The Molecular and Genetic Interactions Between Pax3 and Alx4
by
Golnessa Mojtahedi
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Golnessa Mojtahedi (2009) The Molecular and Genetic Interactions Between Pax3 and Alx4 Master of Science, 2009 Golnessa Mojtahedi, Department of Laboratory Medicine and Pathobiology, University of Toronto
Abstract
Alx4 is a paired-type homeodomain transcription factor that plays a key role in development, strongly expressed in the first branchial arch and craniofacial region. Pax3 also belongs to this family, and it displays a similar pattern of expression to that of Alx4. When Pax3 or Alx4 activity is lost individually, defects arise in an overlapping set of embryonic structures. In addition to their expression patterns, this suggests that these two factors may interact to play a role in normal murine development. We demonstrate an overlapping pattern of expression of Pax3 and Alx4 in the developing embryo and that Pax3 and Alx4 physically interact in vivo and in vitro. Pax3 and Alx4 can activate transcription from a P3 homeodomain consensus site, and preliminary analysis of mice null for both Pax3 and Alx4 show a novel mutant phenotype. We have therefore demonstrated a physical and genetic interaction between Pax3 and Alx4.
ii Acknowledgments
First and foremost, I would like to thank my supervisor, Paul Hamel, for taking a chance and giving me the opportunity to pursue my Masters. I thank him not only for training me to think like a scientist, but also, for giving me the opportunity to share in his interest in health and human rights. The lessons and skills I have learned over these few years will be invaluable in my future endeavors. I would also like to thank my committee members, Dr. Michael Ohh, Dr. Liliana Attisano, and Dr. Maria Rozakis for their valuable input, support and guidance. My sincere thanks to Dr. Tania Christova, Dr. Hong Chang, Dr. Wai Chi Ho, Qing Li and Nadia Mohabir for their support, and helpful advice, and most importantly, for making my experience in the Hamel lab a pleasurable one. In particular, I would like to thank Tania for taking me under her wing from the beginning of my Masters to the end, and for being a mentor in more ways than one. Finally, I would like to thank my friends and family. I thank my friends for making my grad school experience a memorable and truly enjoyable experience. Most importantly, I would like to thank my parents and sisters for their never-ending support. I thank them for always showing a genuine interest in all that I have set out to do and for their encouragement every step of the way. All that I have accomplished is a reflection of their support, patience, love and belief in my abilities, and to them I am forever grateful.
iii Table of Contents
Abstract ii Acknowledgments iii List of Figures vi List of Abbreviations vii Section 1 – Introduction 1 1.1 General Introduction 1 1.2 Transcriptional Regulation of Gene Expression 1 1.2.1 Transcription Factors 1 1.2.2 Mechanisms of Transcriptional Regulation 5 1.3 Homeodomain Containing Transcription Factors 7 1.3.1 Classes of Homeodomain Proteins 8 1.3.2 Structure and DNA Binding 9 1.3.3 Specificity of Homeodomain Protein Action 11 1.4 Pax3 16 1.4.1 Pax Protein Structure 16 1.4.2 Pax3 Paired Domain-Homeodomain Functional Interdependence 19
1.4.3 Pax3 Transcriptional Activity 21
1.4.4 Protein Interactions That Modulate Pax3 Activity and Function 22
1.4.5 Pax3 in Development 24
1.4.6 Pax3 and Disease 27
1.5 Aristaless – like 4 29 1.5.1 Alx4 Structure 29
iv 1.5.2 Alx4 Transcriptional Activity 30 1.5.3 Alx4 in Development and Disease 31 1.6 Rationale and Thesis Summary 34
Section 2 – Materials and Methods 37 Section 3 – Results 40
3.1 Overlapping Expression of Pax3 and Alx4 in the 40 developing embryo
3.2 Alx4 Stably Interacts with Pax3 43
3.3 Transcriptional Effect of Pax3 and Alx4 on a model 48 Promoter
Section 4 – Discussion 51
4.1 Pax3 and Alx4 interact through protein-protein 51 interactions independent of DNA-binding
4.2 Pax3 and Alx4 activate transcription from a P3 element 53
4.3 A model for regulation in vivo by a Pax3-Alx4 regulatory 55 complex
4.4 Preliminary Analysis of the genetic interaction between Pax3 58 and Alx4 during embryonic development using double knockout mice
4.5 Significance 61 4.6 Future Directions 62
Section 5 – References 66
v List of Figures
Figure 1.1 Schematic of Pax3 Protein Structure 17 Figure 1.2 Schematic of Alx4 Proteins Structure 30 Figure 1.3 Defects in Strong’s Luxoid (lstD) Mice 33 Figure 3.1 Overlapping expression between Pax3 and Alx4 in developing embryo 41
Figure 3.2 Validation of Specificity of Antibodies Used For Immunofluoresence 42
Figure 3.3 Interaction Between Pax3 and Alx4 44
Figure 3.4 Pax3 Deletion Mutant Analysis 46
Figure 3.5 Homeodomain of Alx4 mediates interaction with Pax3 47
Figure 3.6 Pax3 and Alx4 Regulate Transcription from the P3 Promoter 49
Figure 4.1 Model of Pax3 and Alx4 Interacting to Regulate Transcriptional Activity 58
Figure 4.2 Preliminary Analysis of Genetic Interaction between Pax3 And Alx4 60
vi List of Abbreviations
Alx4 aristaless-like 4 Antp antennapedia homeobox gene Arx aristaless related homeobox bHLH basic helix-loop-helix bZIP basic-leucine zipper Cart-1 cartilage homeoprotein 1 Dct dopachrome tautomerase E Embryonic day EMSA Electrophoretic mobility shift assay EtdBr Ethidium bromide Grg4 groucho-related gene 4 Gsb gooseberry Gsc goosecoid GST glutathione S-Transferase HD homeodomain HDAC histone deacetylase HP1γ heterochromatin protein-1 gamma HTH helix-turn-helix Lef-1 lymphoid enhancer-binding factor 1 lstD Strong’s luxoid allele MC Meckel’s Cartilage MET mesenchymal-epithelial transition factor Mitf microphthalmia-associated transcription factor Msx1 msh-like 1 Msx2 msh-like 2 Myf5 myogenic factor 5 NCAM neural cell adhesion factor NMR Nuclear Magnetic Resonance P.E.I Polyethylenimine P2 Prd-class homeodomain recognition sequence TAATnnATTA P3 Prd-class homeodomain recognition sequence TAATnnnATTA Pax paired Box protein PD paired domain Pit-1 pituitary-specific positive transcription factor 1 Pitx paired-related homeobox gene pRB retinoblastoma protein PRD paired
vii PRS paired domain recognition sequence RD rhabdomyosarcoma cell line Rx retinal homeobox gene SH3 src homology domain 3 Sox10 sex determining region Y-box 10 Sp splotch allele SpD splotch-delayed allele TAZ transcriptional co-activator with PDZ-binding motif TBP TATA binding protein Trp-1 tyrosinase-related protein 1 ZF zinc finger
viii Section 1 - Introduction
1.1 General Introduction The fundamental basis of the specification of the structural and functional characteristics of the multitude of cell types in an individual organism is the regulation of gene expression. A copy of an organism’s complete genetic material is present in almost every one of its cells, where the activation of a unique combination of genes is responsible for determining cellular identity (Semenza, 1994). Gene expression can be regulated at various levels; 1) RNA processing 2) RNA stability 3) Translational Control and 4) Post- translational modification, where transcriptional regulation is the best understood mechanism of regulating gene expression (Semenza, 1994). In addition to a critical role in cell differentiation, proliferation, survival and pattern formation during development, postnatally, the ability to selectively express specific genes is required for cellular response to stress, infection and response to extra-cellular signals such as hormones (Latchman, 2008; Papavassiliou, 1995).
1.2 Transcriptional Regulation of Gene Expression 1.2.1 Transcription Factors Transcription factors are regulatory factors that function to positively or negatively affect the rate of transcription, thereby activating or repressing gene transcription (Gilbert,
2006; Semenza, 1994). There are two classes of factors directing transcription; 1) the basal transcriptional apparatus and 2) regulatory factors (Locker, 2001). The basal apparatus, consisting of RNA Polymerase II and a number of general transcription factors, directs transcription from the core promoter. This apparatus is not sufficient however for producing significant levels of gene transcription. Additional regulatory factors therefore are necessary to modulate transcription from the core promoter (Locker, 2001). Regulatory transcription factors are of two types; 1) sequence-specific factors that
1 interact directly with cis-regulatory DNA elements upstream of core promoters and 2) cofactors that do not directly interact with DNA, but rather are brought to the DNA through interactions with sequence-specific factors, and function to modulate gene transcription (Locker, 2001). Relevant to this thesis are the sequence-specific DNA- binding transcription factors and they will now be discussed in greater detail. The structure of eukaryotic transcription factors is modular, consisting of independent structural domains (Frankel and Kim, 1991). Mark Ptashne and colleagues first identified this modular structure through domain swapping experiments between the LexA and Gal4 transcription factors (Ma and Ptashne, 1987b). These experiments demonstrated that when the DNA-binding domain of LexA was fused to the activation domain of Gal4, a functional transcriptional activator resulted, operating through the LexA binding site. This led to the conclusion that these factors consist of functionally independent DNA-binding and activation domains. To date, the modular nature of transcription factors has been studied extensively. Consistent with the preliminary studies outlined above, these factors consist of independent DNA-binding domains, and one or more independent regulatory domains, of which activation domains are an example(Frankel and Kim, 1991; Locker, 2001; Ptashne, 2006). There are several well-characterized modular DNA-binding domains including zinc fingers (ZF), basic leucine zippers (bZip), basic helix-loop-helix (bHLH) and homeodomain helix-turn-helix (HTH) (Locker, 2001; Pabo and Sauer, 1984; Semenza, 1994). These domains interact with specific DNA sequences in the major groove of the DNA through hydrogen bonding. Predominantly, these DNA-binding motifs use an
α-helix to make base contacts in the major groove of the DNA; however, β-sheets are also able to make base contacts. Furthermore, although the main interaction between protein and DNA occurs in the major groove, it is important to note that the overall binding specificity is also determined by interactions with DNA by protein regions
2 surrounding the α-helix (Pabo and Sauer, 1984). For example, in homeodomain proteins, the N-terminal tail of the homeodomain DNA-binding domain is able to make base contacts within the minor groove (Wolberger et al., 1991). Additionally, interactions with the DNA backbone play a significant role in site-specific recognition and binding affinity (Pabo and Sauer, 1984). Although these additional interactions are necessary for enhancing DNA-binding affinity, the specificity of the interaction between a single factor and its recognition site is not sufficient to account for the specificity necessary for regulated target gene expression (Locker, 2001). This is complicated further by the fact that sequence-specific regulatory factors bind to relatively short DNA-binding sites, which can occur throughout the genome with high frequency (Locker, 2001). The relative lack of specificity of these DNA-binding domains is in part compensated for by the presence of the regulatory domains. In vivo, the DNA binding domain is not sufficient to modulate transcription of a target gene, and so, the presence of the regulatory domain is also necessary (Ma and ebrary Inc., 2006). There are two types of regulatory domains, activation domains and repression domains. These domains mediate protein-protein interactions, recruiting other factors necessary for transcription to the DNA template (Locker, 2001; Ptashne and Gann, 1997).. Mark Ptashne and colleagues first investigated the function of the activation domain using “activator bypass” experiments. Here, a component of the transcriptional machinery, Srb2, was fused to the DNA-binding domain of the transcriptional activator Gal4. Srb2 interacts with the holoenzyme complex, and when bound to DNA through the Gal4 binding site, recruits the transcriptional machinery to the promoter (Ptashne and Gann, 1997). These experiments suggested the recruitment of transcription machinery is sufficient to activate transcription. Therefore, the activating domain was implicated in recruiting, through protein-protein interactions, additional factors to the gene for transcription. The repressor domain works through the same fashion, recruiting repressor
3 complexes to the DNA (Ptashne, 2007; Renkawitz, 1990). While less is known about the structure of repressor domains, the structure of activation domains has been studied extensively, although their structure is still not well understood. Activator domains can be classified based on their amino acid content. Three general classes have been defined; acidic, proline-rich and glutamine-rich (Latchman, 2008). As opposed to the highly conserved sequence of the DNA-binding domains, the specific protein sequence of the activation domain is not significant. Rather, preliminary analysis of activation domains as well as recent studies suggest that in fact it is the presence of specific bulky hydrophobic amino acids that are most important to activation domain function (Ma and Ptashne, 1987a; Triezenberg, 1995). Furthermore, in contrast to highly structured DNA-binding domains, activation domains lack a well-defined secondary and tertiary structure (Locker, 2001). Since activation domains are responsible for mediating specific interactions with target proteins, the question arises as to how these unstructured regions are able to interact specifically with proteins. Biophysical analyses of activation domains in complex with their target proteins demonstrate that when these domains are in complex they do adopt secondary structure (Frankel and Kim, 1991; Hermann et al., 2001; Locker, 2001). More recently, the interaction between c-Myc protein and its target protein TBP (part of the transcriptional machinery), has been investigated to determine the mechanism by which the activation domain adopts this secondary structure (Hermann et al., 2001). In this study, Hermann et al., propose that initially the unstructured activation domain binds to the target proteins through electrostatic interactions. Once this initial interaction occurs, the activation domain then slowly folds into a defined structure allowing for specific contacts to be made.They also suggest that this model may facilitate the multiple protein contacts that an activation domain generally makes (Hermann et al., 2001). Based on the function of the activation domain in recruiting various target proteins, a high-affinity, high-specificity interaction
4 may not be necessary or desirable. Rather, multiple low-affinity interactions may be sufficient for activator function (Frankel and Kim, 1991; Locker, 2001). Discussed above, sequence-specific transcription factors consist of an independent DNA-binding domain and a regulatory domain, which can either be an activation domain or a repression domain. It is important to note that the classification of a transcription factor as an activator or a repressor is not straightforward (Locker, 2001). This stems from the fact that the activating or repressing activity of a transcription factor is not solely an inherent property of the factor itself. Rather, transcription factor activity is context- dependent. Therefore, factors such as; the nature and distribution of DNA binding sites upstream of target genes, interactions with transcriptional complexes, protein-protein interactions with cofactors, and covalent modifications of DNA can all play a role in determining transcription factor function (Hermanns and Lee, 2001). The ability of transcription factors to adopt different functions based on the binding context plays an important role in differential gene expression during embryonic development. It allows for a single transcription factor to perform multiple functions depending on the stage of development, or the cell-type (Hermanns and Lee, 2001).
1.2.2 Mechanisms of Transcriptional Regulation As discussed above, sequence-specific transcription factors consist of independent modular domains, both of which are necessary for transcriptional regulation. The mechanism by which transcription from a target gene is regulated has been studied for many years, and multiple hypotheses have been proposed. Preliminary studies by Jacob and Monod in 1961 suggested all genes are constitutively “on” unless turned “off” by a repressor protein (Jacob and Monod, 1961). Further investigations in bacteria showing only low basal activity of certain genes, suggested the necessity of an activator of gene transcription (Ptashne, 2005). Studies in Eukaryotes, pioneered by Mark Ptshane
5 and colleagues have led to the current model of gene transcription, that of activation and repression by recruitment. According to this model, transcriptional activators are tethered to specific DNA consensus sequences upstream of target genes, and through their activation domains, they recruit the transcriptional machinery to the transcription start site (Ptashne and Gann, 1997). Additionally, activation domains can also recruit histone modifiers to remove nucleosomes, facilitating DNA access by other complexes (Ma, 2005). Repressors work through the same general mechanism of recruitment, interacting with repressing machines, bringing them to specific genes. They can recruit histone and DNA-modifying enzymes, such as the common Histone Deacetylase (HDAC) corepressor, which modify DNA structure making it inhospitable to the DNA machinery (Ayer, 1999). Based on the recruitment model, multiple proteins are brought to the DNA to modulate transcription from a target gene. As mentioned above the interactions mediating this recruitment are of varying specificity and affinity. Multiple interactions with multiple proteins however stabilize the complex, thereby enhancing specificity (Courey, 2001; Semenza, 1994). This is consistent with the notion that enhancer regions upstream of target genes function on the basis of synergism. DNA binding of individual factors to their respective sites may have little or no enhancer function. Instead, the binding of multiple factors in close proximity to one another, such that they can interact with each other in addition to interactions with their own cofactors, serves to synergistically activate transcription (Courey, 2001; Semenza, 1994). The ultimate decision as to whether a gene is expressed or not is dependent on multiple inputs. Therefore it is generally understood that gene expression is a combinatorial mechanism, dependent on the coordinated action of both positive and negative acting transcription factors (Ptashne, 2005; Semenza, 1994). Based on the fact that multiple interactions are necessary to generate a transcriptional complex with relative
6 high specificity, it is conceivable that loss of even one factor can have a profound affect on the transcription of target genes (Seidman and Seidman, 2002). Within the context of embryonic development, this can have serious implications for the normal development of tissues and organs. Among the families of transcription factors described above, the homeodomain transcription factor family plays a critical role in embryonic development and they will be discussed in the following sections.
1.3 Homeodomain Containing Transcription Factors The Homeobox, a conserved sequence of approximately 180 base pairs, was first discovered in 1984 within the homeotic selector genes of Drosophila (Banerjee-Basu and Baxevanis, 2001; McGinnis et al., 1984; Scott and Weiner, 1984). Subsequently after it’s discovery, homeobox containing genes were discovered across a wide range of species, including sponges (Gehring et al., 1994a) vertebrates (Carrasco et al., 1984), humans (Levine et al., 1984), plants (Vollbrecht et al., 1991) and fungi (Shepherd et al., 1984). The homeobox encodes a 60 amino acid domain, the homeodomain (HD), that functions as a DNA binding motif (Banerjee-Basu and Baxevanis, 2001; Qian et al., 1989). Homeodomain-containing proteins act as regulatory transcription factors regulating a wide array of developmental processes, governed by the differential expression of the genetic information in a precise spatial and temporal pattern (Billeter, 1996; Gehring et al., 1994a) Initially, homeotic genes were studied in Drosophila after they were discovered as being responsible for “homeotic transformations”. A classic example of this transformation is the Antennapedia (Antp) mutation, in Drosophila, in which the antennae of the fly is replaced by a pair of middle legs (Gehring, 1985), or the bithorax mutations which result in transformations of the third thoracic segment towards a second thoracic segment, leading to a phenotype displaying four wings (Lewis, 1978). These
7 phenotypes suggest that the normal functioning homeobox containing gene is responsible for proper segmental identity in the developing embryo. After the identification of the first homeodomain-containing protein, several hundred others were identified in a wide variety of species (Chi, 2005). These proteins are found at all levels of the developmental hierarchy, responsible for establishing morphogenetic gradients, structure formation of groups of body segments, the unique identity of single segments, cell identity and proliferation (Banerjee-Basu and Baxevanis, 2001; Billeter, 1996; Duboule, 1994)
1.3.1 Classes of Homeodomain Proteins Within the mouse genome there are approximately 260 distinct homeodomain proteins. Many of them are conserved across species, having an equivalent human or Drosophila counterpart (Berger et al., 2008). Homeodomain proteins can be classified based on different criteria including 1) sequence similarity and chromosomal clustering (Gehring et al., 1994a) 2) gene class and gene family based on phylogenetic relationships (Holland et al., 2007) as well as 3) residue identities at position 47, 50 and 54 (Banerjee- Basu and Baxevanis, 2001). There exists general inconsistencies in the literature in the way in which homeodomain protein relationships are discussed. Recent studies have attempted to provide a comprehensive review of all homeobox genes and a proper classification scheme for them. Banerjee Basu et al, have used homeodomain sequence alignment and have incorporated the three-dimensional structure of the homeodomain, rather than sequence homology alone, as one example of a way in which homeodomain proteins can be grouped(Banerjee-Basu and Baxevanis, 2001). Although there are multiple classification schemes, the most commonly recognized groups of homeodomain proteins include but are not limited to; ANTP, derived from the Antennapaedia gene (antp), PRD, paired, derived from the paired (prd) gene of Drosophila, LIM, POU, HNF, SINE, zinc finger (ZF), and TALE (Holland et al., 2007). The PRD-class is one of the
8 largest and well studied and is the class of interest to the data presented in this thesis. The PRD-class of homeobox containing genes are closely related to the Drosophila gene paired (prd)(Schneitz et al., 1993). The PRD-class proteins are characterized by a homeodomain, resembling that which is encoded by the Drosophila gene paired (prd)(Bopp et al., 1986). They can be further divided into three subclasses based on the identity of the residue at position 50, with a demonstrated role in determining the DNA binding specificity of the PRD-like homeodomains. Accordingly, the three classes are; the Serine-50 (Ser50) Pax genes, the Lysine-50 (Lys50) paired-like genes, or the Glutamine-50 (Glu50) paired-like genes (Tucker and Wisdom, 1999; Wilson et al., 1993). The Ser50 Pax homeodomain proteins are unique in that they also encode a second DNA-binding domain, the paired domain, which was also first identified in the Drosophila PRD protein(Bopp et al., 1986; Burri et al., 1989). The unique presence of a second DNA-binding motif can serve as an alternate basis for classification for the paired class such that it can also be subdivided into two groups, the Pax proteins and the Pax- like (or Paired-like) proteins, which include all the non-Pax genes (Holland et al., 2007).
1.3.2 Structure and DNA-binding Members of the same class of homeodomain proteins share a highly conserved sequence across a wide variety of species (approximately 80-100%) (Banerjee-Basu and Baxevanis, 2001). Across the classes of homeodomain proteins however, there is very little sequence similarity. However, consistent among all classes of homeodomain proteins, the general three-dimensional primary structure of the homeodomain is conserved (Kissinger et al., 1990; Otting et al., 1990; Qian et al., 1989; Vershon, 1996). The first studies on homeodomain three-dimensional structure were performed using Nuclear Magnetic Resonance (NMR) on the Antp protein homeodomain. These experiments, along with others performed on distantly related homeodomains,
9 demonstrated that the general three-dimensional structure is very similar across most classes of homeodomain proteins. The conserved structure consists of a bundle of three
α-helices, where helix I is joined to helix II by a loop, and helix II and helix III adopt a helix-turn-helix conformation. This three helix bundle is preceded by a flexible N terminal arm, which is unstructured in solution (Qian et al., 1989). The basic structure formed by helices II and III strongly resemble the helix-turn-helix DNA binding motif found in Prokaryotic transcription factors (Billeter, 1996; Wilson et al., 1993). Along with their nearly identical three-dimensional structure, homeodomains interact with DNA in a highly conserved fashion. Homeodomains make sequence- specific contacts with the DNA predominantly through helix III, the recognition helix (Gehring et al., 1994b). Helix III lies in the major groove of the DNA, while helices II and I are aligned anti-parallel perpendicular to the DNA backbone. The N-terminal tail also makes base contacts in the minor groove of the DNA (Gehring et al., 1994b). Studies in which the N-terminal arm of the homeodomain is deleted, indicated that although overall homeodomain structure does not change, DNA binding affinity decreases 10-fold as compared to the unmutated homeodomain (Gehring et al., 1994b). This suggests that interaction between the N-terminal tail and bases in the minor groove play an important role in DNA-binding specificity. A preliminary model of interaction with DNA suggests that conserved residues in the homeodomain interact with a TAAT core in both the major and minor grooves of the DNA. Recent studies have now revealed a breadth of DNA sequences recognized by homeodomains (Noyes et al., 2008; Treisman et al., 1992; Wilson et al., 1993) The preliminary studies on homeodomain structure were performed on classes of homeodomain proteins that interact with the DNA as monomers. In the PRD-class however, the predominant mode of interaction with DNA is cooperative binding on DNA through dimerization (Wilson et al., 1993; Wilson et al., 1995b). Initial studies of
10 PRD- class homeodomain binding to DNA used the P3 recognition site. All members of this class of homeodomain proteins are able to cooperatively dimerize on palindromic DNA sequences, composed of two inverted TAAT half sites, separated by a 3 base pair nucleotide spacer, TAATnnnATTA (P3) (Wilson et al., 1993). On this site, the homeodomains are arranged head-to-head on the palindromic site. When on DNA, the N-terminal arm of one homeodomain interacts with the beginning of helix II of the other. Furthermore, the N-termini of the two recognition helices approach each other, resulting in the formation of a contiguous molecular structure (Wilson et al., 1995b). This ability to dimerize on palindromic P3 sequences is a unique property of the PRD-class homeodomains. This dimerization is also cooperative, where binding of the first homeodomain enhances binding by the second 50-300 fold (Wilson et al., 1993). The magnitude of the cooperativity of binding is dependent on the identity of the residue at position 50, where Gln50 paired-homeodomains bind DNA with the highest cooperativity of approximately 300-fold (Wilson et al., 1993). As will be discussed in the next section, this mechanism of DNA-binding plays a critical role in the specificity of homeodomain binding.
1.3.3 Specificity of homeodomain protein action As described, homeodomain proteins are transcription factors, which are responsible for regulating a diversity of developmental processes. Their ability to carry out this function is dependent on sequence-specific recognition of DNA-binding sites upstream of target genes (Affolter et al., 2008; Gehring et al., 1994a). Much attention has therefore been paid to elucidating the specificity of homeodomain protein action and in determining the target range of individual homeodomains. This is complicated by the fact that early in vitro studies characterizing homeodomain DNA-binding specificity indicated that homeodomains bind DNA with relatively low sequence specificity as
11 compared to other DNA-binding motifs (Hoey and Levine, 1988; Vershon, 1996). A common theme that arises in these studies is the conclusion that homeodomain binding specificity must be considered as a combinatorial effect, determined by a number of different factors (Damante et al., 1996). Recent studies have used high-resolution analysis to determine the breadth of DNA sequences that can be recognized by the homeodomain DNA-binding motif (Berger et al., 2008; Noyes et al., 2008). Within the 5-7 base pair sequence, a wide variety of bases can be preferred at different positions and this accounts for an extensive range of specificities. Moreover, they highlight the fact that many identified binding sites can be recognized by many different distantly related factors (Noyes et al., 2008). Multiple frameworks exist within which the specificity of homeodomain DNA recognition can be considered. There is evidence that the sequence specificity of the individual homeodomains translates into specific recognition target sequences (Ekker et al., 1992). So, for example, the identity of certain residues such as 47, 50, 51, 54 and 55 of the recognition helix can all contribute to DNA binding specificity and a single amino acid change at one of these recognition positions can alter specificity (Ades and Sauer, 1995; Damante et al., 1996). For example, in the bicoid-class, the identity of the residue at position 50 is particularly important. Here, Lys50 targets the bicoid protein to the TAATCC sequence, whereas in the Antp-class, a Gln50 recognizes a TAAT(T/C)(A/G)
DNA sequence (Noyes et al., 2008). Two recent studies have attempted a large-scale analysis where bacterial one-hybrid systems as well as a Protein Binding Microarray were used to deduce a “code” which could predict homeodomain DNA-binding specificity. Although these studies determined monomer-binding preferences, that are evident in vivo, they also recognize the importance of binding context. This is due partly to the fact that closely related homeodomain proteins show different DNA-binding specificity. In the study by Noyes et al., homeodomain proteins were classified to eleven different
12 specificity clusters based on preferred recognition motifs. Sequences of homeodomain proteins within and between these clusters were then compared. A significant correlation between specificity and sequence homology was observed. However, multiple examples did exist where closely related protein, based on sequence homology, were grouped into different specificity clusters (Noyes et al., 2008). The observation that homeodomain binding specificity is not solely dependent on its amino acid sequence, suggests, therefore, that homeodomain proteins employ other mechanisms to specifically target DNA sequences. Many classes of homeodomain proteins contain a second DNA-binding domain, in addition to the homeodomain. It has been suggested that these additional DNA- binding motifs may serve to confer added DNA-binding specificity and affinity (Corry and Underhill, 2005; Vershon, 1996; Wilson et al., 1993). An example of this interaction arises in homeodomain proteins belonging to the POU class. These proteins contain a POU domain, which is a bipartite structural motif consisting of a POU specific domain (POUs) and the POU homeodomain (POUHD) (Ingraham et al., 1990). Pit-1 is a member of this group and through mutational analysis, it has been found that the Pit-1
POUHD alone is required and sufficient for low-affinity DNA-binding with moderate specificity. The POU specific domain however, is necessary to confer site-specific, high- affinity binding (Ingraham et al., 1990). This therefore provides an example by which homeodomain specificity is conferred by a protein sequence outside of the homeodomain itself. Homeodomain proteins can interact with DNA as monomers and both as homodimers and heterodimers. They can also recruit other cofactors to bind to the DNA in a higher order complex. It is these other modes of interaction, which are suggested to also account for DNA binding specificity. In the case ofHox genes, which encode the HOX proteins, the largest family of homeodomain proteins, there has been
13 much attention paid to the roles of cofactors in defining specificity. Hox genes display overlapping regions of expression in the developing embryo, suggesting that within a given cell, more than one HOX protein can be expressed (Chariot et al., 1999). In vitro, many HOX proteins display very similar binding specificities, whereas targeted knockout studies of individual Hox genes result in specific phenotypes suggesting that,in vivo, these proteins interact with target sequences in a specific manner (Lufkin, 2003). To address this issue, therefore, studies have looked at the role of protein-protein interactions in determining in vivo specificity and targeted gene regulation. The TALE class of homeodomain proteins have a well-defined role as interacting partners of HOX proteins (Lufkin, 2003). PBX proteins in vertebrates fall within this family, and they have been shown to form heterodimers with HOX proteins (Chang et al., 1995; van Dijk and Murre, 1994). HOX proteins individually recognize highly related sites with similar affinities, however, through the formation of heterodimer complexes with PBX proteins, greater binding specificity is achieved. This is due in part to specification of target sequence bases by both HOX and PBX proteins (Lufkin, 2003; Sanchez et al., 1997). This interaction is only one of many examples in which HOX proteins interact with cofactors to achieve versatility in gene regulation. The dimerization of homeodomain proteins on DNA is another method by which these proteins achieve DNA binding specificity (Wilson et al., 1993). As discussed above, the ability to cooperatively dimerize on DNA is a unique property of the PRD- class homeodomain proteins. David Wilson and colleagues have demonstrated that members of the PRD class optimally bind to palindromic DNA sequences composed of two 5’TAAT’3 half sites. Furthermore, they have shown that the homeodomain proteins bind these DNA sequences as cooperative dimers, whereas other homeodomain protein classes bind DNA as monomers. These palindromic sequences confer a higher degree of target specificity in that they are longer, and occur less frequently throughout
14 the genome (Wilson et al., 1993). DNA binding specificity of this class is determined by the identity of a single amino acid residue at position 50 within the paired type homeodomain. Specifically, within the PRD class, members can have a Serine, Lysine, or Glutamine at position 50, serving to define three subgroups, each with distinct DNA- binding characteristics. These characteristics can be distinguished on 1) the basis of the specificity of nucleotide spacing between TAAT half sites, 2) the magnitude of the cooperative interaction and 3) the identity of the base pairs in the center of the recognition sequence (Wilson et al., 1993). The formation of heterodimeric complexes between different members of the PRD-Class, provides an additional mechanism of achieving DNA-binding specificity (Tucker and Wisdom, 1999). As transcription factors that play an important role in embryonic development, members of the PRD-Class, along with all homeodomain proteins, may display overlapping expression at any given point in the embryo at different stages of embryogenesis. It has been suggested that PRD-class homeodomain proteins expressed in the same cells may form heterodimers and create transcriptional complexes with functions distinct from the homodimers formed by each individual protein (Tucker and Wisdom, 1999). This is exemplified inDrosophila , where PRD- Class members Paired (Prd) and Gooseberry (Gsb) are expressed in the same cells at the same time during development and furthermore where dimerization of an inactive Prd protein with Gsb on DNA abrogates Gsb function (Morrissey et al., 1991). The additional DNA binding specificity conferred by the formation of a heterodimers is also seen in theAlx4 – Gsc heterodimeric complex(Tucker and Wisdom, 1999). Alx4 (Aristaless-like 4) and Goosecoid (Gsc) are both members of the PRD class of homeodomain proteins, where Alx4 belongs to the Gln50 subgroup, while Gsc belongs to the Lys50 subgroup. Studies by Tucker et al. found that the Alx4 homodimers, the Gsc homodimers, and the Alx4/Gsc heterodimers all exhibit unique DNA binding properties, differentiating between different
15 P3 elements based on the identity of the nucleotides separating the two half sites (Tucker and Wisdom, 1999). The multiple examples of mechanisms by which homeodomain containing transcription factors specify their target range clearly demonstrates the context dependent action of transcription factors. Although many developmental pathways have been delineated to date, there is still a great deal that is unknown about specific interactions and modes of specifying targets of transcriptional regulation. Two transcription factors with critical roles in embryonic development are Pax3 and Alx4 and they will be now be discussed in greater detail.
1.4 Pax3 Pax3 belongs to the Pax gene family, which encodes a group of transcription factors, which play a wide variety of important roles in early embryonic patterning and organogenesis(Goulding et al., 1991; Underhill, 2000). In mammals, the Pax gene family consists of nine members of genes with homologs in worms, flies, frogs, fish and birds(Chi and Epstein, 2002).
1.4.1 Pax Protein Structure All Pax proteins are characterized by the presence of the 128 amino acid paired domain (PD) DNA-binding domain whose activity was first characterized in the
Drosophila protein Paired (Figure 1.1) (Treisman et al., 1991). In addition, the nine mammalian Pax proteins (Pax1-Pax9) can be subdivided on the basis of additional motifs within their protein primary structure (Underhill, 2000). Preliminary studies investigating the DNA- binding activity of the paired domain identified theDrosophila even-skipped (e5) promoter as a binding target (Treisman et al., 1991). Subsequent studies demonstrated that most Pax proteins, through their paired domain, could bind this
16 promoter with similar affinity (Czerny et al., 1993). This observation however, is difficult to reconcile with the specific roles for the individual Pax proteins in development. Extensive studies on Pax proteins to date have identified functional differences in Pax structural motifs that are thought to account for their different specificities. The paired domain (PD) is a bipartite
paired domain octapeptide homeodomain transactivation €1 €2 €3 €4 €5 €6 repeat €1 €2 €3 domain structure, comprised of Pax3 Figure 1.1 Schematic of Pax3. Pax3 consists of 2 DNA-binding two subdomains, PAI domains, the paired domain and the homeodomain. In addition it con- tains an octapeptide repeat and a C-terminal transactivation domain. (N-terminal) and RED The paired domain bipartite structure is represented as two subdomains consisting of three a-helices. The homeodomain also consists of three (C-terminal) (Xu et al., helics regions e 1-3. 1995). Each of these is comprised of three α-helices. The two C-terminal helices of each subdomain fold into a helix-turn-helix (HTH) motif and have the capacity to bind DNA. Specifically, these HTH motifs can make specific base contacts in the major groove of the DNA. The sequence connecting the PAI and RED domains makes base contacts in the minor groove.
Furthermore, the PAI subdomain is preceded by a β-turn and β-hairpin, and these have been demonstrated to also interact with DNA, through the DNA backbone and the minor groove (Xu et al., 1995). Initial studies of PD binding to DNA were performed using the Paired protein. Here, the crystal structure of the PD bound to DNA demonstrated that only the PAI subunit made base pair contacts to the binding site(Xu et al., 1995). Subsequent experiments with different Pax proteins however have shown that Pax proteins differ in their ability to use the C-terminal subdomain (Underhill, 2000). Pax 6 requires both N- and C-terminal subdomains to function in vivo, while isoforms of Pax6 and Pax8 are able to interact with the DNA solely through the RED subdomain (Epstein et al., 1994b). Pax3 is able to recognize sequences using either the PAI subdomain alone or the PAI and RED subunits together (Epstein et al., 1994a; Fortin et al., 1998). This
17 differential use of the C-terminal subdomain for sequence recognition by individual Pax proteins is suggested as a means by which target genes are specified (Underhill, 2000). In addition to the paired domain, Pax3, Pax4, Pax6, and Pax7 harbour a second DNA-binding domain, the paired(prd)-type homeodomain(Chi and Epstein, 2002).The structure of the prd-type homeodomain has been discussed above. In the context of the Pax proteins, the homeodomain is of the Ser50 type. Therefore, these homeodomains have the ability to cooperatively bind on palindromic sequences of the P2 and P3 variety,
5’TAATn(2-3)ATTA’3, with a strong preference for P2 elements (Tucker and Wisdom, 1999). Additionally, residue 50 has been implicated in specifying the two nucleotides immediately 3í to the TAAT core (Wilson et al., 1993). A recent study by Birrane et al., has elucidated the crystal structure of the Pax3 homeodomain (HD) on the P2 element. They have shown that the Pax3 HD adopts a globular structure similar to that of other paired-type homeodomains as described above(Birrane et al., 2009). In addition to the Pax proteins harbouring a full-length homeodomain, Pax2, Pax5 and Pax8 contain only the amino terminal helix I of the homeodomain (Dahl et al., 1997) (Dahl, 1997). This partial homeodomain lacks DNA-binding activity, however it has been shown to mediate protein-protein interactions. Specifically, in Pax5, this region is necessary for its interaction with the retinoblastoma protein, Rb, and TBP. Interestingly, Pax5 interacts with Rb through the same region of the homeodomain as Pax3, suggesting that this may be a conserved interaction between Pax family members and Rb proteins (Eberhard and Busslinger, 1999). The octapeptide is an additional motif conserved in all Pax proteins except Pax4 and Pax6 (Underhill, 2000). This motif can mediate protein-protein interactions, and in Pax2 and Pax8, this motif acts as a transcriptional repressor domain (Chi and Epstein, 2002). Transcriptional repression by Pax5 is mediated by the Groucho family of corepressors, which interact with Pax5 through its octapeptide motif (Eberhard and
18 Busslinger, 1999). This activity has also been confirmed through the deletion of this motif, and is suggested to play a similar role in Pax3 (Hollenbach et al., 1999; Smith and Jaynes, 1996). The Proline-Serine-Threonine-rich carboxy terminus of Pax proteins serves as a transactivating domain. This domain is characteristic of other transcriptional activators (Underhill, 2000). In Pax3, the carboxy-terminal transactivation domain plays a critical role in determining sequence-specific DNA-binding of the homeodomain(Cao and Wang, 2000). The Pax3 C-terminus also contains two (L/P)PXY “PY” domains with a demonstrated capacity for mediating protein-protein interactions, specifically withWW- motifs (Murakami et al., 2006). The Pax proteins therefore can be classified based on the presence or absence of different structural motifs. Four families of Pax proteins emerge; Pax1/Pax9, Pax2/Pax5/ Pax8, Pax3/Pax7 and Pax4/Pax6 (Underhill, 2000). This thesis focuses on Pax3 and so it will now be discussed further.
1.4.2 Pax3 Paired Domain – Homeodomain Interdependence The Pax3 paired domain and the homeodomain are individual DNA-binding motifs found in naturally occurring proteins either separately or together. Therefore, they have each have the capacity to mediate DNA-specific contacts (Jun and Desplan,
1996). In the context of Pax3 however, several studies have demonstrated a functional interdependence between the paired domain and the homeodomain (Apuzzo et al., 2004; Fortin et al., 1997; Fortin et al., 1998). This interdependence is best exemplified by point mutations in either the PD or HD, which serve to affect DNA binding by not only the mutated domain, but also the reciprocal un-mutated DNA binding domain (Underhill et al., 1995). Preliminary studies were performed using the Splotch-delayed (SpD) Pax3 mutation. This mutant is characterized by a glycine to arginine point mutation (G42R) in
19 the β-turn preceding the PAI subdomain of the paired domain. These studies showed that the SpD mutation in Pax3 impaired DNA binding activity to the even-skipped promoter (e5) by a factor of 17. Described above, the e5 promoter can be bound by most PD- containing proteins, and based on the specificity of Pax3 interaction with this promoter, a 17 fold decrease in DNA binding activity represents a defect in paired domain function (Underhill et al., 1995). Simultaneously, these studies also examined homeodomain DNA-binding activity in these mutants. On the P2 element, a known homeodomain recognition sequence, DNA activity was similarly affected (Underhill et al., 1995). Further studies investigating this functional interdependence have demonstrated that the converse is also true. When point mutations occur in the homeodomain, such as R53Q, which occurs in Pax3 in Waardenburg Syndrome patients, DNA-binding is also abrogated by the paired domain (Apuzzo et al., 2004). A number of additional mutations found in Waardenburg Syndrome patients studied by Anouk Fortin and colleagues confirmed this functional interdependence (Fortin et al., 1997). Interestingly, they were able to show that paired domain point mutations could modulate homeodomain activity in three ways, a complete loss, a reduction, or an increase in DNA binding activity. The ability of the paired domain to modulate DNA binding activity through the homeodomain has also been studied through the use of Pax3 chimera proteins (Fortin et al., 1998). In the study by Fortin et al, the Pax3 paired domain was fused to the paired- type homeodomain (HD) of PHOX. While the Pax3 HD contains a serine at position 50, the PHOX HD contains a glutamine at this position, imparting to them different DNA- binding specificities. Their studies demonstrated that when the Pax3 PD was fused to the PHOX HD, this homeodomain recognized and bound to DNA sequences normally recognized by the Pax3 HD. They concluded therefore that the paired domain of Pax3 was able to modulate the DNA binding specificity of the PHOX homeodomain (Fortin et al., 1998). In the G42R paired domain point mutation, deletion of helix 2 of the PAI
20 subdomain restored the dimerization of the Pax3 homeodomain on P2 oligonucleotides. Therefore it suggested that helix 2 is responsible for transmitting the detrimental effect of paired domain mutations to the homeodomain (Fortin et al., 1998). This was speculated to be due in part to the three-dimensional structure of the Pax3-DNA bound complex, in which the paired domain and the homeodomain bind to composite sites on opposite sides of the DNA, and as a result the N-terminal arm of the homeodomain lies in very close proximity to helix 2 of the PAI subdomain (Apuzzo and Gros, 2007).
1.4.3 Pax3 Transcriptional Activity The function of Pax3 as a transcription factor is dependent on its ability to bind to recognition sequences within promoter regions of target genes and to modulate gene expression. In vitro, the most common Pax3 recognition element is derived from the Drosophila even-skipped promoter e5 (Underhill et al., 1995). This sequence contains an upstream ATTA motif and a downstream GTT/CA/C paired domain recognition site (PRS) (Chalepakis et al., 1994a). Consistent with data showing a functional interaction between the homeodomain and the paired domain, both recognition sites are required for Pax3 binding (Chalepakis et al., 1994a). Recently, it was demonstrated that Pax3 employs alternative uses of its paired domain and homeodomain to interact with promoter sequences (Corry and Underhill, 2005). This study by Corry and Underhill demonstrated that Pax3 is able to interact with target sequences using its paired domain only, or both its paired domain and homeodomain. However, the study of downstream targets of Pax3 is complicated by the fact that very few optimal Pax3 recognition sequences have been identified. By investigating the effect of mutations in both the paired domain and homeodomain, Corry and Underhill were able to demonstrate a critical role for the PD- HD interaction in Pax3 target site recognition. For example, they found that in the Mitf promoter, which is comprised of non-canonical recognition motifs for both PD and HD,
21 high affinity binding was achieved and this was attributed to the structural relationship between the PD and HD (Corry and Underhill, 2005). Pax3 transcriptional activity has been demonstrated to be concentration dependent (Chalepakis et al., 1994b). Similar to Pax6, which also demonstrates this property, at low protein concentration levels, Pax3 can activate transcription. When the concentration of Pax3 increases in the cell, it has been demonstrated to inhibit basal promoter activity (Chalepakis et al., 1994b). The biphasic transcriptional activity of Pax3 is suggested to have biological implications specifically at different stages of development. In vivo, Pax3 can regulate transcription of several target genes including myelin basic protein, NCAM, MET, MyoD, Mitf, c-RET, Msx2 and Trp-1(Chalepakis et al., 1994b; Epstein et al., 1996; Galibert et al., 1999; Kwang et al., 2002; Lang et al., 2000; Maroto et al., 1997; Watanabe et al., 1998). Interestingly, for each gene, the Pax3 recognition sequence differs, and furthermore, they do not correspond to an optimized in vitro derived consensus sequence (Corry and Underhill, 2005; Mayanil et al., 2001). Additionally, the activity of Pax3 on these promoters can be either activating or inhibitory. This lack of consistency in Pax3 transcriptional regulation is in part accounted for by factors that physically interact with Pax3 and regulate its transcriptional activity.
1.4.4 Protein Interactions that Modulate Pax3 Activity and Function
The transcriptional activity of Pax3 during development is modulated through interactions with a variety of other factors. Interacting factors can function in different ways to effect Pax3 mediated transcription. Commonly, cofactors interact with Pax3 and modulate transcriptional activity (Kubic et al., 2008). On the Mitf promoter, Pax3 directly interacts with Sox10 to activate Mift expression in a synergistic manner (Potterf et al., 2000). Pax3 and Sox10 also directly interact to synergistically activate transcription from the c-RET promoter (Lang et al., 2000). On the c-RET promoter,
22 Pax3 interacts through a Pax3 binding site, while Sox10 is brought to the DNA through protein-protein interactions with Pax3 (Lang and Epstein, 2003). Pax3 activity on the Mitf promoter is also modulated through its interaction with TAZ, a transcriptional co- activator with a PDZ-binding motif (Murakami et al., 2006). Although Pax3 is able to activate transcription independently, interactions with co-activators allow for synergistic activation of downstream targets. Pax3 can also repress gene expression through interactions with co-repressors. Pax3 can directly interact with Groucho-related protein 4 (Grg4) to repress expression of the Dopachrome tautomerase gene (Dct)(Lang et al., 2005). Pax3 can also interact with KAP1, a known transcriptional repressor, and heterochromatin protein 1 gamma,
HP1γ (Hsieh et al., 2006). In this complex, KAP1 enhances Pax3 repressor activity. Conversely, HP1γ functions to “de-repress” Pax3 transcriptional activity by binding KAP1, attenuating its co-repressor function on Pax3(Hsieh et al., 2006). In addition to cofactors, interacting factors can modulate Pax3 function. In the context of development, Pax3 protein expression and activity must be tightly regulated. This is especially important where Pax3 function must be inhibited for terminal differentiation to occur in Pax3 expressing cells (Kubic et al., 2008). Our lab has shown that pRB family members physically interact with Pax3 and that this interaction represses transcriptional activation of promoters harbouring Pax3 binding sites (Wiggan et al.,
1998). Likewise, the human Daxx protein physically interacts with Pax3, resulting in repression of Pax3 mediated transcriptional activity by 80% (Hollenbach et al., 1999). Pax3 activity is also antagonized by its direct interaction with Msx1, where binding by Msx1 blocks the ability of Pax3 to bind to DNA (Bendall et al., 1999). Furthermore, Pax3 proteins levels can also be regulated by ubiquitination and proteosomal degradation (Boutet et al., 2007). This is particularly important in myoblasts where it is proposed that the degradation of Pax3 allows for terminal differentiation(Kubic et al., 2008).
23 Outlined above are only a few of many examples where Pax3 activity is regulated through its interaction with other proteins. Interestingly, our lab has recently shown a novel role for Pax3 as a co-activator of Lymphoid Enhancer Factor-1 (Lef-1). We have demonstrated that Pax3 can interact with Lef-1 and that this interaction results in the activation of Lef-1 dependent activated transcription (T. Christova, manuscript submitted). 1.4.5 Pax3 in Development The specific regulation of target gene expression by Pax3 plays a critical role in embryonic development. Pax3 expression can be detected by embryonic day 8.5 in the mouse (Engleka et al., 2005; Goulding et al., 1991). Here, Pax3 is first expressed in the dorsal aspect of the neural tube as well as in the region where neural crest cells are specified and delaminate from the neural tube (Engleka et al., 2005). Neural crest cells are a multipotent migratory cell population which differentiate into many cell types in the embryo including; the peripheral ganglia of the peripheral nervous system, melanocytes, enteric ganglia and Schwann cells (Weston, 1970). These cells also contribute to the development of many different organs including the adrenal glad, the thymus and the parathyroid (Conway et al., 1997). The expression of Pax3 in the these neural crest cells is necessary for their proliferation and lack of Pax3 has been demonstrated to affect survival and proliferation of both neural crest precursors as well as the migrating neural crest cells(Kubic et al., 2008). For example, Pax3 is required for the generation of melanoblasts and melanoblast precursors early on in development(Hornyak et al., 2001). It serves to promote melanocyte lineage commitment while preventing their differentiation and promoting their proliferation during migration(Lang et al., 2005). Pax3 also plays a critical role in the migration of cardiac neural crest cells to the developing heart and it is necessary for the proper formation of the heart outflow tract (Conway et al., 1997; Kwang et al., 2002).
24 It is thought to mediate this through the repression of another homeobox gene Msx2, which contains a conserved Pax3 binding site in its promoter (Kwang et al., 2002). As well, Pax3 is required for the development of enteric ganglia, and it is suggested that it exerts it role through a physical interaction with Sox10 to activate the c-RET enhancer, which also plays a critical role in enteric ganglia formation (Lang et al., 2000). Pax3 is widely expressed early on in the paraxial mesoderm flanking the neural tube(Engleka et al., 2005; Goulding et al., 1991). This mesoderm then condenses and segments into somites, which ultimately dissolve and differentiate to give rise to a variety of tissues such as bone, cartilage and dermis. As development proceeds, somites undergo a maturation process. During this process, dorsal cells of the somite develop into dermomyotome, while ventral cells develop into the sclerotome(Yusuf and Brand-Saberi, 2006). Cells from the lateral dermomyotome give rise to hypaxial muscle precursors that will migrate to the ventral body wall, the limbs, the diaphragm and the tongue(Yusuf and Brand-Saberi, 2006). Migrating myoblasts express Pax3. The expression of Pax3 in these cells is also necessary for the initiation of muscle cell differentiation (Wang et al., 2008). In these cells, Pax3 regulates expression of the muscle specific transcription factors, MyoD, Myf5 and Myogenin (Maroto et al., 1997). Once these myogenic markers are expressed however, Pax3 expression is downregulated (Tremblay et al., 1998). The ability of Pax3 to initiate myogenic differentiation has been demonstrated to be antagonized by Msx1 through protein-protein interactions, and this is thought to be necessary during migration of these precursor cells (Bendall et al., 1999). Outlined above is a wide variety of roles for Pax3 in the development of many organs and structures in the developing embryo. As expected, the loss of Pax3 activity during embryogenesis results in a breadth of developmental defects. The splotch (sp) mouse harbours a mutation in intron 3 of the Pax3 gene resulting in the production of four abberantly spliced mRNA transcripts (Epstein et al., 1993). Therefore it serves as a
25 Pax3 knockout mouse, used to study loss of Pax3 activity. Haploinsufficiency arises in the splotch mice such that heterozygous mice also display mutant phenotypes (Zhou et al., 2008). Homozygous splotch mice die in utero at around embryonic day 14.5 and exhibit neural tube defects as well as defects in the derivatives of neural crest cells. Defects are therefore seen in the enteric ganglia, in the outflow tract of the heart, as persistent trunchus arteriosus, and in the absence of the peripheral ganglia(Conway et al., 1997; Epstein et al., 1991; Franz, 1989; Franz et al., 1993; Goulding et al., 1993; Lang et al., 2000; Mansouri et al., 1994). Furthermore, splotch mutants lack hypaxial muscle derivatives (Epstein et al., 1996; Li et al., 1999). Splotch heterozygous mice are characterized by a white belly spot, consistent with the important role for Pax3 in early melanocyte development(Henderson et al., 1997). In the splotch-delayed (SpD) mutant mouse, the phenotype is less severe than in the splotch mouse, with homozygotes surviving to birth (Vogan et al., 1993). The recent use of new mouse models demonstrating loss of Pax3 activity have served to further define the role of Pax3 in development. Engleka et al. have created a new splotch allele of Pax3 that harbours loxP recombination sites. Here, the insertion of cDNA encoding Cre recombinase was inserted into the first exon of Pax3. This results in a null allele of Pax3, and can further be used to drive the expression of B-galactosidase to highlight derivatives of Pax3 expressing cells(Engleka et al., 2005). More recently, in order to overcome the early embryonic lethality in splotch mice, Simon Conway’s group has generated a floxed Pax3 allele, which resulted in a novel hypomorphic Pax3neo allele, resulting in an 80% reduction in Pax3 activity. Resulting defects showed that myogenesis of the limbs and tongue were most sensitive to reduced levels of Pax3 protein, suggesting the presence of a minimum threshold requirement for Pax3, differing in Pax3 expressing lineages(Zhou et al., 2008).
26 1.4.6 Pax3 and Disease Based on the multitude of functions of Pax3 expression, as expected, multiple disease states arise in humans when normal function of Pax3 is disrupted. Mutations in Pax3 can results in Waardenburg Syndromes I and III in which patients exhibit a spectrum of defects (Kubic et al., 2008). These include abnormalities of the central nervous system, eyes, and nose root. They can also exhibit dystopia canthorum, and pigmentation defects affecting skin, hair, and the otic pigment cells necessary for normal hearing (Hoth et al., 1993; Waardenburg, 1951). Defects in the facial region give rise to a recognizable facial appearance in these patients. The presence of these abnormalities is variable among patients and seldom will a patient display all defects (Kubic et al., 2008). These defects are seen in a number of derivatives of the neural crest cell population, such as craniofacial structures and melanocytes (Tassabehji et al., 1992). This is consistent with a role for Pax3 in the proper development of neural crest cells. There are multiple mutations that occur within Pax3 that are associated with Waardenburg Syndrome (Baldwin et al., 1992; Baldwin et al., 1995; Chalepakis et al., 1994a; Fortin et al., 1997). Most of these mutations however, occur as frameshift, or missense mutations in the region of exon 2 that gives rise to the paired domain. This results in loss of function or DNA-binding activity by Pax3 (Vogan et al., 1993).
Mutations in Pax3 can also occur in the homeodomain. Recent structural analysis of the Pax3 homeodomain harbouring Waardenburg Syndrome mutations indicated that these mutations have a destabilizing effect on homeodomain structure (Birrane et al., 2009). In addition, as described above, the Pax3 paired domain and homeodomain are functionally interdependent, and so, mutations in either domain will have a global affect on Pax3 activity. Pax3 mutations have also been implicated in a number of cancers, the most
27 commonly studied, a chromosomal translocation involving Pax3, occurring in 75% of cases of alveolar rhabdomyosarcoma (aRMS). Rhabdomyosarcoma tumor cells generally fail to differentiate into muscle cells as there is a disruption in the regulation of growth and differentiation of skeletal muscle precursor cells (Fredericks et al., 1995; Kikuchi et al., 2008; Wang et al., 2008). A common chromosomal translocation, occurs between the Pax3 gene on chromosome 2 and the Forkhead family protein FKHR, on chromosome 13, t(2:13)(q35:q14), resulting in the Pax3FKHR fusion protein(Barr, 1997; Barr et al., 1993; Shapiro et al., 1993). Pax3FKHR displays oncogenic properties by dysregulating genes involved in differentiation proliferation and apoptosis (Kubic et al., 2008). These activities are thought to arise due to the fact that it exhibits greater transcriptional activity than wild type Pax3, and also its ability to regulate gene expression of factors not previously recognized as targets of Pax3 (Begum et al., 2005; Fredericks et al., 1995). Furthermore, Daxx represses Pax3 transcriptional activity, whereas Pax3FKHR is refractory to Daxx repression; this may play a role in Pax3FKHR oncogenic properties (Hollenbach et al., 1999). As well, Pax3 plays a role in the pathogenesis of melanomas. Most primary melanoma tumors over express Pax3 (He et al., 2005; Scholl et al., 2001). In normal tissue, melanocyte precursor cells express Pax3 until the onset of melanocyte different. The aberrant expression of Pax3 in these tumors may promote melanoma progression by preventing apoptosis (Kubic et al., 2008). The above discussion confirms a critical role for Pax3 dependent transcriptional regulation in normal embryonic development and furthermore a role for abnormal Pax3 expression in oncogenesis. Further insight into mechanisms by which Pax3 transcriptional activity is regulated, as well as potential downstream targets will help to elucidate a role for Pax3 in embryonic development as well as human disease.
28 1.5 Aristaless-like 4 (Alx4) Aristaless-like 4, Alx4, belongs to the family of Aristaless related genes, which constitute a subset of the paired class of homeobox genes (Meijlink et al., 1999). Proteins belonging to this class contain a paired homeodomain of either the Q50 or K50 variety, and are defined by a conserved Paired tail domain located near the c-terminus, and its function is largely unknown(Mathers et al., 1997; Meijlink et al., 1999). The Aristaless genes play a key role in vertebrate embryology and based on biological and structural characteristics, they can be divided further into three groups
(Meijlink et al., 1999). Alx4 falls within the group 1 genes, which are expressed predominantly in the mesenchyme and mesoderm. Group II genes are predominantly expressed in the central and/or peripheral nervous system and its members include, Otp, Rx and Arx genes. Group III Aristaless related genes encode the Pitx homeodomain proteins which are characterized by a lysine residue at position 50 and furthermore by their roles in pituitary development (Meijlink et al., 1999; Szeto et al., 1996). Group I Aristaless related family, and more specifically Alx4 will be the focus of the following discussion.
1.5.1 Alx4 - Structure Alx4 is a member of the structurally highly related group of proteins that also includes Cart1 and Alx3(Qu et al., 1997a; Zhao et al., 1993; Zhao et al., 2006). These three proteins encode nearly identical paired homeodomains, sharing 90% sequence identity, and similar paired tail domains (Qu et al., 1997a). The N-terminal portions of these proteins remain highly divergent (Meijlink et al., 1999). Although their homeodomains share high sequence conservation, individual family members regulate a unique set of processes, although some overlap does exist, for example between Alx4 and Cart1(Qu et al., 1997a; Qu et al., 1999).
29 Alx4 was independently cloned by our lab and Qu and colleagues (Qu et al., 1997a; Wiggan et al., 1998). Alx4 was identified as a novel factor in a two-hybrid screen using the retinoblastoma protein p130 as bait(Wiggan et al., 1998). The Alx4 transcript contains two initiation sites such that translation results in two full-length proteins 399 and 383 amino acids in length(Qu et al., 1997a). The Alx4 protein product contains a Gln50 paired-type
homeodomain (Figure 1.2). The N-terminal region also contains Figure 1.2 Schematic of Alx4 Protein Structure. Alx4 consists of a single DNA-binding domain, the paired-like homeodomain, which can a poly proline motif mediate both protein-protein and protein-DNA interactions. Alx4 is also characterised by the “paired tail” motif at the C-terminus which is (PQPPTPQPPPAPPAPP) highly conserved among members of the Aristaless family of proteins. at residues 92-107(Boras and Hamel, 2002). Unpublished data from our lab have determined that this region complexes SH3 domains, specifically that of c-Abl, and these motifs are known to mediate protein-protein interactions. Furthermore, like other members of the Aristaless related genes, Alx4 protein is distinguished by a 22 amino acid conserved sequence at the carboxy terminus, termed the Paired Tail(Qu et al., 1997a).
1.5.2 Alx4 Transcriptional Activity
The presence of a glutamine at position 50 in the Alx4 homeodomain suggests that it is able to preferentially bind to P3 palindromic sequences. P3 recognition sites consist of two half sites separated by 3 nucleotides, 5’ATTAnnnTAAT’3(Wilson et al., 1993). This DNA binding capacity of Alx4 has been confirmed by Electrophoretic Mobility Shift Assay (EMSA) by different groups(Hudson et al., 1998; Qu et al., 1999; Tucker and Wisdom, 1999). Our lab has also shown that Alx4 can bind to a P2 recognition site, which contains a two-nucleotide spacer between half sites(Hudson et al.,
30 1998). Further investigation of Alx4 binding activity showed a preference for a T or C in the position immediately after the first half site, TAATT or TAATC respectively(Tucker and Wisdom, 1999). Alx4 also contains hydrophobic residues at position 28 (valine) and position 43 (alanine), allowing for cooperative DNA binding as either homo- or heterodimeric complexes (Wilson et al., 1995b). Alx4 is also able to bind to the P3 site as both a monomer and as a dimer. Alx4 also forms a heterodimeric complex in vivo with the closely related Cart-1, as well as with a member of the lysine50 class of paired type homeodomain proteins, Goosecoid (Gsc) (Qu et al., 1999; Tucker and Wisdom, 1999). In both cases, Alx4 preferentially binds to the P3 site whether as a monomer, homodimer, or heterodimer. Transcriptional studies have shown that Alx4 is able to optimally activate transcription from P3 containing promoters(Qu et al., 1999; Tucker and Wisdom, 1999). Furthermore, in vitro, the transcriptional activity of Alx4 is antagonized by Gsc, which normally functions as a repressor, when they are able to form a heterodimeric complex on a P3 promoter(Tucker and Wisdom, 1999). Our lab has demonstrated the ability of Alx4 to bind specifically, although weakly, to the N-CAM promoter, through a P1 consensus sequence, TAATTATTA. In the presence of Lef-1, which binds to an adjacent Lef-1 site, Alx4 binding was enhanced. Furthermore, promoter assays demonstrated that Alx4 and Lef-1 regulate NCAM promoter activity through independent sites(Boras and Hamel, 2002).
1.5.3 Alx4 in Development and Disease Like most homeodomain proteins, Alx4 plays an important role in early embryonic development. Alx4 expression is restricted to mesenchymal cells, and it is largely expressed in a diverse group of tissues whose development is dependent on epithelial mesenchymal interactions(Hudson et al., 1998). Alx4 expression is first apparent at embryonic day 8.25 in the neural crest derived mesenchyme of the
31 prospective craniofacial region of the developing mouse embryo (Qu et al., 1997a). As development proceeds, expression is detected in the first branchial arch and in the dorsal mesoderm flanking the neural tube. The highest level of Alx4 expression is seen in the undifferentiated mesenchymal cells of the developing cranium, craniofacial bones, and ribs(Hudson et al., 1998). At E10.5, a more restricted pattern of Alx4 expression is apparent in the craniofacial region, specifically over the fronto-nasal mass and also in the anterior aspect of the fore and hind limb buds(Qu et al., 1997a). A reduction in Alx4 transcripts is apparent between E15.5 and E18.5 in the structures that form the bones, while its level remain high in the mesenchymal condensations of the hair and whisker follicles(Hudson et al., 1998; Qu et al., 1997a). This mesenchymal expression of Alx4 throughout the embryo, prominently in the craniofacial region and the developing limb bud suggests a role for Alx4 in the morphogenesis and patterning of these structures. The function of Alx4 in the mouse embryo is studied using the Strong’s Luxoid
(lstD) mutant mouse (Figure 1.3) (Qu et al., 1998). These mice harbour a single R206Q point mutation, abrogating Alx4 DNA-binding capacity, such that it behaves as a functional null mutant incapable of binding target DNA elements and regulating the transcription of target genes(Qu et al., 1998). The lstD allele exhibits semi-dominant inheritance as heterozygotes also present abnormal phenotypes, most often preaxial polydactyly characterized by a single additional digit on the hind limb (Forsthoefel,
1962). Complete loss of Alx4 in the lstD/lstD homozygous mutant results in a more severe phenotype which includes craniofacial defects, extensive preaxial polydactyly of all four limbs, hemimelia (absence of the tibia), dorsal alopecia and weakness of the ventral body wall(Forsthoefel, 1962; Forsthoefel, 1963; Forsthoefel et al., 1966; Qu et al., 1998). Almost all Alx4lstD/lstD embryos die in utero due to gastrochinesis, a ventral body wall closure defect. Only 2% of these mice are viable after birth and are fertile (Boras-Granic et al., 2006; Qu et al., 1997b).
32
Dorsal Alopecia
Cranio-facial defects
Preaxial polydactyly
Weakness of the ventral body wall
Figure 1.3 Defects in Strong’s Luxoid (lstD) Mice. Strong’s luxoid mice harbour a point mutation (R206Q) in the homeodomains of Alx4, resulting in loss of DNA-binding activity. Heterozygous lstD/+ mice exhibit preaxial polydactyly on their hindlimbs only, while homozygous lstD/lstD mice exhibit preaxial polydactyly on all limbs. In addition, these mice exhibit dorsal alopecia, cranio-facial defects and weakness of the ventral body wall. Only 2% of homozygotes survive to term (far right), with the rest dying in-utero due gastrochisis. (Figure reproduced from Joshi P. “Alx4, a Stromally Restricted Homeodomain Protein, is Required for Normal Mammary Epithelial Interactions” MSc. Thesis. 2006) The polydactyly associated with loss of Alx4 activity is consistent with its demonstrated role in the anterior-posterior patterning of the developing vertebrate limb (Chan et al., 1995; Qu et al., 1997b; Takahashi et al., 1998). Alx4 expression in the anterior aspect of the developing limb bud serves to confine Sonic HedgeHog (Shh) expression to the posterior aspect of the limb bud. This is necessary for correct limb bud outgrowth and anterior posterior patterning(Qu et al., 1997b). As mentioned above, our lab has demonstrated a physical interaction between Alx4 and Lef-1 and their ability to simultaneously bind to and regulate transcription from the N-CAM promoter(Boras and Hamel, 2002). Furthermore, we have demonstrated a genetic role for this interaction, such that simultaneous loss of Alx4 and Lef1 in doubly homozygous knockout mice results in early embryonic lethality associated with defective vasculogenesis(Boras-Granic et al., 2006). In humans, loss of function of Alx4 is associated with ossification defects of the skull vault. Heterozygous mutation of Alx4 is implicated in the skull vault defect Parietal Foramina (PFM), although this phenotype is attributed to heterozygous mutations of the homeobox gene Msx2 as well(Antonopoulou et al., 2004; Mavrogiannis et al., 2001;
33 Mavrogiannis et al., 2006). More recently, epigenetic studies have identifiedAlx4 gene methylation in the early stages of carcinogenesis in colon cancer, therefore serving as a potential marker for colon cancer detection (Ebert et al., 2006). The above discussion suggests a myriad of function for Alx4 and its proper function as a homeodomain transcription factor. What remains unknown however are mechanisms by which Alx4 transcriptional activity is regulated, which ultimately determines its role in embryogenesis.
1.6 Rationale and Thesis Summary The rationale for this thesis stems from the nature of Pax3 and Alx4 as transcription factors. Pax3 and Alx4 belong to the paired-type family of homeodomain transcription factors. Factors containing the paired-type homeodomain are unique in that they have demonstrated a capacity for forming heterodimeric complexes with other paired-type homeodomain family members(Qu et al., 1999; Tucker and Wisdom, 1999). Examples of these complexes include Gsb-Prd, Alx4-Gsc and Alx4-Cart 1, and interestingly, these interactions are not limited to family members with identical residues at position 50; i.e Alx4 contains a glutamine at position 50, whereas this residue is a lysine in Gsc(Morrissey et al., 1991; Qu et al., 1999; Tucker and Wisdom, 1999). These heterodimeric complexes modulate the DNA binding specificity of partner factors such that they are able to bind to as novel sequences, other than those that they would bind to as monomers and homodimers. Emphasized in Chapter 1 is the critical role for transcriptional regulation of gene expression in the specification of cell types and proliferation, as well as in the patterning of organs and tissues during embryogenesis. The specificity of transcription factor action is therefore imperative in the activation of target genes. Currently however, there exists very little information on the way in which Alx4 transcriptional activity is regulated. Although progress has been made
34 in elucidating roles for Alx4 since its discovery 10 years ago, much work remains in characterizing its transcriptional specificity as well as its interacting partners. Along with being a candidate for forming heterodimeric complexes through its paired-type homeodomain, Pax3 also shares a similar pattern of expression with Alx4 in embryonic development. More importantly however, when Pax3 or Alx4 activity is lost, overlapping processes appear to be disturbed, specifically in the craniofacial region and in the ventral body wall, suggesting that these two factors may play a role in regulating the same processes; (Asher et al., 1996; Beverdam et al., 2001; Forsthoefel, 1962; Qu et al., 1997b; Tremblay et al., 1998; Wu et al., 2008). Therefore this thesis investigates a physical interaction between Pax3 and Alx4 with the goal of determining whether Alx4 transcriptional activity is modulated through a physical interaction with Pax3. In Section 3, I demonstrate the presence of Pax3 and Alx4 in the same cells at different stages of embryogenesis in the developing embryo. Further more, I show the physical interaction between Pax3 and Alx4 in vitro and in vivo. Additionally, the ability of Pax3 and Alx4 to coordinately regulate transcription from a model promoter is tested. Lastly in Section 4, a preliminary analysis of the genetic interaction between Pax3 and Alx4 is presented.
35 Section 2 - Materials and Methods Plasmids, Constructs and Antibodies The Alx4lstD point mutation plasmid was a gift from Dr. R. Wisdom (Departments of Biochemistry and Medicine, Vanderbilt University, School of Medicine, Nashville, TN). The GST-Pax3(HD) fusion protein was generated by PCR amplification of the Pax3 homeodomain (-200 to -283) from the full length Pax3 cDNA, which was subsequently subcloned into the PGEX2T (GE Healthcare Life Sciences) vector at the BamH1/EcoR1 sites. The homeodomain of Alx4 was cut out of the pcDNA3 Alx4 plasmid at Apa (724) and Bspe1(1001) and ligated into PGEX2T at the BamH1 site to produce the GST-Alx4 HD fusion protein. The pGL3-(P3)1 (P3-Luc) construct was generated by inserting the single P3 site oligo (5’ TCG ACA GCT AAT TAA ATT AGC TT’3) into the Kpn1 and BglII sites, upstream of a CMV immediate early promoter in pGL3-Basic (Promega). Alx4 deletion mutants were previously available in the lab (Boras and Hamel, 2002; Wiggan et al., 1998), and generated by cutting at specified restriction sites and religating the vectors. The pMT2-
Pax3G42R and pMT2-Pax3G42R∆65-79 Pax3 mutants were a kind gift from Dr. P. Gros (McGill University, Montreal, Canada). The GST fusion construct of Alx4 was generated by restriction digest of pcDNA3-HA-Alx4 (BamH1/Sma1) and cloned into the BamH1/ Sma1 sites of the PGEX2T vector (Wiggan et al. 1998). The mouse monoclonal antibody against Alx4 was previously prepared in the lab (Hudson et al. 1998). The monoclonal
α-Flag, α-GFP antibodies were used to detect flag and GFP tagged proteins (Applied Biological Materials Inc., Canada). Antibody to mouse Pax3 (α-Pax3) was obtained from the Developmental Studies Hybridoma bank (maintained by the University of Iowa, Iowa, IA).
Cell Culture, DNA Transfections Cos-1 and HEK 293 cells were cultured in Dulbecco’s minimal essential medium
36 supplemented with 10% fetal bovine serum (Sigma) and 2mmol/L L-glutamine. Cos-1 cells were transfected at 90% confluency with either Lipofectamine 2000 (Invitrogen) or polyethylenimine (PEI) (Sigma) as per manufacturers instructions. Cells were harvested 48 hrs after transfection.
GST Fusion Proteins and Binding Assay Bacteria expressing GST fusion proteins were cultured in 2ml LB medium overnight with
100mg/ml Ampicillin. 200µl of this culture was used to inoculate 20ml LB medium with ampicillin and this was grown overnight with shaking at 37°C. The 20ml culture was then added to 200ml LB with ampicillin and grown for 1 hour at 37°C with shaking. Bacterial growth was then induced with 200mM IPTG for 3 hours at 30°C. Flasks were then placed on ice for 10mins and then cultures were spun at 4°C, 5000RPM, 15 mins. The pellet was then resuspended in 5ml FPLB (500mM NaCl, 1% Triton X, 50mM Hepes, 2mM EDTA, 10% Glycerol) plus protease inhibitors and cell suspension was sonicated 4 times, for 30secs.
This was then spun at 10,000RPM for 10mins at 4°C. GST proteins were then purified on glutathione-Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. An equivalent amount of GST and GST-fusion proteins bound to glutathione beads were incubated with COS-1 whole cell lysates containing over-expressed proteins. These were rocked at 4oC for 2 hrs. The glutathione beads were then washed in a Nonidet
P-40 lysis buffer (1% Nonidet P-40, 120 mM NaCl, 50mM Tris pH 8.0). Bound proteins were eluted from beads by boiling in 2x SDS buffer for 5mins before loading onto 10% SDS-polyacrylamide gels.
Immunoprecipitation and Western Blot COS-1 cells were transfected as described above with various expression constructs and cells were lysed 48hrs post-transfection in a Nonidet P-40 lysis buffer. Immunoprecipitation
37 was performed with mouse anti-Alx4 hybridoma cell supernatant at a dilution of 1:5. 20ul of protein G-sepharose beads were then added and incubated for 2 h. Immunocomplexes were washed three times with ice-cold 1%NP40 lysis buffer and separated on 10% SDS- Polyacrylamide gels. Nitrocellulose membranes were blocked for 1h with 5% skim milk power in PBS + 0.1%Tween. Blots were incubated with primary antibody overnight at a dilution of 1:40 for Pax3 Hybridoma cell supernatant antibody, followed by another hour of incubation with the appropriate secondary horseradish peroxidase antibody. Co- immunoprecipitated complexes were detected using the Mouse True Blot Secondary Antibody® (EBioscience) to minimize interference of signal of heavy chain at 55kDA with protein signal at same approximate size. Blots were developed using ECL fluorescent detection kit according to the manufacturers instructions (GE Healthcare).
Luciferase Reporter Assays and B-Galactosidase Assays COS-1 cells were transfected using Polyethylenimine (PEI) (Sigma) as per the manufacturers instructions. Cells were seeded onto 6-well plates and transfected with the appropriate combination of plasmids in triplicate. 48 hours post transfection, cells were lysed and luciferase activity was measured using the Luciferase Reporter Assay System (Promega).
Cells were co-transfected with a CMV-β-galactosidase expression plasmid as an internal control and Luciferase activity was normalized to β-galactosidase levels. Total amounts of transfected DNA were kept constant by the addition of an empty expression vector. Equal concentrations of lysates proteins were prepared for Western blotting as explained above to detect levels of protein expression.
Immunofluorescence Immunofluorescence was performed on paraffin embedded sagittal sections of paraformaldehyde fixed embryos at different days post coitus. Embryos were paraffin
38 embedded and re-hydrated and antigen retrieval was performed 3 times 5 mins in 10mM Sodium Citrate. Pax3 Hybridoma Cell Supernatant was used at a dilution of 1:2 and mouse anti-Alx4 monoclonal antibody (Hudson et al., 1998) was used at a dilution of
1:5 and incubated at 4°C overnight. Alexa Fluor 488 specific to IgG1 and Alexa Fluor
568 (Molecular Probes) specific to IgG2a secondary bodies were incubated for 1 hour at room temperature. Slides were mounted using VectaShield mounting medium (Vector, Burlingame, CA, USA).
Animals and Genotyping The functionally null allele of Alx4, lstD, from the inbred strain, Strong’s luxoid (Forsthoefel, 1963) was back crossed (>10 generations) onto C57Bl/6 mice. Mice homozygous for the lstD allele phenocopies mice with a complete knockout of Alx4 (Qu et al. 1997). Mice heterozygous for the null Pax3 allele, C57BL/6J-Pax3Sp/J, were aquired from The Jackson Laboratory. Mice heterozygous for both the null allele of Pax3 (Pax3sp-j/+) and the lstD allele of Alx4 (lstD/+) were then crossed to generate animals homozygous for both mutant alleles. Alx4 genotyping has been described previously (Boras-Granic et al., 2006). For genotyping of the sp-j allele, two PCR reactions were performed. The mutant sp allele arises from a single nucleotide deletion near the 3’ end of intron 3. The forward primer in intron 3 was 5’ –GAG AGG GTT GAG TAC GTT AGC-3’. The wild-type reverse primer was 5’-CGG CTG ATA GAA CTC ACA CAC-3’ and the mutant reverse primer was 5’-
CGG CTG ATA GAA CTC ACA CAC-3’ ( 94°C, 60°C, 72°C, 35 cycles). A mutant and wild-type reaction were performed for each sample, and the PCR product was run out on a 2% agarose gel. The presence of a 110 bp band in both reaction lanes was indicative of a heterozygous phenotype.
39 Section 3 - Results 3.1 Overlapping Expression of Pax3 and Alx4 in the developing embryo Previous studies from our lab and others have characterized the expression pattern of Alx4 during specific stages of embryogenesis (Hudson et al., 1998; Qu et al., 1997a). Interestingly, Alx4 is expressed in developing somites and in specific structures such as the first branchial arch, from which the craniofacial aspects of the embryo are derived. Cells in these same structures also express Pax3 and both of these transcription factors play important roles in early embryonic development(Goulding et al., 1991). In order to determine whether they are expressed in the same cells of the developing embryo, the overlapping expression pattern of Pax3 and Alx4 was determined by indirect immunofluorescence using mice at various embryonic stages (Figure 3.1) As Figure 3.1 illustrates, progressively greater coincident expression of Pax3 and Alx4 occurs between
9.5 and 12.5 dpc. For example, both factors were observed in the dermomyotome (DM; Figure 3.1A-C), Pax3 being expressed in all of the cells in this transient structure while Alx4 expression was restricted to a subset of cells in the central region of the dermomyotome, the latter reminiscent of the expaxial-hypaxial boundary of this structure(Borycki et al., 1999). Greater overlapping expression can be observed 24 hours later in, for example, the first branchial arch (BA; Figure 3.1D-F). This overlap continues in the derivatives of the first branchial arch. Specifically, the apparent mandible at 11.5 continues to express Pax3 and
Alx4 in the same cells, although Meckle’s cartilage (MC) shows staining for both of these factors at levels approaching background. Significant overlapping expression for Pax3 and Alx4 is also observed in the maxillary mesenchyme adjacent the nasal epithelium at 12.5
(Figure 3.1J-L). Thus, progressively greater co-expression of Pax3 and Alx4 is observed in a number of developing tissues during embryogenesis, beyond those presented here, but particularly in a number of elements in the mesenchymal portion of the craniofacial region.
40 Alx4 Pax3 Merge A B C
9.5 DPC DM
DR E F
10.5 DPC BA
G H I
11.5 DPC
MC
J K L
12.5 DPC NM
Mx NE
Figure 3.1 - Overlapping expression between Pax3 and Alx4 in the developing mouse embryo. Embryos between 9.5 and 12.5 dpc were sectioned and stained for co-immunofluorescence using istoype specific antibodies to Pax3 and Alx4. A-C) Shows a restricted region of co-expression at 9.5 dpc in a small region of the presumptive hypaxial-expaxial boundary in the dermomyotome. Increasing overlapping ex- pression arises at later stages of development. Panels D-F represent the first branchial arch at 10.5dpc and overlapping expression is seen in F in the distal mesenchyme. I shows overlapping expression in the lower mandible at 11.5 dpc and L shows overlapping expression at 12.5 dpc in the maxillary mesenchyme adja- cent the nasal epithelium. DM – dermomyotome; BA – 1st Branchial Arch; MC – Meckle’s Cartilage; MX- Mesenchyme of Maxillary Region; NE – Nasal Epithelium; NM – mesenchyme rostral to nasal epithelium.
41 To validate the specificity of the antibodies used in these immunofluorescence experiments, further staining was performed. In the splotch (spJ) Pax3 knockout mouse, a mutation occurs within intron 3 of the Pax3 gene, and this results in the production of four aberrantly spliced mRNA transcripts(Epstein et al., 1993). The resulting protein products lack the C-terminus recognized by the Pax3 antibody. Therefore, in these mice, Pax3 is not detected by immunofluorescence. Therefore immunofluorescence was performed on spJ/spJ lstD/+ mice, in the distal mesenchyme of the first branchial arch, where Pax3 expression has been demonstrated in wild type embryos (Figure 3.1 E, Figure 3.2 D). In
A Alx4 Pax3 Merge A B C
B Merge
Alx4 Pax3 Merge D E F
Figure 3.2 - Validation of Specificity of Antibodies used for Immunofluorescence Embryos taken at 9.5 and 10.5dpc were paraffin embedded and sectionned for immunofluorescence.A-C Shows expression for Alx4 and Pax3 in the distal tip of the first branchial arc at E10.5 in a wild type embryo and overlap- ping expression in cells of this region. D-F Staining for Alx4 and Pax3 in spJ/spJ lstD/+ embryos taken at E10.5. Splotch embryos (spJ) lack a functional Pax3 protein that can be reconized by the Pax3 antibody. B) Overlapping expresion of Pax3 and Alx4 at the proximal limb bud at the dermomyotome. Cells exclusively expressing Pax3 are seen in a field of Alx4, presumably migrating myoblast precursors known to express Pax3.
42 the spJ/spJ lstD/+ embryos, as expected, Pax3 expression is not observed, and in the merged fields, only Alx4 expression is visible in these cells, suggesting that the Pax3 antibody is specific for the full length Pax3 protein. Furthermore,Figure 3.2 B exhibits a region in the developing embryo, at the limb bud, where Alx4 and Pax3 expression occurs in mutually exclusive cells, suggesting that the overlapping expression patterns are a function of their expression in the same cells and not the antibodies. Previously our lab has demonstrated the specificity of the Alx4 antibody through the use of a blocking peptide directed against the Alx4 antibody (N. Mohabir, Alx4 Expression in the Normal Breast and in Breast Cancer, MSc. Thesis)
3.2 Alx4 Stably Interacts with Pax3 Immunofluorescence data showing overlapping Pax3 and Alx4 expression ina subset of cells in the developing embryo suggested that these two transcription factors might interact at the molecular level. Individual loss of Pax3 or Alx4 activity in the developing embryo results in developmental defects that arise in the same embryonic structures. This suggests that these factors may be regulating the same developmental processes. Furthermore, Pax3 and Alx4 belong to the family of PRD-type homeodomain proteins, which demonstrate the capacity to form heterodimeric complexes between different family members (Tucker and Wisdom, 1999). Therefore, a physical interaction between Pax3 and
Alx4 was investigated (Figure 3.3). The interaction between endogenous Pax3 and Alx4 was tested in vivo in the Rhabdomyosarcoma (RD) cell line. RD cells were harvested and whole cell lysates were immunoprecipitated with the Alx4 antibody and the interaction was detected by immunoblotting with the Pax3 antibody (Figure 3.3A). Endogenous levels of both Pax3 and Alx4 were detected in input lysates recognized by the relevant antibody.
Whole cell lysates were also treated with an irrelevant α-GFP antibody, and the lack of complex formation confirms the specificity of the interaction.
43 Both Pax3 and Alx4 are DNA binding proteins, suggesting that their interaction may be mediated by DNA interactions. Furthermore, members of the PRD-type family of homeodomain proteins, to which Pax3 and Alx4 belong, have the capacity to from heterodimeric complexes with other family members, and this dimerization has been shown to occur on DNA (Qu et al., 1999; Tucker and Wisdom, 1999).
A B
Pax3 + Alx4 + Pax3 Pax3 + Alx450ug/ml EtdBr Pax3 Pax3 + Alx4 72 72 10% Input IP: α Alx4IP: α −GFP 10% Input IP: α Alx4 55 55 IgG 43 43 55 10% Input IgG IP: αAlx4 WB: α−Pax3 WB: αPax3 WB: α Pax3 WB: α Alx4
50ug 200ug /ml /ml C EtdBr concentration
72
55 IgG
43
Pax3 + + + + Alx4 + + + + IP: αAlx4 - + + +
WB: αPax3 Figure 3.3 - Interaction between Pax3 and Alx4 A) Coimmunoprecipitation of endogenous Alx4 and Pax3, in the human derived Rhabdomyosarcoma cell line, RD. Whole cells lysates of RD cells were sub- jected to immunoprecipitation using mouse monoclonal a-Alx4 antibody or a-GFP as a negative control. Immunoblotting with a-Pax3 monoclonal antibody reveal co-immunoprecipitation between endogenous Pax3 and endogenous Alx4 B-C) Immunoprecipitation of Pax3 and Alx4 in the presence of DNA interca- lator EtdBr. Cell lysates transfected with Pax3 and co-transfected with Pax3 and Alx4, were immunopre- cipitated by a-Alx4 in the presence of 50ug/ml or 200ug/ml EtdBr. Immunoblotting with a-Pax3 reveals retained complex formation in the presence of increasing concentrations of EtdBr. Arrow with * denotes IgG heavy chain. True Blot(r) mouse secondary antibody was used for coimmunoprecipitation to diminsh signal of heavy chain to prevent interference with signal of Pax3 at approximately the same size ~ 55kDA.
In order to test whether the interaction between Pax3 and Alx4 is DNA dependent, co-immunoprecipitation was performed in the presence of ethidium bromide (EtDBr), which disrupts protein-DNA interactions (Lai and Herr, 1992). Cos-1 cells were transiently transfected with either Pax3 alone or co-transfected with Pax3 and Alx4, and whole cell lysates were treated with either 50µg/ml or 200µg/ml EtdBr before the expressed proteins
44 were immunoprecipitated with the Alx4 antibody (Figure 3.3 B&C). Immunoblotting with the Pax3 antibody detected complex formation between Pax3 and Alx4. In the presence of 200ug/ml EtdBr complex formation is still detected indicating that in vitro, Pax3 and Alx4 can form a complex that is not dependent on the presence of DNA (Figure 3.3C). In order to determine the specific domains through which Pax3 and Alx4 interact, deletion mutants were expressed and used in binding assays. Figure 3.4A shows a schematic of the Pax3 deletion mutants. Large deletions in either the paired domain or the homeodomain are present in the Pax3∆Xma and Pax3∆C/H variants respectively. In addition, a more subtle point mutation is present in the Pax3G42R mutant, specifically in the β-turn preceding the paired domain, abrogating DNA binding through the paired domain while simultaneously strongly reducing binding by the homeodomain (Fortin et al., 1998). Introduction of the small deletion in helix 2 of the paired domain in Pax3 G42R∆65/79 maintains the block to paired domain DNA-binding but rescues the ability of Pax3 to bind DNA through its homeodomain. (Fortin et al., 1998). Cos-1 cells were transiently transfected with Pax3 and the Pax3 deletion mutants, and whole cell lysates were used in GST pull down assay with bacterially expressed GST and a GST-Alx4 fusion protein (Figure 3B). Complex formation was detected by immunoblotting with the Pax3 antibody, and while the wild-type Pax3 protein demonstrates binding to the GST-Alx4 fusion protein, binding is lost in the Pax3∆C/H mutant. Furthermore, only an apparently weak interaction between Alx4 and the paired domain deletion mutant, Pax3∆Xma, was observed. In contrast, both the G42R and the G42R∆65/79 mutants avidly associated with Alx4. These results suggested that the homeodomain of Pax3 is necessary for its interaction with Alx4. In order to verify that the homeodomain of Pax3 is necessary and sufficient for the interaction, a GST-fusion protein harboring only the homeodomain of Pax3 (Figure 3.4A schematic illustrates GST-Pax3(HD)) was bacterially-expressed and used in GST-pull
45 A paired domain octapeptide homeodomain transactivation €1 €2 €3 €4 €5 €6 repeat €1 €2 €3 domain wild type
∆Xma
G42 R∆65-79 G42 R G42 R G42 R ∆C/H GST-Pax3 (HD) GST
B
C GST GST -Alx4 kDA
72 untransfectedPax3 Pax3 G42RPax3 G42R∆65/79Pax3 ∆C/HPax3 ∆Xma 55 10% Input 55kDA 43 WB: α-Pax3
Figure 3.4 -Pax3 Deletion Mutant Analysis a) Schematic of Pax3 deletion mutants b) GST Pulldown assay with Cos-1 cells transiently transfected with Pax3 and Pax3 deletion untransfectedPax3Pax3 G42RPax3 G42R∆65/79Pax3 ∆C/HPax3 ∆Xma untransfectedPax3 Pax3 G42RPax3 G42R∆65/79Pax3 ∆C/HPax3 ∆Xma mutants. Whole cell lysates were used in Pull down assays with bacterially expressed GST 55kDA and the GST-Alx4 fusion protein and binding was detected by immunoblotting with m-aPax3. Interaction is lost between Alx4 and Pax3 C/H, while Pax3 Xma shows decreased + GST + GST-Alx4 binding to GST-Alx4 c) Coomassie stain of WB: α-Pax3 WB: α-Pax3 10ug GST and GST-Alx4, where the GST protein is expressed to a greater extent than GST-Alx4 d) GST Pulldown assay with Cos-1 transiently transfected with Alx4 and the homeodomain mutant of Alx4, Alx4lstD. D Whole cell lysates were used in pull down Alx4 Alx4 lstD Alx4 Alx4 lstDAlx4 Alx4 lstDE GST GST -Pax3(HD) assays with GST and GST -Pax3 (HD) and kDA binding was detected by immunoblotting with m-aAlx4 55kDA 72 55
43 10% Input GST GST- Pax3(HD) WB:α- Alx4
46 A B GST- Alx-4(HD) Alx-4 Alx-4 D Alx-4 Alx-4 Alx-4 pcDNA3 Alx-4 lstD ∆Sma ∆WW ATG Bam
Alx4 Nco 5%Input Control
10% Input Alx4 ATG Pax3 ∆WW ATG ATG Alx4 Pax3 ΔC/H ∆Sma
Alx4 GST GST Bam SH3-binding Alx4 domain Control
Nco GST Alx4 GST
Pax3 R206 Q lstD
Pax3 ΔC/H GST -Alx4(HD) GST Control pcDNA3 €1
WB: α - Alx4 paired-like Alx4 Pax3 €2 homeodomain GST-Pax3
Alx4 €3 Pax3 ΔC/H ∆WW Alx4
WB: α - Pax3 ∆Sma Pro-rich Alx4 domain Bam Alx4 Nco Alx4 lstD
pcDNA3 paired tail
Alx4 E in the Alx4lstD and Binding was detected by immunoblotting with down assays with bacterially expressed GST and the GST-Pax3 fusion protein. Whole cell lysates transfected with Alx4 deletion mutants were used in Pull Schematic of Alx4 mutants B) GST Pulldown assays with Alx4 deletion mutants. FIgure 3.5 - Homeodomain of Alx4 mediates interaction with Pax3 E) Coomassie stain of 10mg GST and GST Alx4(HD) immunoblotting with a- Pax3 detects binding of to the Alx4 homeodomain GST Pulldown assay of Pax3 and GST-Alx4(HD) and GST fusion proteins, and GST-Pax3 in which full length GST is expressed more than GST-Pax3 D) Alx4
GST ∆WW Alx4 GST ∆Sma Alx4 GST -Alx4(HD) Bam
Alx4 Alx4 Nco 26 34 43 55 72 kDa Alx4 ∆WW lstD variants. C) Coomassie stain of 10mg GST C kDa α -Alx4, showing loss of binding 26 34 43 55 72
GST
GST-Pax3 A)
47 down assays. As Figure 3.4D illustrates, wild type Pax3 bound to GST Pax3(HD) but not to GST alone, supporting the conclusion that the homeodomain of Pax3 mediates its interaction with Alx4. Deletion analysis of Alx4 also revealed a complex pattern of binding characteristics
(Figure 3.5). The point mutation in the lstD Alx4 mutant derived from Strong’s luxoid mice blocked Alx4 binding to Pax3 suggesting that a functional homeodomain was important for binding to Pax3. Indeed, significant deletions of either the N-terminus or C-terminus of Alx4, as seen in the Alx4∆Bam and Alx4∆Sma mutants, respectively, had little apparent effect on Pax3 binding. However, the small deletion in Alx4∆WW, that removes the region containing polyproline motifs and that we previously demonstrated facilitates binding to factors harbouring SH3-domains (unpublished observation), abrogated Alx4- binding to Pax3(Figure 3.5B). These data suggested that, while the homeodomain appears to mediate Alx4 binding to Pax3, intramolecular mechanisms might play an important role in regulating this binding activity. In order to support the data implicating the Alx4 homeodomain in mediating its interaction with Pax3, a GST-fusion protein harbouring the
Alx4 homeodomain GST-Alx4(HD) was used in a GST pulldown assay with Pax3 (Figure 3.5D). The binding of Pax3 to the GST-Alx4(HD) protein and not to GST indicates that the homeodomain is sufficient for this interaction.
3.3 Transcriptional Effect of Pax3 and Alx4 on a model Promoter The physical interaction between Pax3 and Alx4 suggested that their interaction may play a regulatory role in the transcription of target genes. In order to test this, the effect of Pax3 on Alx4 transcriptional activity was determined in reporter assays using a single P3 (TAATnnnATTA) homeodomain consensus as an enhancer of a CMV Immediate Early
Promoter, to drive luciferase activity, (P3)1 –Luc (Figure 3.6A). Glutamine-50 Paired-type homeodomain proteins, such as Alx4, preferentially bind to this sequence as cooperative
48 dimers (Tucker and Wisdom, 1999). Expression of Pax3 alone weakly increased basal activation of the of the (P3)1-Luc reporter in Cos-1 cells, where increasing concentrations of Pax3 did not significantly increase promoter activity(Figure 3.6B). In accordance with previous studies, Alx4 significantly enhanced basal activity six- fold. Co-expression of
Pax3 with Alx4 enhanced Alx4-dependent activation of (P3)1 -Luc, in a strictly additive manner. Increased concentrations of Pax3 appeared to have no affect on transcription from this promoter. Lysates used for luciferase assays were also prepared for Western analysis to verify relative levels of protein expression (Figure 3.6 C and D).
B
12
10 * P<0.05 A 8 TAATTAAATTA 6 LUC 4
Relative Luciferase Units 2
pcDNA3 1μg Pax3 0.5μg Pax3 1.5μg Pax3 1.5μg Alx4 1.5μg Alx4 +1.5 μg Alx4 + 1.5μg Alx4 + 0.5μg Pax3 1μg Pax3 1.5μg Pax3
+ 0.5μg HD-Luc
C D pcDNA31.5μg Pax31μg Pax31.5μg Alx41.5μg Alx41.5 μ+g 0.5 Alx4μ1.5g +Pax3μ 1gμ Alx4g Pax3 + 1.5μg Pax3 pcDNA31.5μg Pax31μg Pax31.5μg Alx41.5μg Alx41.5 μ+g 0.5 Alx4μ1.5g +Pax3μ 1gμ Alx4g Pax3 + 1.5μg Pax3 55kDA 55kDA
WB: αPax3 WB: αAlx4
Figure3.6 - Pax3 and Alx4 regulate transcription from the P3 promoter A) Schematic of (P3)1-Luc reporter construct. A single P3 site is placed upstream of the CMV Immediate Early Promoter driving luciferase activity. B) Cos -1 cells were transfected with the (P3)1-Luc construct and co-transfected with pcDNA3, pcDNA3-Pax3, or pcDNA3-Alx4 and or in combination. To control for transfection efficiency, all samples were co-transfected with the CMV-b-galactosidase reporter (0.2mg). 48hrs after transfection cells were as- sayed for luciferase and b-galactosidase activity. Error bars indicate SEM from three independent experi- ments done performed in triplicate. Values were considered statistically significant if P<0.05. C) Western blot showing relative levels of expression of Pax3 and Alx4 in luciferase samples. 30mg protein from lysates used for reporter assays were prepared for Western analysis, and expression levels of Pax3 and Alx4 were detected using their respective antibodies
49 Taken together, these results demonstrate that Pax3 and Alx4 display overlapping patterns of expression in the developing embryo. Furthermore we observe the in vivo and in vitro interaction between Pax3 and Alx4 mediated by their respective homeodomains, and that their interaction is independent of DNA-binding. Lastly, Pax3 and Alx4 are able to regulate transcription from a P3 response element in a strictly additive manner.
50 Section 4 – Discussion
4.1 Pax3 and Alx4 interact through protein-protein interactions independent of DNA binding Our data has revealed that Pax3 and Alx4 expression patterns overlap in multiple regions in the developing embryo. Furthermore we observe that while only a small number of fields cells co-express Pax3 and Alx4 at 9.5dpc, as development progresses, an apparent increase in the extent of overlap is seen, specifically in the mesenchymal cells of the distal tip of the first branchial arch and the fronto nasal prominence. This increase in overlap continues as the craniofacial structures develop further. Given that the individual loss of Pax3 or Alx4 activity results in developmental defects that arise in the same tissues, we suspected that Pax3 and Alx4 may physically interact. We have demonstrated the in vivo and in vitro interaction between Pax3 and Alx4, and furthermore that this interaction is independent of DNA binding. Initially, we hypothesized that the PRD-like homeodomains of Pax3 and Alx4 may mediate their heterodimerization on DNA. PRD-like homeodomain proteins have the unique capacity to dimerize on palindromic sequences, consisting of two TAAT half sites separated by a variable number of nucleotides (Wilson et al., 1993). The dimerization of members of the PRD-class is a cooperative binding event, where cooperativity is defined as the extent to which binding to the first site enhances binding to the second site (Wilson et al., 1993). This suggests that for members of this class, DNA binding is necessary for dimerization. Additionally, previous examples of heterodimerization between members of the paired class suggest that these proteins do not complex in solution (Qu et al., 1999). For example, while Alx4 and Gsc are observed to dimerize on a P3 element, co-immunoprecipitation studies failed to demonstrate an interaction in solution (Tucker and Wisdom, 1999). The observation that Pax3 and Alx4 complex in solution and that this is independent of DNA
51 binding suggests that their interaction differs from the characteristic dimerization of prd- class homeodomain proteins. The possibility that Pax3 and Alx4 may form a heterodimer that can then bind to DNA can still be considered. Several examples exist in other classes of homeodomain proteins where heterodimers are formed as a means of conferring additional specificity in target recognition. Members of the HOX class of homeodomain proteins predominantly form heterodimers with cofactors to achieve greater DNA binding affinity and specificity (Jabet et al., 1999). For example, Hoxb-8 and Pbx-1 form a heterodimer in solution. Upon DNA binding, a conformational change is induced, increasing affinity for the DNA, greater than for either factor individually. Additionally, the heterodimer complex recognizes DNA sites different than those recognized by each factor individually (Sanchez et al., 1997). Pax3 and Alx4 contain several domains with demonstrated roles in mediating protein-protein interactions. Therefore, we sought to characterize the domains through which these proteins interact. GST pulldown assays with expressed deletion mutants suggest that the interaction between Pax3 and Alx4 is mediated by their respective homeodomains. GST fusion proteins harbouring their individual homeodomains appeared to be sufficient for binding to the reciprocal full-length protein, further strengthening a role for the homeodomain in their interaction. In contrast to other classes of homeodomain proteins, PRD-like homeodomains have the intrinsic capacity to dimerize independent of any extrinsic protein domains(Wilson et al., 1993). For example, HOX class proteins, in addition to their homeodomains, require the presence of a hexapeptide cooperativity motif to mediate their heterodimerization with cofactors (Chang et al., 1995). Therefore, although we have shown that the single homeodomain of Alx4 is able to bind to the full-length Pax3 protein, it is difficult to discern whether this is due to the propensity of PRD-like homeodomains to interact. Indeed, when the N-terminal proline rich region of Alx4 is deleted, as in the Alx4∆WW mutant, interaction
52 with Pax3 is also lost. This suggests that in addition to their respective homeodomains, additional motifs may also be necessary for the Pax3 – Alx4 interaction. It has yet to be determined however whether the N-terminal proline rich region of Alx4 in itself is sufficient for binding to Pax3. Studies have identified multiple examples where Pax3 interacts through its homeodomain with other regulatory factors. Where Pax3 interacts with Rb (Wiggan et al., 1998) and Daxx (Hollenbach et al., 1999), these interactions serve to attenuate Pax3 activation of target genes. Interestingly, Pax3 interacts with Rb, and Rb related proteins p130 and p107 through the first two helices of its paired-like homeodomain (Wiggan et al., 1998). Similarly, loss of helix I and helix II in the Pax3 deletion mutant Pax3∆C/H disrupts binding of Pax3 to Alx4, as well as to another identified Pax3 binding partner, Lef-1 (T. Christova, manuscript submitted). This is in contrast to Pax6, a closely related family member, where protein-protein interactions are mediated through helix III (Bruun et al., 2005). This suggests an important role for helices I and II of the Pax3 homeodomain, although it is conceivable the recognition helix, helix III, may also play a role in protein- protein interactions.
4.2 Pax3 and Alx4 activate transcription from a P3 promoter Based on the initial hypothesis that Pax3 and Alx4 heterodimerize to form a regulatory complex, we tested their transcriptional activity on a P3 element. As described, paired- like homeodomain proteins cooperatively dimerize on palindromic elements, TAATn(2-3) ATTA. The glutamine 50 paired-like homeodomain preferentially binds to a P3 element (Wilson et al., 1993; Wilson et al., 1995b). Reporter assays using a single P3 site upstream of a CMV-immediate early promoter showed that although Pax3, with a serine homeodomain preferentially binds to P2 sites, it was also able to activate transcription from the P3 site, albeit activation was relatively weak. As expected, Alx4 activated transcription 6-fold
53 from this site. The addition of Pax3 to Alx4 on this promoter activated transcription further, although the increase in transcription was not significant. This suggests that rather than modulating the transcriptional activity of Alx4, Pax3 is acting independently. The dose dependent effect of Pax3 on Alx4-dependent activation ceases after a certain Pax3 concentration however, and transcriptional activity is attenuated and begins to decrease. This observation is consistent with previous studies indicating that at higher concentrations, Pax3 serves an inhibitory function, most plausibly through “squelching”(Chalepakis et al., 1994b). This is the process by which over-expression of an activating transcription factor, results in the repression of transcription by sequestering limited components of the transcriptional machinery required for transcriptional activation (Cahill et al., 1994). At higher concentrations a Pax3 variant lacking its DNA-binding domain is able to repress basal activity from the model TK-PRS9-CAT promoter, suggesting a non-specific repressive role (Chalepakis et al., 1994b).
The independent actions of Pax3 and Alx4 on the (P3)1-Luc promoter are consistent with previous studies. Our lab has previously demonstrated that Pax3 is able to activate transcription from a reporter construct harbouring three P3 sites, and that this activation is repressed when Rb is co-expressed (Wiggan et al., 1998). Alx4 has also demonstrated an ability to activate transcription from a P3 site (Qu et al., 1999; Tucker and Wisdom, 1999). Their independent actions and the additive transcriptional effect suggest that Pax3 and
Alx4 are not interacting on the P3 site, but rather they are each occupying different P3 sites. What remains to be tested however is whether Alx4 and Pax3 are interacting while bound to a single P3 site. Electrophoretic Mobility Shift Assays (EMSA’s) (not shown) suggest that this is not the case, however, further studies are needed. The demonstrated interaction between Pax3 and Alx4 in vivo and in vitro however, suggests that their interaction may be biologically relevant to their role as transcription factors. Therefore, in the next section we propose alternative models by which Pax3 and Alx4 may cooperate to regulate
54 transcription.
4.3 A model for regulation in vivo by a Pax3-Alx4 regulatory complex. While less is known about downstream targets of Alx4, extensive studies have been done to elucidate Pax3 recognition targets (Begum et al., 2005; Mayanil et al., 2001; Phelan and Loeken, 1998). Pax3 can regulate transcription through the alternative use of its two DNA binding domains; the paired domain and the homeodomain. Consequently, Pax3 can interact with recognition targets through its paired domain individually, its homeodomain individually, or through both its paired domain and its homeodomain(Corry and Underhill, 2005). The focus of the following discussion will be on Pax3 targets containing consensus sites for either the paired domain, or a composite site consisting of adjacent paired domain and homeodomain binding sites. Several in vivo examples exist in which Pax3 regulates transcription from a paired domain binding site. These include promoter regions upstream of; dct, c-RET, c-MET, Trp-1 (Epstein et al., 1996; Galibert et al., 1999; Lang and Epstein, 2003; Lang et al., 2005). The identification of downstream targets of Pax3 is difficult in that a unique paired domain binding for Pax3 site is not available (Mayanil et al., 2001). As expected therefore, recognition elements upstream of target genes, although similar, do not contain perfect consensus sequences for the Pax3 paired domain (Corry and Underhill, 2005). An example of an optimized site is the paired domain binding consensus site, Nf3’, identified as a high affinity Pax3 paired domain binding site derived from an in vitro binding site selection assay (Epstein et al., 1994a). Phelan and Loeken have also demonstrated that although Pax3 is able to interact with identified paired domain binding sequences, these interactions are almost always of low affinity (Phelan and Loeken, 1998). As mentioned in Section 1, transcription factors regularly interact through protein-protein interactions to increase affinity for binding sites. Thus, it is plausible that Pax3 interacts with Alx4 through their
55 respective homeodomains and then can bind to Pax3 recognition elements through its paired domain. This interaction may increase Pax3 affinity for binding sites; thereby enhancing Pax3 mediated transcriptional activity of the promoter. Pax3 is also able to interact with promoter sequences containing binding sites for both the paired domain and the homeodomain. The Drosophila even-skipped promoter, e5, contains a single homeodomain recognition site, ATTA (P1/2). This P1/2 site is upstream of a paired domain recognition site (Goulding et al., 1991). The Drosophila PRD protein is able to recognize a PH0 site, consisting of tethered PD and HD recognition sites(Jun and Desplan, 1996). Furthermore, a consensus site upstream of Mitf, a recognized downstream target of Pax3, adopts a similar structure(Corry and Underhill, 2005; Watanabe et al., 1998). Interestingly, PRD-like homeodomains, in addition to their ability to dimerize on palindromic sites, have demonstrated the capacity to bind to P1/2 sites as monomers, albeit with reduced affinity compared to P3 sites (Tucker and Wisdom, 1999). It is suggested therefore that in vivo, paired-like homeodomain proteins bind to P1/2 sites that are juxtaposed to the target site of DNA binding partners. For example, the Phox1 homeodomain protein is able to bind to a P1/2 site, and this is enhanced by its interaction with serum response factor (Grueneberg et al., 1992). Similarly, our lab has shown that in concert with Lef-1, Alx4 is able to mediate transcriptional affects a P1 site (TAATnATTA) in the NCAM promoter.
This P1 site is adjacent to a Lef-1 binding site, where Lef-1 enhances Alx4 binding to DNA. In this case, Alx4 must interact with Lef-1, in addition to their DNA binding, in order mediate its effect on the NCAM promoter (Boras and Hamel, 2002). Therefore, it is plausible that Pax3 and Alx4 are interacting on adjacent sites, where their interaction is necessary for enhanced binding affinity. The binding of Pax3 and Alx4 to adjacent DNA binding sites would be consistent with the ability of transcription factors to interact in composite regulatory elements. Composite
56 regulatory elements consist of pairs of closely situated transcription factor binding sites (Kel et al., 1995). Transcription factors binding to these sites interact with each other resulting in gene expression patterns provided by combinatorial control. Components of composite regulatory elements may act synergistically or antagonistically, ultimately exerting their effect as one functional unit. In the case of a synergistic interaction, it is proposed that an increase in transcriptional activity arises as one factor stabilizes binding by the other(Kel et al., 1995; Kel-Margoulis et al., 2000; Wissmuller et al., 2006). For example, Pax3 and Sox10 physically interact on adjacent binding sites in a conserved c-RET enhancer, resulting in synergistic activation (Lang and Epstein, 2003). The importance of protein- protein interactions is highlighted by a Sox10 point mutation in which DNA-binding activity is compromised. This mutant retains its capacity to bind to Pax3, and therefore can still be recruited to the DNA through protein-protein interactions. In this case, activation of the c-RET gene is largely unaffected (Lang and Epstein, 2003). Pax3 downstream targets have already been identified in which a paired domain recognition site is adjacent to a P1/2 site. These include Mitf, as well as the myogenic factor Myf5(Buchberger et al., 2007; Watanabe et al., 1998). Interestingly, in the Myf5 promoter, adjacent homeodomain and paired domain binding sites are present, however Pax3 activity alone is not sufficient for transactivation (Buchberger et al., 2007). The proposed models of Pax3 and Alx4 interacting to regulate transcription are summarized in Figure 4.1. Palindromic recognition elements of the P3 type are highly conserved in the promoters of most opsin genes and are required for opsin expression in Drosophila (Sheng et al., 1997; Wilson et al., 1995a). It is expected however, that these genes are downstream targets of Pax6, which also contains a paired-like homeodomain, and furthermore plays a key role in the development of the eye. Studies investigating downstream targets of Pax3 have predominantly identified genes regulated by promoter regions containing a single paired domain recognition site (Phelan and Loeken, 1998). Fewer
57 examples have also been found in promoter regions of Pax3 downstream targets where a paired domain recognition site is juxtaposed to a P1/2 site. Therefore we can speculate that Pax3 and Alx4 may be interacting on promoters other than those solely containing the P3 recognition element. What remain to be determined however are functional downstream targets that are dependent on a Pax3-Alx4 interaction.
A B
P P
A A
X X
3 3 PD PD AX3 PD P HD 4 HD A A lx lx lx
A 4 4 HD HD HD HD
NNNNNNNN P3 NNNNNNNNNN P3 NNNNNNN
4 C x D
l A A
lx
4 HD
PA HD X HD 3 HD PD PAX3 PD NNNNNNNN PD NNNNNNNNNNNNNNNNNNNNNN NNNNNNNN PD NNNNNNNNNN TAAT NNNNNN
Figure 4.1 Model of Pax3 and Alx4 interacting to regulate transcriptional activity. A) Pax3 and Alx4 interact through their respective homeodomains (HD) to form a heterodimer in solution B) Pax3 and Alx4 individually bind to P3 recognition sequences. The prd-type homeodomains of both Pax3 and Alx4 have the capacity to cooperatively dimerize on palindromic P3 elements. Pax3 and Alx4 independently interact with P3 sites to activate transcription in a strictly additive manner C) The paired domain (PD) of Pax3 can bind to paired domain recognition sites through the PD alone, however this is a low affinity binding event. It is proposed that Alx4 can interact with Pax3 through its free homeodomain to stabilize the complex on DNA D) Pax3 and Alx4 can interact on a composite regulatory element consisting of a PD binding site uptream of a TAAT P1/2 site. Alx4 can bind to P1/2 as a monomer with weak affinity. The interaction between Pax3 and Alx4 while bound to their respective binding sites can serve to enhance binding of both factors.
4.4 Preliminary analysis of the genetic interaction between Pax3 and Alx4 during embryonic development using double knockout mice Pax3 and Alx4 are expressed in overlapping regions in the developing embryo and furthermore, the individual loss of either Pax3 or Alx4 activity in the mouse, results
58 in developmental defects that arise in the same embryonic structures. The demonstrated physical interaction of Pax3 and Alx4 therefore predicted that they might cooperate to regulate processes necessary for normal embryonic development. To complement the study outlined in this thesis, preliminary analysis using double knockout mice were undertaken to ascribe a genetic role for the interaction between Pax3 and Alx4. Thus, mice heterozygous for both the spJ allele of Pax3 and the lstD allele of Alx4 were backcrossed to generate a spectrum of genotypes, including double homozygotes for both alleles
(Figure 4.2). Phenotypically, mice simultaneously heterozygous for both spJ and lstD are indistinguishable from individual heterozygotes of either splotch of lstD. As mentioned in Chapter 1, homozygous splotch mice alone exhibit characteristic defects of an open neural tube and limb defects (Epstein et al., 1996; Goulding and Paquette, 1994). In addition to the expected splotch defects as well as the preaxial polydactyly observed in lstD mice (Qu et al., 1998), a spectrum of defects were observed in the compound mutant mice. When Alx4 activity is lost on the background of these splotch embryos, the most typical of these defects is severe facial clefting, in which both the lip and palate were unfused (Figure 4.2D). In addition to the severe craniofacial defects, approximately 30% of embryos with these mutant genotypes were not viable at E13.5, displaying early embryonic lethality. This is in contrast to the Alx4 lstD/lstD homozygous mice, 2% of which survive to term and the Pax3 spJ/spJ mice, which survive up to 14.5dpc (Boras-Granic et al., 2006; Engleka et al., 2005). One of the embryos taken at E13.5, figure 4.2E, was sectioned and upon histological analysis, most cellular structures appeared completely degraded. As described above, the splotch-delayed allele of Pax3 results in less severe defects in mice, due to reduced, rather than complete loss of Pax3 activity(Vogan et al., 1993).
Binding assays in Figure 3.4 also demonstrate an ability of Alx4 to interact with the splotch-delayed variant of Pax3, G42R. Surprisingly, it is apparent that these embryos more closely resemble the wild type, where the spD/spD lstD/+ embryos do not exhibit
59 the facial clefting seen in the majority of their counterparts on the splotch background. Therefore, the partial activity of the G42R variant of Pax3, and its ability to participate in protein-protein interactions, appears to mitigate the effects of loss of Alx4 on a Pax3 null background. This is a very preliminary analysis of a small population of mutant embryos. In order to elucidate a potential role for Pax3 and Alx4 in embryonic development, a more exhaustive genetic analysis is required. Based on the craniofacial defects that arise however, it is proposed that the coordinated actions of Pax3 and Alx4 may play a role in craniofacial morphogenesis. This is consistent with two studies in which an important role for Alx4 has been implicated in normal craniofacial development. For example, Beverdam and
Wt spJ/spJ lstD/+
wt A B C
13.5dpc
Wt spJ/spJ lstD/+ spJ/spJ lstD/lstD
D E
15.5dpc
Wt spD/spD lstD/+spD/spD lstD/+ Figure 4.2 - Preliminary analysis of genetic interaction between Pax3 and Alx4 Heterozygotes for the Alx4 lstD allele and the splotch or the splotch delayed allele were backcrossed and embryos were taken at different stages of development and formalin fixed. A-C Embryos taken at 13.5 dpc. B and C in which Alx4 activity is lost on the splotch homozygous background display severe facial clefting. C was not viable and displays reduced mandible as well as clefting in the facial region. DandE) Splotch-delayed heterozy- gotes were backcrossed with lstD embryos. E, mutant embryo displays almost proper closure of the facial region, and is very similar to the wild type embryo in D.
60 colleagues have shown that when Alx4 activity is lost simultaneously with Alx3 activity, severe facial clefting is observed, not seen in the Alx3 null homozygotes. They suggest that Alx4 compensates for the loss of Alx3 activity in the craniofacial region (Beverdam et al., 2001). Similarly, loss of Alx4 activity in combination with loss of Cart-1, results in a wide range of craniofacial phenotypes including facial clefting, cleft palate and mandibular truncations, defects not seen in either homozygote individually (Qu et al., 1999). These observations suggest that Alx4 does play a role in regulating gene expression governing craniofacial morphogenesis. It is conceivable therefore that Alx4 may be interacting with Pax3 to modulate its regulatory activity. The above discussion postulates a role for coordinated Pax3 and Alx4 activities in the embryonic development. It must also be considered however, that the novel mutant phenotypes seen in these double mutant mice, may be a result of independent Pax3 and Alx4 roles. It is plausible that Alx4 and Pax3 regulate expression of target genes at consecutive points in a signaling pathway, or that these two factors act on separate pathways which ultimately impinge on the same developmental process. The observation that Pax3 and Alx4 interact through protein-protein interactions in vivo and in vitro, and display overlapping patterns of expression however, suggests that it is likely that they interact to regulate transcription of a downstream target necessary for proper embryonic development.
4.5 Significance The study presented above outlines a novel interaction between paired-type homeodomain transcription factors Pax3 and Alx4. Furthermore, it is suggestive of a functional role in vivo based on their coordinated effect in transcriptional activation from a model reporter, as well as a preliminary observation that loss of their activity simultaneously results in a novel phenotype and early embryonic lethality in a small percentage of embryos. Although Pax3 has been studied extensively, Alx4 is a relatively new factor, and the
61 literature available on its characterization in vivo remains limited. Nevertheless, studies have investigated its role in development, and particularly mechanisms by which Alx4 can regulate downstream target gene expression. More recently, it has been identified as a player in human disorder, such as Parietal Foramina(Mavrogiannis et al., 2001), as well as breast and colon cancers(Ebert et al., 2006). The identification of Pax3 as an interacting factor with Alx4 provides another example by which Alx4 can interact with other factors in the developing embryo to exert its regulatory effects. Furthermore, the suggestion that Pax3 and Alx4 may be acting in conjunction with each other to regulate a process important in craniofacial morphogenesis futher defines a role for Alx4 here. Other studies by Beverdam et al, and Qu et al, have also identified a role for Alx4 in craniofacial morphogenesis, and have postulated that its function may be due to a redundant relationship with closely related family members Cart-1 and Alx3 individually(Beverdam et al., 2001; Qu et al., 1999). The identification of Pax3 as a potential mediator of Alx4 related regulation of craniofacial development suggests a novel mechanism by which Alx4 controls a developmental process. Further defining the Pax3-Alx4 interaction and its role in craniofacial morphogenesis, can serve as a basis for determining underlying causes of human craniofacial disorders. Recently, animal models with craniofacial defects have identified genes, which, through human genetic analysis, have proved to be the genetic cause of human congenital malformations (Thyagarajan et al. 2003, Murray and Schutte, 2004). Alx4 is already implicated in a genetic mechanism by which Human Parietal Foramina arises in humans(Mavrogiannis et al., 2006; Wu et al., 2000), and therefore it serves as a good candidate to study in other human genetic defects.
4.6 Future Directions A significant portion of this thesis is dedicated to identifying and characterizing
62 the protein-protein interactions between Pax3 and Alx4. What remains to be determined however is if additional Alx4 regions are necessary for this interaction, such as the proline rich sequence, termed WW-binding motif, as alluded to at the beginning of this discussion. It is necessary to determine whether this Alx4 motif is also sufficient for an interaction with Pax3. In order to determine this, a GST-fusion protein of this motif would also need to be generated and used in GST Pulldown assays with Pax3. Furthermore, the above discussion postulates several possible mechanisms through which coordinated Pax3 and Alx4 transcriptional regulation can play a role in normal embryonic development. In order to further define a role for this interaction, a more extensive analysis of the phenotypes of double knockout mice as well as other genotypic variants is necessary. Defects seen in the small sample size in this study could be due to polymorphisms of other alleles and genetic variations not related to Pax3 and Alx4. A comprehensive phenotypic analysis in knockout mouse studies requires a large sample size, which allow for quantitative statistical analysis as well, as opposed to a purely qualitative description. Furthermore, although gross anatomical defects are visible, careful examination of the craniofacial region would serve to better define the defects and the developmental processes being disrupted. The ultimate goal of this study is to determine a downstream target regulated by coordinated Pax3 and Alx4 activity in order to provide functional significance to this interaction. There are two methods by which this could be accomplished. The first is the candidate gene approach, in which potential target genes are tested for their susceptibility to regulation by Pax3 and Alx4. Previously our lab published the results of a microarray in which downstream targets of Pax3 were identified during a Pax3 induced -mesenchymal to epithelial transition in SaOS-2 cells (Begum et al., 2005). Among this list of downstream targets, CITED-1, Dlx3, and BMP-4 are all candidate genes with demonstrated roles in craniofacial morphogenesis. The insertion of promoter regions of these genes upstream
63 of a pGL3 Luciferase reporter gene would provide a useful method for determining transcriptional activity of these genes upon induction by Pax3 and Alx4. Furthermore, immunofluorescence, as well as whole mounts using riboprobes directed against these transcripts in mutant and wild type embryos would be indicative of changes in expression due to loss of Pax3 and Alx4 activity. The second approach is more global in scope. Overlapping expression of Pax3 and Alx4 in the first branchial arch at E10.5 and in the frontonasal process at E12.5, suggests that their interaction may be playing a role in these structures. Studies have been done in which branchial arches for example have been micro dissected and tissues have been used to detect changes in gene expression by microarray analysis (Ivins et al., 2005). This technique, performed on both the first branchial arch and in the frontonasal process, on both wild type and spJ/spJ lstD/+ and double homozygote embryos at both 10.5 and 12.5, would serve to identify potential downstream targets of Pax3 and Alx4. These candidates would then be tested further using the techniques described above. Lastly, Alx4 was identified as a novel factor approximately ten years ago, and to date, although it has been the focus of numerous studies, there is still much progress to be made on the identity of downstream targets of Alx4. Here we have studied the action of Alx4 in conjunction with Pax3, as just one example in which Alx4 action may be modulated during development. It would be interesting however to investigate further the scope of
Alx4 action in vivo, and to determine downstream targets that it may regulate, with or without Pax3. New tools in genome wide analysis allow for this, and in particular, ChIP-on chip, would be a useful way in which to study downstream targets of Alx4. Based on this technique, chromatin immunoprecipitation (ChIP) assays targeting Alx4 would be used in order to generate a multitude of genomic fragments to which Alx4 is bound. The next step would hybridize genomic fragments pulled out by ChIP to a microarray. Alx4 is a DNA binding transcription factor and so the main interest here is to look at the protein/DNA
64 interaction in mouse promoter regions. The GeneChip® Mouse Promoter 1.0R Array from Affymetrix would be used to identify Alx4 interactions with promoter regions across the entire mouse genome. Based on these results then, candidate genes could be investigated further to determine modes of Alx4 regulation of gene expression (Affymetrix). The three processes outlined above used to determined downstream targets of Pax3, of Alx4 or of Pax3 and Alx4 together, could be combined in order to generate a consolidated list of possible downstream targets which could then be tested further. The results of these experiments would provide a clear picture of a regulatory mechanism by which Pax3 and Alx4 interact to regulate gene expression in normal embryonic development.
65 Section 5 - References Ades, S. E. and Sauer, R. T. (1995). Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex. Biochemistry 34, 14601-8. Affolter, M., Slattery, M. and Mann, R. S. (2008). A lexicon for homeodomain-DNA recognition. Cell 133, 1133-5. Antonopoulou, I., Mavrogiannis, L. A., Wilkie, A. O. and Morriss-Kay, G. M. (2004). Alx4 and Msx2 play phenotypically similar and additive roles in skull vault differentiation. J Anat 204, 487-99. Apuzzo, S., Abdelhakim, A., Fortin, A. S. and Gros, P. (2004). Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding. J
Biol Chem 279, 33601-12. Apuzzo, S. and Gros, P. (2007). Cooperative interactions between the two DNA binding domains of Pax3: helix 2 of the paired domain is in the proximity of the amino terminus of the homeodomain. Biochemistry 46, 2984-93. Asher, J. H., Jr., Harrison, R. W., Morell, R., Carey, M. L. and Friedman, T. B. (1996). Effects of Pax3 modifier genes on craniofacial morphology, pigmentation, and viability: a murine model of Waardenburg syndrome variation. Genomics 34, 285-98. Ayer, D. E. (1999). Histone deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol 9, 193-8. Baldwin, C. T., Hoth, C. F., Amos, J. A., da-Silva, E. O. and Milunsky, A. (1992). An exonic mutation in the HuP2 paired domain gene causes Waardenburg’s syndrome [see comments]. Nature 355, 637-8. Baldwin, C. T., Hoth, C. F., Macina, R. A. and Milunsky, A. (1995). Mutations in PAX3 that cause Waardenburg syndrome type I: ten new mutations and review of the literature. Am J Med Genet 58, 115-22.
66 Banerjee-Basu, S. and Baxevanis, A. D. (2001). Molecular evolution of the homeodomain family of transcription factors. Nucleic Acids Res 29, 3258-69. Barr, F. G. (1997). Fusions involving paired box and fork head family transcription factors in the pediatric cancer alveolar rhabdomyosarcoma. Curr Top Microbiol Immunol
220, 113-29. Barr, F. G., Galili, N., Holick, J., Biegel, J. A., Rovera, G. and Emanuel, B. S. (1993). Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet 3, 113-7. Begum, S., Emani, N., Cheung, A., Wilkins, O., Der, S. and Hamel, P. A. (2005). Cell-type-specific regulation of distinct sets of gene targets by Pax3 and Pax3/FKHR. Oncogene.
Bendall, A. J., Ding, J., Hu, G., Shen, M. M. and Abate-Shen, C. (1999). Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.
Development 126, 4965-76. Berger, M. F., Badis, G., Gehrke, A. R., Talukder, S., Philippakis, A. A., Pena- Castillo, L., Alleyne, T. M., Mnaimneh, S., Botvinnik, O. B., Chan, E. T. et al. (2008). Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell 133, 1266-76. Beverdam, A., Brouwer, A., Reijnen, M., Korving, J. and Meijlink, F. (2001). Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice.
Development 128, 3975-86. Billeter, M. (1996). Homeodomain-type DNA recognition. Prog Biophys Mol Biol 66, 211-25.
Birrane, G., Soni, A. and Ladias, J. A. (2009). Structural basis for DNA recognition by the human PAX3 homeodomain. Biochemistry 48, 1148-55. Bopp, D., Burri, M., Baumgartner, S., Frigerio, G. and Noll, M. (1986). Conservation
67 of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell 47, 1033-40. Boras, K. and Hamel, P. A. (2002). Alx4 binding to LEF-1 regulates N-CAM promoter activity. J Biol Chem 277, 1120-7. Boras-Granic, K., Grosschedl, R. and Hamel, P. A. (2006). Genetic interaction between Lef1 and Alx4 is required for early embryonic development. Int J Dev Biol 50, 601-10.
Borycki, A. G., Brunk, B., Tajbakhsh, S., Buckingham, M., Chiang, C. and Emerson, C. P., Jr. (1999). Sonic hedgehog controls epaxial muscle determination through Myf5 activation. Development 126, 4053-63. Boutet, S. C., Disatnik, M. H., Chan, L. S., Iori, K. and Rando, T. A. (2007). Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors. Cell 130, 349-62. Bruun, J. A., Thomassen, E. I., Kristiansen, K., Tylden, G., Holm, T., Mikkola, I., Bjorkoy, G. and Johansen, T. (2005). The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions. Nucleic Acids Res 33, 2661-75. Buchberger, A., Freitag, D. and Arnold, H. H. (2007). A homeo-paired domain-binding motif directs Myf5 expression in progenitor cells of limb muscle. Development 134, 1171-80.
Burri, M., Tromvoukis, Y., Bopp, D., Frigerio, G. and Noll, M. (1989). Conservation of the paired domain in metazoans and its structure in three isolated human genes. EMBO
J 8, 1183-90. Cahill, M. A., Ernst, W. H., Janknecht, R. and Nordheim, A. (1994). Regulatory squelching. FEBS Lett 344, 105-8. Cao, Y. and Wang, C. (2000). The COOH-terminal transactivation domain plays a key
68 role in regulating the in vitro and in vivo function of Pax3 homeodomain. J Biol Chem
275, 9854-62. Carrasco, A. E., McGinnis, W., Gehring, W. J. and De Robertis, E. M. (1984). Cloning of an X. laevis gene expressed during early embryogenesis coding for a peptide region homologous to Drosophila homeotic genes. Cell 37, 409-14. Chalepakis, G., Goulding, M., Read, A., Strachan, T. and Gruss, P. (1994a). Molecular basis of splotch and Waardenburg Pax-3 mutations. Proc Natl Acad Sci U S A
91, 3685-9. Chalepakis, G., Jones, F. S., Edelman, G. M. and Gruss, P. (1994b). Pax-3 contains domains for transcription activation and transcription inhibition. Proc Natl Acad Sci U S
A 91, 12745-9. Chan, D. C., Laufer, E., Tabin, C. and Leder, P. (1995). Polydactylous limbs in Strong’s Luxoid mice result from ectopic polarizing activity. Development 121, 1971-8. Chang, C. P., Shen, W. F., Rozenfeld, S., Lawrence, H. J., Largman, C. and Cleary, M. L. (1995). Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev 9, 663-74. Chariot, A., Gielen, J., Merville, M. P. and Bours, V. (1999). The homeodomain- containing proteins: an update on their interacting partners. Biochem Pharmacol 58, 1851-7.
Chi, N. and Epstein, J. A. (2002). Getting your Pax straight: Pax proteins in development and disease. Trends Genet 18, 41-7. Chi, Y. I. (2005). Homeodomain revisited: a lesson from disease-causing mutations. Hum Genet 116, 433-44. Conway, S. J., Henderson, D. J. and Copp, A. J. (1997). Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant.
Development 124, 505-14.
69 Corry, G. N. and Underhill, D. A. (2005). Pax3 target gene recognition occurs through distinct modes that are differentially affected by disease-associated mutations. Pigment
Cell Res 18, 427-38. Courey, A. J. (2001). Cooperativity in transcriptional control. Curr Biol 11, R250-2. Czerny, T., Schaffner, G. and Busslinger, M. (1993). DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev 7, 2048- 61.
Dahl, E., Koseki, H. and Balling, R. (1997). Pax genes and organogenesis. Bioessays 19, 755-65. Damante, G., Pellizzari, L., Esposito, G., Fogolari, F., Viglino, P., Fabbro, D., Tell, G., Formisano, S. and Di Lauro, R. (1996). A molecular code dictates sequence-specific DNA recognition by homeodomains. EMBO J 15, 4992-5000. Duboule, D. (1994). Guidebook to the homeobox genes. Oxford ; New York: Oxford University Press.
Eberhard, D. and Busslinger, M. (1999). The partial homeodomain of the transcription factor Pax-5 (BSAP) is an interaction motif for the retinoblastoma and TATA-binding proteins. Cancer Res 59, 1716s-1724s; discussion 1724s-1725s. Ebert, M. P., Model, F., Mooney, S., Hale, K., Lograsso, J., Tonnes-Priddy, L., Hoffmann, J., Csepregi, A., Rocken, C., Molnar, B. et al. (2006). Aristaless-like homeobox-4 gene methylation is a potential marker for colorectal adenocarcinomas.
Gastroenterology 131, 1418-30. Ekker, S. C., von Kessler, D. P. and Beachy, P. A. (1992). Differential DNA sequence recognition is a determinant of specificity in homeotic gene action.EMBO J 11, 4059-72. Engleka, K. A., Gitler, A. D., Zhang, M., Zhou, D. D., High, F. A. and Epstein, J. A. (2005). Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives. Dev Biol 280, 396-406.
70 Epstein, D. J., Vekemans, M. and Gros, P. (1991). Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell 67, 767-74. Epstein, D. J., Vogan, K. J., Trasler, D. G. and Gros, P. (1993). A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch
(Sp) mouse mutant. Proc Natl Acad Sci U S A 90, 532-6. Epstein, J., Cai, J., Glaser, T., Jepeal, L. and Maas, R. (1994a). Identification of a Pax paired domain recognition sequence and evidence for DNA-dependent conformational changes. J Biol Chem 269, 8355-61. Epstein, J. A., Glaser, T., Cai, J., Jepeal, L., Walton, D. S. and Maas, R. L. (1994b). Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev 8, 2022-34. Epstein, J. A., Shapiro, D. N., Cheng, J., Lam, P. Y. and Maas, R. L. (1996). Pax3 modulates expression of the c-Met receptor during limb muscle development. Proc Natl
Acad Sci U S A 93, 4213-8. Forsthoefel, P. F. (1962). Genetics and manifold effects of Strong’s luxoid gene in the mouse, including its interactions with Green’s luxoid and Carter’s luxate genes. J
Morphol 110, 391-420. Forsthoefel, P. F. (1963). The Embryological Development of the Effects of Strong’s Luxoid Gene in the Mouse. J Morphol 113, 427-51. Forsthoefel, P. F., Fritts, M. L. and Hatzenbeler, L. J. (1966). The origin and development of alopecia in mice homozygous for strong’s luxoid gene. J Morphol 118, 565-80.
Fortin, A. S., Underhill, D. A. and Gros, P. (1997). Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain.
Hum Mol Genet 6, 1781-90.
71 Fortin, A. S., Underhill, D. A. and Gros, P. (1998). Helix 2 of the paired domain plays a key role in the regulation of DNA- binding by the Pax-3 homeodomain. Nucleic Acids
Res 26, 4574-81. Frankel, A. D. and Kim, P. S. (1991). Modular structure of transcription factors: implications for gene regulation. Cell 65, 717-9. Franz, T. (1989). Persistent truncus arteriosus in the Splotch mutant mouse. Anat Embryol (Berl) 180, 457-64. Franz, T., Kothary, R., Surani, M. A., Halata, Z. and Grim, M. (1993). The Splotch mutation interferes with muscle development in the limbs. Anat Embryol (Berl) 187, 153- 60.
Fredericks, W. J., Galili, N., Mukhopadhyay, S., Rovera, G., Bennicelli, J., Barr, F. G. and Rauscher, F. J., 3rd. (1995). The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol 15, 1522-35. Galibert, M. D., Yavuzer, U., Dexter, T. J. and Goding, C. R. (1999). Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter. J Biol Chem
274, 26894-900. Gehring, W. J. (1985). The homeo box: a key to the understanding of development? Cell 40, 3-5. Gehring, W. J., Affolter, M. and Burglin, T. (1994a). Homeodomain proteins. Annu Rev Biochem 63, 487-526. Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G. and Wuthrich, K. (1994b). Homeodomain-DNA recognition. Cell 78, 211-23. Gilbert, S. F. (2006). Developmental biology. Sunderland, Mass.: Sinauer Associates, Inc. Publishers.
72 Goulding, M. and Paquette, A. (1994). Pax genes and neural tube defects in the mouse. Ciba Found Symp 181, 103-13. Goulding, M., Sterrer, S., Fleming, J., Balling, R., Nadeau, J., Moore, K. J., Brown, S. D., Steel, K. P. and Gruss, P. (1993). Analysis of the Pax-3 gene in the mouse mutant splotch. Genomics 17, 355-63. Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R. and Gruss, P. (1991). Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. Embo J
10, 1135-47. Grueneberg, D. A., Natesan, S., Alexandre, C. and Gilman, M. Z. (1992). Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor. Science 257, 1089-95. He, S. J., Stevens, G., Braithwaite, A. W. and Eccles, M. R. (2005). Transfection of melanoma cells with antisense PAX3 oligonucleotides additively complements cisplatin- induced cytotoxicity. Mol Cancer Ther 4, 996-1003. Henderson, D. J., Ybot-Gonzalez, P. and Copp, A. J. (1997). Over-expression of the chondroitin sulphate proteoglycan versican is associated with defective neural crest migration in the Pax3 mutant mouse (splotch). Mech Dev 69, 39-51. Hermann, S., Berndt, K. D. and Wright, A. P. (2001). How transcriptional activators bind target proteins. J Biol Chem 276, 40127-32. Hermanns, P. and Lee, B. (2001). Transcriptional dysregulation in skeletal malformation syndromes. Am J Med Genet 106, 258-71. Hoey, T. and Levine, M. (1988). Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature 332, 858-61. Holland, P. W., Booth, H. A. and Bruford, E. A. (2007). Classification and nomenclature of all human homeobox genes. BMC Biol 5, 47. Hollenbach, A. D., Sublett, J. E., McPherson, C. J. and Grosveld, G. (1999). The
73 Pax3-FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx. Embo
J 18, 3702-11. Hornyak, T. J., Hayes, D. J., Chiu, L. Y. and Ziff, E. B. (2001). Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf. Mech Dev 101, 47-59. Hoth, C. F., Milunsky, A., Lipsky, N., Sheffer, R., Clarren, S. K. and Baldwin, C. T. (1993). Mutations in the paired domain of the human PAX3 gene cause Klein- Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I). Am J
Hum Genet 52, 455-62. Hsieh, M. J., Yao, Y. L., Lai, I. L. and Yang, W. M. (2006). Transcriptional repression activity of PAX3 is modulated by competition between corepressor KAP1 and heterochromatin protein 1. Biochem Biophys Res Commun 349, 573-81. Hudson, R., Taniguchi-Sidle, A., Boras, K., Wiggan, O. and Hamel, P. A. (1998). Alx-4, a transcriptional activator whose expression is restricted to sites of epithelial- mesenchymal interactions. Dev Dyn 213, 159-69. Ingraham, H. A., Flynn, S. E., Voss, J. W., Albert, V. R., Kapiloff, M. S., Wilson, L. and Rosenfeld, M. G. (1990). The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions. Cell 61, 1021-33. Ivins, S., Lammerts van Beuren, K., Roberts, C., James, C., Lindsay, E., Baldini, A., Ataliotis, P. and Scambler, P. J. (2005). Microarray analysis detects differentially expressed genes in the pharyngeal region of mice lacking Tbx1. Dev Biol 285, 554-69. Jabet, C., Gitti, R., Summers, M. F. and Wolberger, C. (1999). NMR studies of the pbx1 TALE homeodomain protein free in solution and bound to DNA: proposal for a mechanism of HoxB1-Pbx1-DNA complex assembly. J Mol Biol 291, 521-30. Jacob, F. and Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3, 318-56.
74 Jun, S. and Desplan, C. (1996). Cooperative interactions between paired domain and homeodomain. Development 122, 2639-50. Kel, O. V., Romaschenko, A. G., Kel, A. E., Wingender, E. and Kolchanov, N. A. (1995). A compilation of composite regulatory elements affecting gene transcription in vertebrates. Nucleic Acids Res 23, 4097-103. Kel-Margoulis, O. V., Romashchenko, A. G., Kolchanov, N. A., Wingender, E. and Kel, A. E. (2000). COMPEL: a database on composite regulatory elements providing combinatorial transcriptional regulation. Nucleic Acids Res 28, 311-5. Kikuchi, K., Tsuchiya, K., Otabe, O., Gotoh, T., Tamura, S., Katsumi, Y., Yagyu, S., Tsubai-Shimizu, S., Miyachi, M., Iehara, T. et al. (2008). Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma. Biochem Biophys Res Commun
365, 568-74. Kissinger, C. R., Liu, B. S., Martin-Blanco, E., Kornberg, T. B. and Pabo, C. O. (1990). Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions. Cell 63, 579-90. Kubic, J. D., Young, K. P., Plummer, R. S., Ludvik, A. E. and Lang, D. (2008). Pigmentation PAX-ways: the role of Pax3 in melanogenesis, melanocyte stem cell maintenance, and disease. Pigment Cell Melanoma Res 21, 627-45. Kwang, S. J., Brugger, S. M., Lazik, A., Merrill, A. E., Wu, L. Y., Liu, Y. H., Ishii, M., Sangiorgi, F. O., Rauchman, M., Sucov, H. M. et al. (2002). Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest.
Development 129, 527-38. Lai, J. S. and Herr, W. (1992). Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A 89, 6958-62. Lang, D., Chen, F., Milewski, R., Li, J., Lu, M. M. and Epstein, J. A. (2000). Pax3 is required for enteric ganglia formation and functions with Sox10 to modulate expression
75 of c-ret. J Clin Invest 106, 963-71. Lang, D. and Epstein, J. A. (2003). Sox10 and Pax3 physically interact to mediate activation of a conserved c-RET enhancer. Hum Mol Genet 12, 937-45. Lang, D., Lu, M. M., Huang, L., Engleka, K. A., Zhang, M., Chu, E. Y., Lipner, S., Skoultchi, A., Millar, S. E. and Epstein, J. A. (2005). Pax3 functions at a nodal point in melanocyte stem cell differentiation. Nature 433, 884-7. Latchman, D. S. (2008). Eukaryotic transcription factors. Amsterdam ; Boston: Elsevier/ Academic Press.
Levine, M., Rubin, G. M. and Tjian, R. (1984). Human DNA sequences homologous to a protein coding region conserved between homeotic genes of Drosophila. Cell 38, 667- 73.
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276, 565-70. Li, J., Liu, K. C., Jin, F., Lu, M. M. and Epstein, J. A. (1999). Transgenic rescue of congenital heart disease and spina bifida in splotch mice.Development 126, 2495-503. Locker, J. (2001). Transcription factors. Oxford San Diego, CA: BIOS ; Academic Press.
Lufkin, T. (2003). Murine homeobox gene control of embryonic pattering and organogenesis. Amsterdam ; Boston: Elsevier Science.
Ma, J. (2005). Crossing the line between activation and repression. Trends Genet 21, 54-9.
Ma, J. and ebrary Inc. (2006). Gene expression and regulation, (ed., pp. 582 p. Beijing [New York]: Higher Education Press ; Springer.
Ma, J. and Ptashne, M. (1987a). A new class of yeast transcriptional activators. Cell 51,
76 113-9.
Ma, J. and Ptashne, M. (1987b). Deletion analysis of GAL4 defines two transcriptional activating segments. Cell 48, 847-53. Mansouri, A., Stoykova, A. and Gruss, P. (1994). Pax genes in development. J Cell Sci Suppl 18, 35-42. Maroto, M., Reshef, R., Munsterberg, A. E., Koester, S., Goulding, M. and Lassar, A. B. (1997). Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell 89, 139-48. Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387, 603-7. Mavrogiannis, L. A., Antonopoulou, I., Baxova, A., Kutilek, S., Kim, C. A., Sugayama, S. M., Salamanca, A., Wall, S. A., Morriss-Kay, G. M. and Wilkie, A. O. (2001). Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects. Nat Genet 27, 17-8. Mavrogiannis, L. A., Taylor, I. B., Davies, S. J., Ramos, F. J., Olivares, J. L. and Wilkie, A. O. (2006). Enlarged parietal foramina caused by mutations in the homeobox genes ALX4 and MSX2: from genotype to phenotype. Eur J Hum Genet 14, 151-8. Mayanil, C. S., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B., McLone, D. G. and Bremer, E. G. (2001). Microarray analysis detects novel Pax3 downstream target genes. J Biol Chem 276, 49299-309. McGinnis, W., Levine, M. S., Hafen, E., Kuroiwa, A. and Gehring, W. J. (1984). A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308, 428-33. Meijlink, F., Beverdam, A., Brouwer, A., Oosterveen, T. C. and Berge, D. T. (1999). Vertebrate aristaless-related genes. Int J Dev Biol 43, 651-63. Morrissey, D., Askew, D., Raj, L. and Weir, M. (1991). Functional dissection of the
77 paired segmentation gene in Drosophila embryos. Genes Dev 5, 1684-96. Murakami, M., Tominaga, J., Makita, R., Uchijima, Y., Kurihara, Y., Nakagawa, O., Asano, T. and Kurihara, H. (2006). Transcriptional activity of Pax3 is co-activated by TAZ. Biochem Biophys Res Commun 339, 533-9. Noyes, M. B., Christensen, R. G., Wakabayashi, A., Stormo, G. D., Brodsky, M. H. and Wolfe, S. A. (2008). Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell 133, 1277-89. Otting, G., Qian, Y. Q., Billeter, M., Muller, M., Affolter, M., Gehring, W. J. and Wuthrich, K. (1990). Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution. EMBO J 9, 3085-92.
Pabo, C. O. and Sauer, R. T. (1984). Protein-DNA recognition. Annu Rev Biochem 53, 293-321.
Papavassiliou, A. G. (1995). Molecular medicine. Transcription factors. N Engl J Med 332, 45-7. Phelan, S. A. and Loeken, M. R. (1998). Identification of a new binding motif for the paired domain of Pax-3 and unusual characteristics of spacing of bipartite recognition elements on binding and transcription activation. J Biol Chem 273, 19153-9. Potterf, S. B., Furumura, M., Dunn, K. J., Arnheiter, H. and Pavan, W. J. (2000). Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3. Hum Genet 107, 1-6. Ptashne, M. (2005). Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci 30, 275-9. Ptashne, M. (2006). Lambda’s switch: lessons from a module swap. Curr Biol 16, R459- 62.
Ptashne, M. (2007). Repressors. Curr Biol 17, R740-1.
78 Ptashne, M. and Gann, A. (1997). Transcriptional activation by recruitment. Nature 386, 569-77.
Qian, Y. Q., Billeter, M., Otting, G., Muller, M., Gehring, W. J. and Wuthrich, K. (1989). The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors. Cell 59, 573-80. Qu, S., Li, L. and Wisdom, R. (1997a). Alx-4: cDNA cloning and characterization of a novel paired-type homeodomain protein. Gene 203, 217-23. Qu, S., Niswender, K. D., Ji, Q., van der Meer, R., Keeney, D., Magnuson, M. A. and Wisdom, R. (1997b). Polydactyly and ectopic ZPA formation in Alx-4 mutant mice. Development 124, 3999-4008. Qu, S., Tucker, S. C., Ehrlich, J. S., Levorse, J. M., Flaherty, L. A., Wisdom, R. and Vogt, T. F. (1998). Mutations in mouse Aristaless-like4 cause Strong’s luxoid polydactyly. Development 125, 2711-21. Qu, S., Tucker, S. C., Zhao, Q., deCrombrugghe, B. and Wisdom, R. (1999). Physical and genetic interactions between Alx4 and Cart1. Development 126, 359-69. Renkawitz, R. (1990). Transcriptional repression in eukaryotes. Trends Genet 6, 192-7. Sanchez, M., Jennings, P. A. and Murre, C. (1997). Conformational changes induced in Hoxb-8/Pbx-1 heterodimers in solution and upon interaction with specific DNA.Mol Cell
Biol 17, 5369-76. Schneitz, K., Spielmann, P. and Noll, M. (1993). Molecular genetics of Aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila. Genes Dev 7, 911. Scholl, F. A., Kamarashev, J., Murmann, O. V., Geertsen, R., Dummer, R. and Schafer, B. W. (2001). PAX3 is expressed in human melanomas and contributes to tumor cell survival. Cancer Res 61, 823-6. Scott, M. P. and Weiner, A. J. (1984). Structural relationships among genes that control
79 development: sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila. Proc Natl Acad Sci U S A 81, 4115-9. Seidman, J. G. and Seidman, C. (2002). Transcription factor haploinsufficiency: when half a loaf is not enough. J Clin Invest 109, 451-5. Semenza, G. L. (1994). Transcriptional regulation of gene expression: mechanisms and pathophysiology. Hum Mutat 3, 180-99. Shapiro, D. N., Sublett, J. E., Li, B., Downing, J. R. and Naeve, C. W. (1993). Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res 53, 5108-12. Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D. S. and Desplan, C. (1997). Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev 11, 1122-31. Shepherd, J. C., McGinnis, W., Carrasco, A. E., De Robertis, E. M. and Gehring, W. J. (1984). Fly and frog homoeo domains show homologies with yeast mating type regulatory proteins. Nature 310, 70-1. Smith, S. T. and Jaynes, J. B. (1996). A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141-50. Szeto, D. P., Ryan, A. K., O’Connell, S. M. and Rosenfeld, M. G. (1996). P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development. Proc Natl Acad Sci U S A 93, 7706-10. Takahashi, M., Tamura, K., Buscher, D., Masuya, H., Yonei-Tamura, S., Matsumoto, K., Naitoh-Matsuo, M., Takeuchi, J., Ogura, K., Shiroishi, T. et al. (1998). The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development. Development 125, 4417-25. Tassabehji, M., Read, A. P., Newton, V. E., Harris, R., Balling, R., Gruss, P. and
80 Strachan, T. (1992). Waardenburg’s syndrome patients have mutations in the human homologue of the Pax-3 paired box gene. Nature 355, 635-6. Treisman, J., Harris, E. and Desplan, C. (1991). The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev 5, 594-604. Treisman, J., Harris, E., Wilson, D. and Desplan, C. (1992). The homeodomain: a new face for the helix-turn-helix? Bioessays 14, 145-50. Tremblay, P., Dietrich, S., Mericskay, M., Schubert, F. R., Li, Z. and Paulin, D. (1998). A crucial role for Pax3 in the development of the hypaxial musculature and the long-range migration of muscle precursors. Dev Biol 203, 49-61. Triezenberg, S. J. (1995). Structure and function of transcriptional activation domains. Curr Opin Genet Dev 5, 190-6. Tucker, S. C. and Wisdom, R. (1999). Site-specific heterodimerization by paired class homeodomain proteins mediates selective transcriptional responses. J Biol Chem 274, 32325-32.
Underhill, D. A. (2000). Genetic and biochemical diversity in the Pax gene family. Biochem Cell Biol 78, 629-38. Underhill, D. A., Vogan, K. J. and Gros, P. (1995). Analysis of the mouse Splotch- delayed mutation indicates that the Pax-3 paired domain can influence homeodomain
DNA-binding activity. Proc Natl Acad Sci U S A 92, 3692-6. van Dijk, M. A. and Murre, C. (1994). extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 78, 617-24. Vershon, A. K. (1996). Protein interactions of homeodomain proteins. Curr Opin Biotechnol 7, 392-6. Vogan, K. J., Epstein, D. J., Trasler, D. G. and Gros, P. (1993). The splotch-delayed (Spd) mouse mutant carries a point mutation within the paired box of the Pax-3 gene.
Genomics 17, 364-9.
81 Vollbrecht, E., Veit, B., Sinha, N. and Hake, S. (1991). The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature 350, 241-3. Waardenburg, P. J. (1951). A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair and with congenital deafness. Am J Hum Genet 3, 195-253. Wang, Q., Fang, W. H., Krupinski, J., Kumar, S., Slevin, M. and Kumar, P. (2008). Pax genes in embryogenesis and oncogenesis. J Cell Mol Med 12, 2281-94. Watanabe, A., Takeda, K., Ploplis, B. and Tachibana, M. (1998). Epistatic relationship between Waardenburg syndrome genes MITF and PAX3. Nat Genet 18, 283-6. Weston, J. A. (1970). The migration and differentiation of neural crest cells. Adv Morphog 8, 41-114. Wiggan, O., Taniguchi-Sidle, A. and Hamel, P. A. (1998). Interaction of the pRB- family proteins with factors containing paired- like homeodomains. Oncogene 16, 227- 36.
Wilson, D., Sheng, G., Lecuit, T., Dostatni, N. and Desplan, C. (1993). Cooperative dimerization of Paired class homeo domains on DNA. Genes & Dev. 7, 2120-2134. Wilson, D. S., Guenther, B., Desplan, C. and Kuriyan, J. (1995a). High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA. Cell
82, 709-19. Wilson, D. S., Guenther, B., Desplan, C. and Kuriyan, J. (1995b). High resolution structure of a paired (Pax) class cooperative homeodimer on DNA. Cell 82, 709-719. Wissmuller, S., Kosian, T., Wolf, M., Finzsch, M. and Wegner, M. (2006). The high- mobility-group domain of Sox proteins interacts with DNA-binding domains of many transcription factors. Nucleic Acids Res 34, 1735-44. Wolberger, C., Vershon, A. K., Liu, B., Johnson, A. D. and Pabo, C. O. (1991). Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general
82 model for homeodomain-DNA interactions. Cell 67, 517-28. Wu, M., Li, J., Engleka, K. A., Zhou, B., Lu, M. M., Plotkin, J. B. and Epstein, J. A. (2008). Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice. J Clin Invest 118, 2076-87. Wu, Y. Q., Badano, J. L., McCaskill, C., Vogel, H., Potocki, L. and Shaffer, L. G. (2000). Haploinsufficiency of ALX4 as a potential cause of parietal foramina in the
11p11.2 contiguous gene-deletion syndrome. Am J Hum Genet 67, 1327-32. Xu, W., Rould, M. A., Jun, S., Desplan, C. and Pabo, C. O. (1995). Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations. Cell 80, 639-50. Yusuf, F. and Brand-Saberi, B. (2006). The eventful somite: patterning, fate determination and cell division in the somite. Anat Embryol (Berl) 211 Suppl 1, 21-30. Zhao, G. Q., Zhou, X., Eberspaecher, H., Solursh, M. and de Crombrugghe, B. (1993). Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.
Proc Natl Acad Sci U S A 90, 8633-7. Zhao, H., Bringas, P., Jr. and Chai, Y. (2006). An in vitro model for characterizing the post-migratory cranial neural crest cells of the first branchial arch.Dev Dyn 235, 1433- 40.
Zhou, H. M., Wang, J., Rogers, R. and Conway, S. J. (2008). Lineage-specific responses to reduced embryonic Pax3 expression levels. Dev Biol 315, 369-82.
83 84