A twist of insight - the role of Twist-family bHLH factors in development

Abstract

Members of the Twist-family of bHLH proteins play a pivotal role in a number of essential developmental programs. Twist-family bHLH proteins function by dimerizing with other bHLH members and binding to cis- regulatory elements, called E-boxes. While Twist-family members may simply exhibit a preference in terms of high-affinity binding partners, a complex, multilevel cascade of regulation creates a dynamic role for these bHLH proteins. We summarize in this review information on each Twist-family member concerning expression pattern, function, regulation, downstream targets, and interactions with other bHLH proteins. Additionally, we focus on the phospho-regulatory mechanisms that tightly control posttranslational modification of Twist-family member bHLH proteins.

Fig. 1. Identity and conservation among Twist-family member bHLH's

(A) Predicted Protein structure of all six Twist-family bHLH members with the basic-helix-loop-helix (bHLH) domain indicated (Red Bar). The entire Twist-family shares a very high degree of conservation across the functional bHLH domain, including serine and threonine residues located in the first α-helix which undergo phosphorylation, directly regulating dimerization and functionality (blue stars). Many Twist-family members have unique stretches of amino acids located outside of the bHLH domain in either the N- or C- terminus (red boxes). (B) A phylogenetic tree of a CLUSTALW alignment of bHLH domains across Twist-family members and other bHLH and HLH domain containing proteins. Twist-family members show relatively high conservation between the functional bHLH domain when compared with other bHLH sub-families.

Fig. 2. Regulation and function of Twist-family bHLH members

Twist-family bHLH's undergo several layers of regulation to tightly regulate their function. Principally, transcriptional regulation establishes the spatial and temporal expression within the cell, thereby establishing the potential bHLH dimer pool within each cell (1). Hand2 transcriptional regulation has been well documented and serves as a prime example of the degree of transcriptional regulation associated with Twist-family members. Multiple tissue specific enhancer elements, such as the separate branchial arch enhancer and the heart enhancer, directly regulate spatial and temporal transcription of Hand2. The Hand2 cardiac enhancer is directly bound by Gata4, regulating ventricular expression (McFadden et al., 2000). Availability of these enhancers is dependent on their accessibility, which is tightly regulated by chromatin conformation. m-Bop, recruits HDACs which deacytlate specific lysine residues promoting chromatin condensation and gene silencing of Hand2 (Gottlieb et al., 2002). Hand2 mRNA can be regulated post transcriptionally by microRNAs. Mir-1 is capable of binding to the 3'UTR of the Hand2 mRNA, resulting in the silencing of the transcript and having a direct impact on the Hand2 dosage (Zhao et al., 2007; Zhao et al., 2005). Following translation, modifications can occur to the protein resulting in changes to the behavior of bHLH's (2). Protein kinase's, including PKA (pictured) and PKC, and the β56δ subunit of Protein Phosphatase 2A (PP2A) are capable of regulating the phosphorylation state of conserved serine and threonine residues of Twist-family bHLH's (Firulli et al., 2003). Phosphorylation of Hand1 has been shown to result in alterations of cellular localization and directly affecting the activity and accessibility of the bHLH protein (Martindill et al., 2007). Dephosphorylated Hand1 is sequestered away in the nucleolus, remaining inactive. Upon phosphorylation, Hand1 is released from the nucleolus allowing it to dimerize and bind to cis- regulatory units on target genes. Although they have not been explored, multiple forms of posttranslational modification, such as ubiquitination and protein degradation, are possible and likely regulate both protein behavior and the equilibrium of the bHLH dimer pool. Once the pool of bHLH proteins is established, an antagonistic competition for dimer partners is closely regulated by the transitional phosphorylation state of the protein (3). Phosphorylation state has a direct affect on dimer partner choice of bHLH proteins within the bHLH dimer pool (Firulli et al., 2003). FRET analysis shows that a phosphorylated form of Twist1 has the highest affinity for Hand2 while having a low affinity for Twist1-homodimerization (Firulli et al., 2005). In contrast, a dephosphorylated form of Twist1 has the lowest affinity for dimerization with Hand2 and instead has a preference for Twist1-homodimers (Firulli et al., 2005). While the phosphoregulatory circuitry closely regulates bHLH dimer choice, ID-proteins, a class of HLH passive inhibitory proteins, can sequester away E-proteins, altering the bHLH dimer pool and potentially establishing a preference for homodimers among Class B bHLH's (Massari and Murre, 2000). Once dimerized, bHLH proteins can bind tightly to E-Boxes (CANNTG) located in target genes. Target genes may contain multiple E-Boxes, which can be transactivated by select dimer pairs. Dimer partner choice is particularly important since it can directly alter the function of bHLH proteins. In the fly, Twist-homodimers are capable of directing a mesodermal fate while Twist-E-Proteinheterodimers can directly antagonize the mesodermal lineage (Castanon et al., 2001).