While Hox proteins may function in many settings, indeed, they interact directly with factors involved in processes as diverse as vesicular trafficking and cell migration (Lambert et al., 2012), this review focuses on the role of Hox proteins in transcriptional regulation. In this context, the current view is that Hox proteins act in multi-component regulatory complexes. For the purpose of this review, we will focus on factors that have been shown to act as part of Hox complexes by biochemical assays. However, it should be noted that genetic screens have identified factors that cooperate with Hox proteins and it is likely that some such factors also bind Hox complexes, perhaps in a context-specific manner (for a recent review, see Mann et al., 2009). Given these criteria, the components of Hox complexes can, somewhat simplistically, be divided into three groups: 1. The Hox proteins themselves; 2. Cofactors with DNA binding domains that interact with Hox proteins and bind adjacent DNA elements; 3. General factors that do not bind DNA directly, but are recruited by Hox proteins and/or cofactors (Fig. 1A).
In the context of multi-component complexes, Hox proteins provide several key functions. First, Hox proteins are responsible for targeting the complex to the appropriate gene regulatory elements. The DNA binding specificities of homeodomain-containing proteins have recently been characterized in some detail (Berger et al., 2008; Noyes et al., 2008), identifying at least 65 different specificities for murine homeodomain proteins and 11 for homedomain proteins in D. melanogaster. Because there are many types of homeodomain proteins in addition to Hox proteins, the first level of classification parsed Hox proteins into only two groups: an Abd-B group (corresponding to paralog groups 9–13 in chordates) and an Antp group containing Hox proteins from the remaining paralog groups (reviewed in Affolter et al., 2008). However, closer examination of DNA-binding specificities revealed that Hox proteins can be further subdivided based on their preferences for lower affinity target sites. Consequently, Hox proteins as a family bind a relatively broad range of DNA sequences, thereby allowing individual Hox proteins to selectively target transcription complexes to distinct regulatory elements, although it is not fully clear how the lower affinity sites are selected in vivo (see next section for a potential role for cofactors in this process). Second, Hox proteins provide interaction surfaces that recruit additional factors to the complex. For instance, Hox proteins interact with Extradenticle (Exd)/Pbx family proteins by means of a short motif (YPWM) found N-terminal to the homeodomain (Fig. 1B; reviewed in Mann and Chan, 1996). This motif is present in many Hox proteins and the central tryptophan residue makes contact directly with a loop in the Exd/Pbx homeodomain. Some Hox proteins that lack the YPWM motif nevertheless contain a tryptophan in a similar position that may be involved in contacting Exd/Pbx proteins (Shen et al., 1997b). Notably, regions in Hox proteins other than the YPWM motif (such as the homedomain and C-terminal residues; Chan et al., 1994) are also likely to be required for binding to Exd/Pbx, particularly in the presence of Homothorax (Hth)/Meis/Prep factors (Hudry et al., 2012). In addition, some Hox proteins interact directly with Hth/Meis/Prep factors (Fig. 1B). This interaction appears to be restricted primarily, but not exclusively, to the Abd-B paralogs (Shen et al., 1997a; Williams et al., 2005) and may be independent of the conserved tryptophan motif, instead requiring N-terminal sequences in the Hox protein. Hox proteins also contain domains able to interact with components of the general transcription machinery, as well as regions that recruit chromatin-modifying enzymes (Fig. 1B). For instance, the N-terminus of several Hox proteins binds the CBP/p300 acetyltransferases (Chariot et al., 1999; Saleh et al., 2000; Shen et al., 2001; Choe et al., 2009), that function as co-activators by acetylating histones (Bannister and Kouzarides, 1996; Ogryzko et al., 1996) and other transcription components (e.g., p53; Gu and Roeder, 1997). Lastly, a recent search for Hoxa1-binding proteins identified more than forty interacting factors, suggesting that numerous additional components are recruited to Hox complexes (Lambert et al., 2012). Hence, Hox proteins play a central role in the targeting of transcription complexes to specific genomic regulatory elements, as well as in the recruitment of key components to such complexes.
Notably, Hox proteins are expressed in a tissue restricted manner such that any given cell expresses only a subset of Hox proteins. This arrangement is reinforced by Hox proteins activating and repressing each other's expression in cross-regulatory patterns, as well as by the action of microRNAs targeting Hox transcripts (reviewed in Yekta et al., 2008). The tissue restricted expression indicates that Hox function is partially regulated at the level of transcription to ensure that Hox complexes of a particular specificity are only present in a subpopulation of cells, although there is also evidence that Hox activity may be modulated posttranslationally (e.g., by phosphorylation; see below). The restricted expression of Hox proteins is particularly apparent during embryogenesis when Hox proteins are found in restricted domains along the anteroposterior, dorsoventral, and proximodistal axes (reviewed in Kmita and Duboule, 2003). For instance, Hox proteins show collinear expression along the anteroposterior axis, in the sense that Hox genes located at the 3′ end of the Hox clusters are expressed earlier and further anteriorly than genes located further 5′. These Hox expression domains overlap, causing several Hox genes to be expressed in the same region. In these cases, it appears that there is a functional hierarchy where the activity of posterior Hox proteins dominates over the activity of anterior ones. This phenomenon is known as phenotypic suppression, or posterior dominance (reviewed in Duboule and Morata, 1994), and the mechanism is unclear, but may involve posterior Hox proteins competing with anterior ones for Exd/Pbx factors (Noro et al., 2011). Hence, the tissue restricted expression of Hox proteins, coupled with processes such as posterior dominance, serve to limit Hox function in vivo.
As discussed, factors that interact with Hox proteins can be divided into two classes: cofactors and general factors (Fig. 1A). The first class, discussed in this section, are cofactors that bind DNA, indeed, these cofactors contain homeodomains, albeit of a slightly different type than that found in Hox proteins, and have important roles in the modulation of Hox function. There are two types of Hox cofactors: Extradenticle/Pbx (herein referred to as PBC proteins) and Homothorax/Meis/Prep (herein referred to as HMP proteins).
extradenticle (exd) was identified in Drosophila as a mutation that causes homeotic phenotypes without affecting the expression of Hox genes, suggesting that it is required for Hox function (reviewed in Mann and Chan, 1996). Pbx proteins were initially discovered as fusion proteins resulting from chromosome translocations that cause pre-B cell leukemia in humans (reviewed in Korsmeyer, 1992). Subsequently, Pbx proteins were shown to be required for Hox function in several biological systems (Pöpperl et al., 2000; DiMartino et al., 2001; Selleri et al., 2001; Waskiewicz et al., 2002; Manley et al., 2004). While there is only one exd gene in Drosophila, there are at least four Pbx genes in vertebrates. Sequence analyses demonstrated that Exd and Pbx proteins are orthologous. Indeed PBC genes appear broadly conserved and have been found also in C. elegans (Burglin and Ruvkun, 1992; Burglin, 1997; Van Auken et al., 2002). Sequence analyses also revealed the presence of a homeodomain in PBC proteins. The PBC homeodomain is unique in that it contains an additional three amino acids in the loop between helix one and helix two. This arrangement is emblematic of the “three amino acid loop extension” (TALE) subclass of homedomains (Bertolino et al., 1995). Notably, these additional amino acids form a pocket that constitutes the contact for the YPWM motif in Hox proteins (Passner et al., 1999; Piper et al., 1999) and are, therefore, essential for the interaction between PBC and Hox proteins that possess this motif, primarily Antp class Hox proteins (Shen et al., 1997b). Pbx also makes contact with Hox proteins by means of an additional helix immediately C-terminal to the PBC homeodomain and by means of residues in the linker between the homeodomain and the YPWM motif (Piper et al., 1999; Saadaoui et al., 2011). Indeed, different Hox:PBC interaction modes may be used in different contexts (Saadaoui et al., 2011).
The interaction with PBC proteins is critical for Hox function (reviewed in Mann and Chan, 1996). First, several studies have demonstrated that Hox proteins bind DNA with greater affinity in complexes with PBC proteins than alone. In fact, in many instances this effect appears to be cooperative such that the affinity of the complex is greater than the summed affinities of the two monomers (Chan et al., 1994; Chang et al., 1995; Popperl et al., 1995). Second, PBC proteins affect DNA target selection by Hox proteins (Chan et al., 1994; Chang et al., 1996; Lu and Kamps, 1997; Joshi et al., 2007; Slattery et al., 2011). This increased selectivity is mediated, at least in part, by the N-terminal arm of the homeodomain and the adjacent linker region making contacts in the minor groove of DNA. These interactions are thought to be favored in the dimer due to PBC binding to the YPWM motif and fixing the region N-terminal to the Hox homeodomain in a conformation permissive for minor groove contacts. In contrast, there are relatively minor changes in PBC DNA binding preference upon dimerization with Hox proteins (Chang et al., 1995; Slattery et al., 2011), consistent with Hox proteins contributing target selectivity while PBC proteins are required to fully reveal that selectivity. Third, PBC proteins bind factors other than Hox proteins and play important roles in recruiting such factors to Hox transcription complexes (Fig. 1B). For instance, Pbx binds both histone deacetylases (HDACs) and histone acetyltransferases (HATs) (Saleh et al., 2000; Choe et al., 2009), as well as N-CoR/SMRT co-repressors (Asahara et al., 1999), suggesting a role for Pbx in regulating the activity of Hox transcription complexes. Additional Pbx interacting factors, such as the zinc finger Pbx1-interacting protein (ZFPIP; Laurent et al., 2007) and hematopoietic Pbx-interacting protein (HPIP; Abramovich et al., 2000), continue to be identified, but their function is poorly understood. Lastly, there have been some reports of Pbx homodimers (Neuteboom and Murre, 1997; Calvo et al., 1999), but their potential roles in vivo are not clear. Hence, PBC proteins bind Hox proteins to affect affinity and selectivity for DNA target sequences, as well as to recruit additional factors to the Hox transcription complex. Importantly, PBC proteins fulfill this function not only for Hox proteins, but also for other transcription factors, such as Engrailed, Pdx1, Emx2, Smads and MyoD (Peers et al., 1995; Serrano and Maschat, 1998; Knoepfler et al., 1999; Kobayashi et al., 2003; Bailey et al., 2004; Capellini et al., 2010).
Notably, some functions appear to be conserved within the PBC family, for instance, Drosophila exd can rescue embryonic development of a zebrafish pbx4 mutant (Pöpperl et al., 2000). However, all functions may not be conserved. In particular, several Pbx genes are alternatively spliced, leading to the formation of distinct isoforms (Nourse et al., 1990; Monica et al., 1991; Milech et al., 2001). Whereas such isoforms do not differ in DNA binding specificity, they may contribute different activities to transcription complexes. For instance, the Pbx1a isoform binds the N-CoR/SMRT co-repressors, while the Pbx1b isoform, that lacks a C-terminal extension, does not (Fig. 1B; Asahara et al., 1999).
In contrast to Hox genes, whose expression is tightly controlled both spatially and temporally during development and differentiation, exd is broadly expressed in Drosophila and Pbx genes are expressed in broad, often overlapping, domains in vertebrates (Monica et al., 1991; Rauskolb et al., 1993; Roberts et al., 1995; Pöpperl et al., 2000; Vlachakis et al., 2000; Maeda et al., 2002). Hence, it is unlikely that restricted PBC expression represents a regulatory step in controlling their availability as Hox cofactors. However, there is evidence that both nuclear localization and protein stability of PBC factors is controlled by means of interactions with other factors, such as Hth/Meis/Prep (Aspland and White, 1997; Rieckhof et al., 1997; Pai et al., 1998; Abu-Shaar et al., 1999; Berthelsen et al., 1999; Vlachakis et al., 2001; Waskiewicz et al., 2001), and Pbx nuclear localization is also regulated by PKA-mediated phosphorylation (Kilstrup-Nielsen et al., 2003), suggesting that PBC availability may be controlled posttranslationally.
The second family of Hox cofactors consists of the Homothorax, Meis, and Prep proteins. These proteins also belong to the TALE family, but form a subgroup distinct from the PBC proteins. The Drosophila ortholog is known as homothorax (hth; Rieckhof et al., 1997) and orthologous genes are found also in C. elegans (Burglin, 1997; Van Auken et al., 2002). As in the case of exd, disrupting hth produces a homeotic phenotype without affecting Hox gene expression (Rieckhof et al., 1997). Meis genes were originally identified as proto-oncogenes co-activated with Hox genes in leukemias (Moskow et al., 1995; Nakamura et al., 1996b), while Prep proteins were discovered as transcription factors regulating expression of the urokinase plasminogen activator gene (Berthelsen et al., 1998b). Subsequent analyses revealed the existence of three vertebrate Meis genes (Meis1, 2, 3; (Nakamura et al., 1996a); four in zebrafish; Waskiewicz et al., 2001) and two vertebrate Prep genes (Prep1, 2; Imoto et al., 2001). Many groups have demonstrated that HMP proteins form complexes with PBC and Hox proteins (reviewed in Mann and Affolter, 1998). In particular, HMP proteins interact with PBC proteins by means of conserved domains in the N-termini of HMP and PBC family proteins (Fig. 1B; Chang et al., 1997; Knoepfler et al., 1997; Berthelsen et al., 1998a, 1999; Abu-Shaar et al., 1999; Jaw et al., 2000; Vlachakis et al., 2001). While PBC:HMP dimers have been observed to form on regulatory elements in vitro (e.g., Berthelsen et al., 1998b; Bischof et al., 1998) and such dimers may have functions in vivo, they will not be considered further here because they would act independently of Hox complexes. Meis/Prep proteins are also able to bind some, but not all, Hox proteins. Specifically, Meis proteins interact directly with Hox proteins of the Abd-B class (paralog groups 9–13) by means of a C-terminal domain containing the Meis homeodomain (Shen et al., 1997a). In contrast, Meis proteins do not interact with Antp class Hox proteins (paralog groups 1–8), at least not when tested in gel shift assays (Shen et al., 1997a; Williams et al., 2005). As discussed above, Pbx proteins appear to have a complementary pattern of Hox interactions such that Pbx binds Antp, but not Abd-B, class Hox proteins (Shen et al., 1997b). Because PBC and HMP proteins interact with each other by means of N-terminal domains, while both families interact with Hox proteins by means of residues within and near their homeodomains, it is plausible that HMP:PBC:Hox complexes might form. Indeed, numerous studies have demonstrated the existence of such trimeric complexes (Berthelsen et al., 1998a; Jacobs et al., 1999; Ryoo et al., 1999; Shanmugam et al., 1999; Shen et al., 1999; Ferretti et al., 2000; Vlachakis et al., 2000) and ChIP experiments have identified the presence of all three protein families at Hox-regulated enhancers in developing embryos (Choe et al., 2009; Penkov et al., 2013). Because all Hox proteins appear to interact with either PBC or HMP proteins, it is formally possible that Hox proteins from all paralog groups act in such trimeric complexes, but this has not been tested exhaustively.
HMP proteins are required for Hox function in several contexts (Rieckhof et al., 1997; Jacobs et al., 1999; Ferretti et al., 2000; Waskiewicz et al., 2001; Choe et al., 2002). However, HMP proteins do not appear to modify Hox DNA binding specificity the way PBC proteins do. Instead, HMP may modulate Hox function indirectly by regulating the stability and nuclear localization of PBC, as discussed above (Aspland and White, 1997; Rieckhof et al., 1997; Pai et al., 1998; Abu-Shaar et al., 1999; Berthelsen et al., 1999; Vlachakis et al., 2001; Waskiewicz et al., 2001), as well as by modulating the interaction between PBC and Hox proteins (Hudry et al., 2012). As for PBC proteins, HMP proteins may also regulate the recruitment of transcriptional co-regulators (Fig. 1B). In particular, Pbx:Hox complexes bind HDACs and repress transcription, at least under some conditions (Saleh et al., 2000). This repression can be overcome by blocking HDAC activity (with TSA) or by inclusion of Meis proteins (Saleh et al., 2000; Huang et al., 2005; Choe et al., 2009), suggesting that Meis proteins function by counteracting HDAC activity. Indeed, Meis proteins compete with HDAC for binding to Pbx such that in the presence of Meis, HDACs are displaced from the complex (Choe et al., 2009). In addition, the Meis1 protein contains a C-terminal activation domain that is activated by PKA and/or GSK-3 signaling to interact with TORC and CBP (Huang et al., 2005; Goh et al., 2009; Wang et al., 2010). Hence, HMP proteins may function by controlling co-regulator recruitment to Hox complexes, thereby modulating the function of these complexes.
The two Prep genes are essentially ubiquitously expressed (Ferretti et al., 1999; Imoto et al., 2001; Waskiewicz et al., 2001), similar to Pbx genes, and hth is broadly expressed in Drosophila (Rieckhof et al., 1997). In addition, while expression of the Meis genes is spatially and temporally restricted (Nakamura et al., 1996a; Waskiewicz et al., 2001), there are multiple Meis genes (three in vertebrates, except four in zebrafish), suggesting that most cells express one or several Meis/Prep proteins. There have been a few reports of functional differences between Meis and Prep proteins, for instance, Meis1 accelerates HoxA9-dependent leukemia (Thorsteinsdottir et al., 2001) and responds to PKA signaling (Huang et al., 2005), but Prep1 does neither. However, in other assays Meis and Prep proteins appear interchangeable (Choe et al., 2002). There may also be functional differences among the Meis proteins. In particular, the Meis C-terminus harbors an activation domain (Huang et al., 2005; Goh et al., 2009). C-terminal sequences vary considerably among Meis family members, but zebrafish Meis3 (that has a truncated C-terminus) is nevertheless capable of functioning as a Hox cofactor to activate transcription (Vlachakis et al., 2001; Choe et al., 2009). Hence, Meis/Prep family members may differ in some functional features, but also share several key features. Hox complexes that contain different Meis/Prep family members may, therefore, share many activities, but may also have some distinct functions, as supported by recent genome wide ChIP data (Penkov et al., 2013).
Lastly, as in the case of PBC proteins, HMP proteins act as cofactors not only to Hox proteins, but also to other transcription factors such as MyoD and Engrailed (Kobayashi et al., 2003; Berkes et al., 2004).
The second class of factors that associates with Hox complexes has more general roles in transcription. Most of these factors differ from the PBC and HMP cofactors in that they lack DNA-binding domains and must, therefore, be recruited to Hox complexes exclusively by means of protein–protein interactions. In general, these factors are ubiquitously expressed and, accordingly, are available for recruitment to Hox complexes in most cell types. This group of factors includes chromatin-modifying enzymes mentioned previously. Specifically, Hox and Pbx proteins interact with CBP (Chariot et al., 1999; Saleh et al., 2000; Shen et al., 2001; Choe et al., 2009), as does Meis1 (Huang et al., 2005; Wang et al., 2010), but apparently not Meis3 (Choe et al., 2009). CBP acts as a histone acetyltransferase (HAT) that acetylates nucleosomes, thereby promoting open chromatin and facilitating transcription (Goodman and Smolik, 2000). CBP can also acetylate nonhistone proteins, but, to date there is no evidence that acetylation of Hox complex components affects the activity of the complex. In addition, Pbx proteins interact with HDAC1 and HDAC3 co-repressors, either directly or by means of NCoR/SMRT, (Asahara et al., 1999; Saleh et al., 2000; Choe et al., 2009). HDACs function as histone deacetylases to oppose the activity of CBP, thereby repressing transcription. The recruitment of co-repressors appears to be mediated by means of both N- and C-terminal domains in Pbx (Asahara et al., 1999; Saleh et al., 2000), while the CBP co-activator interacts with N-terminal activation domains in Hox proteins and a C-terminal domain in Meis1 (Chariot et al., 1999; Saleh et al., 2000; Huang et al., 2005; Goh et al., 2009).
Importantly, these co-activators and co-repressors have themselves been found to be part of larger regulatory complexes. For instance, HDAC1 is part of the Sin3, NuRD, and CoREST complexes, while HDAC3 is part of the NCoR/SMRT complex (reviewed in Hayakawa and Nakayama, 2011). Hence, while experiments to date have examined only recruitment of individual co-activators/co-repressors, it is likely that they are recruited as components of larger complexes that contribute additional activities. For instance, both the NuRD and CoREST complexes contain enzymes that regulate nucleosome positioning and modification (Tong et al., 1998; Xue et al., 1998; Shi et al., 2004).
Ultimately RNA Polymerase II must be recruited to Hox-regulated promoters and activated to drive transcription. As in other systems, this is likely to be accomplished by recruitment of the Mediator complex (reviewed in Conaway and Conaway, 2011). Mediator is commonly recruited to enhancers by sequence specific transcription factors and Mediator then bridges the distance to the transcription start site where it facilitates RNA Polymerase II loading. Hence, it is likely that Hox, PBC, and/or HMP proteins recruit Mediator. However, interactions between these factors and Mediator have not been well characterized and it is unclear how recruitment and activity of Mediator is regulated by Hox transcription complexes.