly understood. We previously showed that Scute (Sc) and Atonal (Ato) target genes have different cognate E box motifs, suggesting that specific DNA interactions contribute to differences in their target gene specificity. Here we show that Sc and Ato proteins (in combination with Daughterless) can activate reporter gene expression via their cognate E boxes in a non‐neuronal cell culture system, suggesting that the proteins have strong intrinsic abilities to recognize different E box motifs in the absence of specialized cofactors. Functional comparison of E boxes from several target genes and site‐directed mutagenesis of E box motifs suggests that specificity and activity require further sequence elements flanking both sides of the previously identified E box motifs. Moreover, the proneural cofactor, Senseless, can augment the function of Sc and Ato on their cognate E boxes and therefore may contribute to proneural specificity.

Assembly of EcoKI DNA methyltransferase requires the C-terminal region of the HsdM modification subunit

The methyltransferase component of type I DNA restriction and modification systems comprises three subunits, one DNA sequence specificity subunit and two DNA modification subunits. Limited proteolysis of the EcoKI methyltransferase shows that a 55-kDa N-terminal fragment of the 59-kDa modification subunit is resistant to degradation. We have purified this fragment and determined by mass spectrometry that proteolysis removes 43 or 44 amino acids from the C-terminus. The fragment fails to interact with the other subunits even though it still possesses secondary and tertiary structure and the ability to bind the S-adenosylmethionine cofactor. We conclude that the C-terminal region of the modification subunit of EcoKI is essential for the assembly of the EcoKI methyltransferase.

The SUMO pathway promotes basic helix-loop-helix proneural factor activity via a direct effect on the Zn finger protein senseless

ABSTRACT During development, proneural transcription factors of the basic helix-loop-helix (bHLH) family are required to commit cells to a neural fate. In Drosophila neurogenesis, a key mechanism promoting sense organ precursor (SOP) fate is the synergy between proneural factors and their coactivator Senseless in transcriptional activation of target genes. Here we present evidence that posttranslational modification by SUMO enhances this synergy via an effect on Senseless protein. We show that Senseless is a direct target for SUMO modification and that mutagenesis of a predicted SUMOylation motif in Senseless reduces Senseless/proneural synergy both in vivo and in cell culture. We propose that SUMOylation of Senseless via lysine 509 promotes its synergy with proneural proteins during transcriptional activation and hence regulates an important step in neurogenesis leading to the formation and maturation of the SOPs.

Atoh1 in sensory hair cell development: constraints and cofactors

The proneural gene, Atoh1, is necessary and in some contexts sufficient for early inner ear hair cell development. Its function is the subject of intensive research, not least because of the possibility that it could be used in therapeutic strategies to reverse hair cell loss in deafness. However, it is clear that Atoh1s function is highly context dependent. During inner ear development, Atoh1 is only able to promote hair cell differentiation at specific developmental stages. Outside the ear, Atoh1 is required for differentiation of a variety of other cell types, for example in the intestine and cerebellum. The reasons for this context dependence are poorly understood. So far, the pathways and key players that instruct Atoh1 to act as a mechanosensory cell fate determinant in the context of the inner ear are largely unknown. Here we review evidence that suggests that Atoh1 function in hair cell differentiation is modulated by interaction with other transcription factors. We particularly focus on the possible roles of Gfi1 and Pou4f3, drawing from studies in mouse, Drosophila and C. elegans.

Analysis of DNA-Protein Interactions Using PAGE: Band-Shift Assays

The band-shift assay using polyacrylamide gel electrophoresis is a powerful technique used to investigate DNA-protein interactions. The basis of the method is the separation of free DNA from DNA-protein complexes by virtue of differences in charge, size, and shape. The band-shift assay can be used to determine thermodynamic and kinetic binding constants and also to analyze the composition and stoichiometries of DNA-protein complexes.

SUMOs Mediate the Nuclear Transfer of p38 and p-p38 during Helicobacter Pylori Infection

The p38 mitogen activated protein kinase (MAPK) signaling pathway has been suggested to play a significant role in the gastric mucosal inflammatory response to chronic Helicobacter pylori (H. pylori) infection. Nuclear translocation is thought to be important for p38 function, but no nuclear translocation signals have been found in the protein and no nuclear carrier proteins have been identified for p38. We have investigated the role of small ubiquitin-related modifier (SUMO) in the nuclear transfer of p38 in response to H. pylori infection. Exposure of human AGS cells to H. pylori induced the activation of p38 and the expression of SUMOs, especially SUMO-2. SUMO knockdown counteracted the effect of H. pylori infection by decreasing the resulting p38 mediated cellular apoptosis through a reduction in the nuclear fraction of phosphorylated p38. We identified a non-covalent interaction between SUMOs and p38 via SUMO interaction motifs (SIMs), and showed that SUMO-dependent nuclear transfer of p38 was decreased upon mutation of its SIMs. This study has identified a new pathway of p38 nuclear translocation, in response to H. pylori infection. We conclude that in the presence of H. pylori SUMO-2 has a major role in regulating nuclear levels of p38, through non-covalent SUMO-p38 interactions, independent of the p38 phosphorylation state.