Journal of Molecular Biology
Review articleThe virtuoso of versatility: POU proteins that flex to fit1
Introduction
With the recent advances in whole-genome analyses, the molecular details that underlie the diversity of life are emerging. One salient example concerns the complexity of modern multi-cellular organisms, which appears to have arisen in concert with the fusion of simple building-blocks to create sophisticated regulatory components (Rubin et al., 2000). POU domains are a classic example of such a modular molecular construction, taking ancient folds used in transcriptional control in prokaryotes and early eukaryotes and adapting them to regulation in highly specialised tissues associated with complex organisms. The modular structure imparts a functional versatility that allows the domain to participate in transcriptional regulation of a variety of ubiquitous and tissue-specific genes.
Transcription is a highly organised process that is tightly regulated by specific recruitment of regulatory cofactors into large assemblies. These in turn affect transcription rates by interacting, sometimes over several kilobases, with components of the preinitiation complex Kornberg and Baker 1992, Naar et al 1999, Rachez et al 1999. The preinitiation complex is at the core of the assembly and it recruits and activates an RNA polymerase molecule at the gene start site to transcribe the DNA template. Structural studies of the preinitiation components is advanced, with crystal complexes available for many of them. Now a catalytically active form of RNA polymerase II (pol II) has been solved to a resolution of 3.5 Å (Cramer et al., 2000). This has shed light on the molecular interactions which lead pol II to form the transcription complex. The complexity of the polymerase, the pre-initiation complex, the associated regulatory assembly, and in addition the essential role of chromatin organisation, indicates that transcription involves many interactions at different structural levels and that hierarchical organisation is a feature of eukaryotic transcription regulation.
Another hallmark of transcription in eukaryotes is that individual regulatory components have a modular architecture, comprised of discrete functional and structural domains. For instance, domains encompassing DNA binding, transcription activation or repression functions can be fused together to create chimeric transcription factors with novel characteristics. The family of POU domain proteins is an example of this principle with a diverse assortment of domain-defined functions found outside the POU motif.
The POU domain was first identified 12 years ago and takes its name after the first four transcription factors in which it was found: mammalian it-1, ct-1, Oct-2 and Caenorhabditis elegansnc86 Clerc et al 1988, Finney et al 1988, Herr et al 1988, Ingraham et al 1988, Sturm et al 1988. A sub-region of the domain was shown to contain the previously well-characterised DNA-binding motif known as the homeodomain. Homeodomain proteins comprise a superfamily of highly conserved DNA-binding factors that are involved in the transcriptional regulation of key developmental processes. The POU domain was distinguished from other homeodomains by a region found N-terminal to it, which when first identified had no known homologues. The two sub-domains are separated by a segment of variable sequence (Sturm & Herr, 1988) suggesting that the domain was bipartite in nature.
Today, there are over 150 entries for POU domains in the SMART database of protein domains involved in signalling events (Schultz et al., 2000). Although this is a mere fraction of the 2000 homeodomain proteins identified so far, the importance of the POU proteins is undeniable, as they are known to regulate many fundamental developmental and homeostatic processes, such as embryogensis and histone gene expression.
POU domain proteins have been grouped according to properties of the linker that separates the two DNA-binding domains Ryan 1997, Wegner et al 1993, Xi et al 1989. At present, seven distinct classes have been elaborated on the basis of linker length and composition (Spaniol et al., 1996). The neuronally expressed Pit proteins define the type I class, and are the sole example. The ubiquitously expressed Oct-1 and tissue-specific Oct-2 are representative of the second category. Brn1,2,4 and Tst-1, which are involved in neural development, comprise a third category, while the Brn3.x class with Unc86, also involved in development, comprise a fourth category. Type V POU domains tend to be involved in early embryogenesis and type VI are expressed in the central nervous system, while type VII POU proteins are critical for early developmental stages Ryan and Rosenfeld 1997, Spaniol et al 1996. Despite this classification scheme, the evolutionary relationship between the different POU families is not clear.
The pivotal role that the relatively small domain plays in transcriptional regulation has been dissected by molecular genetics in parallel with stereochemical analysis. Over the last six years, a small but steadily growing body of structural data has become available on the POU sub-domains, the complete domains in complex with their promoter sites and, more recently, a POU protein and co-activator assembly. This review addresses briefly the particular developmental pathways that are governed by POU proteins, the underlying structural characteristics of the POU domain, and its versatile role in recruitment of other proteins to affect transcription of different genes.
Section snippets
Oct 1 and 2, two POU archetypes
Both Oct-1 (Sturm et al., 1988) and Oct-2 (Clerc et al., 1988) were identified as the proteins which target the ubiquitous octamer regulatory element, ATGCAAAT Falkner and Zachau 1984, Parslow et al 1984. While Oct-1 is found in all tissue types, Oct-2 is expressed specifically in the immune system’s B cells. Oct-1 is known to interact with a variety of other transcriptional regulators to activate the transcription of small nuclear RNA (snRNA) genes Segil et al 1991, Yang et al 1991, histone
Anatomy of the POU domain
After the initial identification of POU domains, it was already clear that the fold could be divided into two distinct sub-domains separated by a variable linker. These sub-domains were named the POU specific domain (POUS) at the N terminus of the motif, and the POU homeodomain (POUH) at the C terminus. The larger POUS domain (75 amino acid residues) was thought to be largely helical, while the POUH domain (60 amino acid residues) had all the features of the DNA-binding motif found in the
How does POU recognise the octamer?
The crystal structure of the Oct-1 POU domain complexed with the octamer site from the H2B promoter has revealed the detailed interactions underlying recognition of the DNA element (Figure 1)(Klemm et al., 1994). This has shown that the two sub-domains make independent interactions with the two half-sites of the operator, and that they are bound on opposite faces of the DNA. Only part of the linker that joins the sub-domains is visible and this segment is found to track along the minor groove.
Linker function in promoter recognition and co-operative effects
Although structural biology is an incredibly powerful tool, providing detailed information about stereochemistry, it gives little information about conformationally variable regions. For instance, the crystal structure of the Oct-1/H2B promoter (Klemm et al., 1994) provided no structural information about the role that the linker might play in sequence-specific recognition. More subtle approaches had to be used to begin to unravel the linker’s contribution to the assembly process Klemm and Pabo
Pit-1, POU regulation in pituitary development and function
Pit-1 is expressed exclusively in the central nervous system during development and later in neurons and in the pituitary for regulation of hormone secretion (reviewed by McEvilly & Rosenfeld, 2000). Once differentiated, the pituitary gland is comprised of six distinct cell types, each of which is dedicated to the secretion of specific hormones (Dasen & Rosenfeld, 1999). Pit-1 was first identified as the transcription factor that bound a regulatory region common to the prolactin and growth
Recognition of DNA through dimerisation
The Pit-1 POU domain binds an assortment of targets, and the variation makes it difficult to achieve anything more then a weak consensus sequence for the majority of Pit regulatory elements. Pit-1 has accommodated this variation through the high degree of conformational flexibility conferred by the POU domain, which endows it not only with an ability to re-orientate the sub-domains to recognise different sites, but also to co-operatively oligomerise (Figure 2)Holloway et al 1995, Ingraham et al
Structural versatility in central nervous system development
The best-studied system for neuronal development is that of Unc-86 in the nematode C. elegans (reviewed by Sengupta & Bargmann, 1996). Unc-86 was first identified as a protein involved in control of cell lineage fate and neuronal terminal differentiation (Chalfie et al., 1981) and was later found to be expressed exclusively in neuronal cells. After asymmetric cell division, Unc-86 is expressed in only one of the daughter cells. The cell that lacks Unc-86 follows the differentiation pattern of
Viruses and Oct
Viral transcriptional regulation has evolved so that the virus itself has few of its own endogenous transcription factors and, instead, appropriates those of the host cell. In the case of HSV, the virion releases the viral protein, VP16 (αTIF) into the cell, which in turn associates with a host cellular protein, called host cell factor (HCF) or C1 (Herr, 1998). HCF is synthesised as a large polypeptide, of 300 kDa which becomes post-translationally modified by cleavage into fragments which
Octamer-regulated transcription
As Oct-1 and Oct-2 stimulate transcription by interacting with transcription promoter sites, an obvious question is whether the proteins actually interact directly with components of the preinitiation complex, such as TBP, TFIIB, TFIIA, or other components found at the transcription start site. Both Oct-1 and Oct-2 have been shown to associate through their POU domains with the conserved core domain of TBP in vitro. This interaction was confirmed in vivo by co-precipitation, and co-transfection
Regulation of the immune response: Bob1 and its interaction with Oct-1/2
How gene transcription becomes tissue-specific has been an intriguing question. When the first octamer-binding factors were isolated, Oct-2 was found to be expressed exclusively in B cells and was clearly very important in immunological development, as mice deficient in Oct-2 die at birth (Corcoran et al., 1993). This led to the suggestion that this transcription factor alone was responsible for B-cell limited expression of immunoglobulin genes. However, two groups also identified another
Oct-1/Bob1/DNA crystal structure: how does Bob fit in?
What does a POU domain/co-activator complex look like, and does the addition of the third party affect the other two components in any way? As Bob1 is known to have little intrinsic structure, what structural transitions accompany the assembly? The crystal structure of the Oct-1/Bob1/octamer element, with two copies of the complex in the asymmetric unit, provided the first opportunity to address these questions Figure 1, Figure 4(Chasman et al., 1999).
The structure of the ternary complex showed
Summary and perspectives
Lefstin & Yamamoto (1998) have suggested that some transcription factors have the potential to assume a variety of conformations, and the choice is governed by the bound DNA element itself. Therefore, the target can be thought of as an allosteric modulator, serving to present different interfaces for the recruitment of element specific co-factors to regulate a wide variety of transcriptional events (Lefstin & Yamamoto, 1998). In the case of the POU domain, it is the linker which permits the
Acknowledgements
We are very grateful to Matthias Wilmanns for sharing results with us before publication, and the referees for all their extremely helpful and constructive comments on the manuscript. We also thank Martyn Symmons, Larissa Lee, Elliott Stollar and all members of the Luisi/Blundell groups (past and present) for helpful discussions. K.P. and B.L are supported by the Wellcome Trust.
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2022, Journal of Biological ChemistryCitation Excerpt :The 30-residue POUS-POUH linker tethers the two homeodomains together and allows for distance variation between them. This intrinsic conformational flexibility is a general feature of the POU transcription factor family, enabling the proteins to adopt diverse quaternary structures during DNA recognition (29–31). Two well-characterized exemplary POU transcription factors are Oct-1 and Pit-1, which are both able to bind to DNA motifs in different arrangements, depending on the nature of the DNA response element (32).
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Edited by P. Wright