To better understand the sequence requirements for Hox factors to bind DNA with Exd and Hth, we selected a set of Hox target sites from Drosophila
and vertebrates for comparative analysis (). The Drosophila
target genes include: the Distal-less
element (DMXR) that is repressed by the Abdominal-A (Abd-A) and Ultrabithorax (Ubx) Hox factors (A/U) in abdominal segments (Gebelein et al., 2002
; Gebelein et al., 2004
); a rhomboid
) activation element (RhoA) that is stimulated by Abd-A in the abdominal nervous system (Li-Kroeger et al., 2008
); a labial
) auto-regulatory element (Lab48-95) and an element (EVIII) from CG11339 that are activated by Lab within the gut endoderm (Ryoo et al., 1999
). The vertebrate target sites include a Hoxb1
auto-regulatory element (Hoxb1-R3-PM2) and elements within other anterior Hox genes (Hoxb2-PP2; Hoxa2-PM-PH2; and Hoxa3-PHP1), which are all regulated by HoxB1 (a lab
homologue) and the Pbx and Meis co-factors within the hindbrain (Ferretti et al., 2005
; Ferretti et al., 2000
; Manzanares et al., 2001
; Popperl et al., 1995
; Tumpel et al., 2007
). Importantly, each cis
-element has been experimentally verified as a Hox target using a combination of genetic, DNA binding, and transgenic reporter assays.
Sequence comparisons between the eight cis-regulatory elements in reveals that while each contains an Exd(Pbx), Hth(Meis), and at least one Hox binding site, the order, orientation, and spacing among sites vary. For example, the two abdominal Hox targets contain neighboring Hth/Hox sites whereas the Lab and HoxB1 targets contain Exd(Pbx)/Hox sites with a distant Hth(Meis) site. Moreover, no specific order and/or orientation of the Hth(Meis) site relative the Exd(Pbx)/Hox site is favored as all four possibilities are represented in the six Lab/HoxB1 targets. These data indicate that a great deal of flexibility exists in forming Hox transcriptional complexes. However, as each site was studied independently, it is unclear how Hox complex formation compares between sites and if there are significant differences between the formation of anterior and posterior Hox complexes. In this study, we use a combination of protein and DNA binding site mutations to compare and contrast the ability of the Abd-A and the Lab Hox factors to form transcription factor complexes with Exd and Hth on their respective target sites.
Characterization of Hox tetramer formation on DMXR
Compared to other Hox-regulated elements, DMXR is unusual in that it contains Hox binding sites close to both an Exd site (Hox1) and a Hth site (Hox2). Previous studies revealed each independent Hox site is cooperatively bound by an Abd-A, Exd, and Hth complex, and that altogether, an abdominal Hox/Exd/Hth/Hox tetramer forms on DMXR to repress Dll
and suppress leg development (Gebelein et al., 2004
). However, it is unclear whether the two Hox sites synergize to further enhance binding cooperativity during Hox complex formation. To address this question, we compared the ability of Abd-A to form complexes with Exd and Hth on probes containing all four binding sites (DMXR), the Hox1/Exd/Hth sites (DMXR1), or the Exd/Hth/Hox2 sites (DMXR2, ). Using a defined quantity of Exd and Hth with two concentrations of Abd-A, we found the amount of DMXR bound (68% and 81%) was approximately the sum of DMXR1 (47% and 62%) and DMXR2 (24% and 33%) (). Next, we analyzed the relative strength of Hox binding to each probe using unlabeled DMXR, DMXR1, and DMXR2 to compete with labeled DMXR. As shown in , the ability to compete for Hox complexes is in the following order: DMXR ≥ DMXR1 > DMXR2 with DMXR2 being significantly weaker than both DMXR and DMXR1. These findings suggest the Hox1 site mediates most of the cooperative binding to DMXR. Consistent with this idea, mutation of the Hox1 but not the Hox2 site significantly compromised binding to DMXR (). However, Hox1/Hox2 double mutations resulted in additional loss in competition, demonstrating both sites can mediate Hox complex formation (). Importantly, these DNA binding assays correlate well with transgenic reporter assays in Drosophila
showing that Hox-mediated repression on DMXR is more sensitive to mutations in Hox1 than Hox2 but double mutations fully compromise gene repression (Gebelein et al., 2004
). Nevertheless, these data indicate that the binding of either Abd-A/Exd/Hth on DMXR1 or Exd/Hth/Abd-A on DMXR2 is sufficient to mediate significant repression in vivo
(Gebelein et al., 2004
Comparing RhoA with DMXR2 – the role of Exd binding on Hth/Hox targets
While Abd-A directly regulates the expression of several other genes, only one has a confirmed set of Exd, Hth, and Hox sites. The RhoA element within rhomboid
contains Exd/Hth/Hox sites that are bound by an Abd-A complex to stimulate gene expression in abdominal sensory cells () (Gebelein et al., 2004
; Li-Kroeger et al., 2008
). Like DMXR2, RhoA has adjacent Hth/Hox sites that closely match a consensus sequence bound by the vertebrate Meis/HoxA9 factors (, (Shen et al., 1997
)). However, the RhoA site more closely matches this consensus, as DMXR2 contains a single nucleotide difference at a constrained position within the Hth site. RhoA and DMXR2 also differ in regards to the location of their Exd binding sites. The Exd site in RhoA is directly adjacent to the Hth site, a configuration found in an optimal Pbx/Meis binding site defined using selection assays in the absence of Hox factors (TGATTGACAG
, Pbx site is italicized; Meis site in bold (Chang et al., 1997
)). In contrast, DMXR2 has the same Exd site (TGAT), but in the opposite orientation and separated from the Hth site by seven nucleotides.
To compare transcription factor binding to RhoA and DMXR2, we performed competition assays with Exd/Hth in the absence and presence of Abd-A. As shown in , RhoA has much higher affinity than DMXR2 for both Exd/Hth and Exd/Hth/Abd-A complexes (). To determine if the single nucleotide change within the DMXR2 Hth site is sufficient to alter binding, we performed gel shift assays using probes containing reciprocal nucleotide changes: DMXR2 G->T and RhoA T->G. As shown in , DMXR2 can be made into a significantly better Exd/Hth site by changing the G to T, but not to wild type RhoA levels. Changing the T to G in RhoA also significantly compromises Exd/Hth binding, but again the RhoA T>G change competes better than DMXR2. Surprisingly, however, neither RhoA T>G nor DMXR2 G>T results in significant changes in competition for the Exd/Hth/Abd-A complex (). Thus, enhanced Exd/Hth binding to RhoA is not sufficient to explain high affinity Hox complex formation relative to DMXR2.
Prior biochemical studies showed that the vertebrate Meis/HoxA9 factors cooperatively bind DNA in the absence of Pbx (Shen et al., 1997
). To determine if Abd-A complex formation on DMXR2 and RhoA depends on Exd binding, we generated DNA binding compromised Exd and Hth proteins by mutating the highly conserved asparagine 51 residue of each of their homeodomains to alanine (N51A) (Gehring et al., 1994
). Using equimolar amounts of wild type Exd/Hth, Exd51A/Hth, or Exd/Hth51A with Abd-A, we found that Exd binding was largely dispensable for Abd-A complex formation on RhoA and DMXR2 (). Moreover, point mutations in the Exd binding sites (Exdm) of RhoA and DMXR2 also have a negligible effect in competing Abd-A complexes compared to wild type probes (). In sharp contrast, the Hth protein mutation (Hth51A) abolished Hox complexes on DMXR2 and greatly diminished Hox complexes on RhoA (). To more easily visualize these results, we assigned the amount of wild type Exd/Hth/Abd-A bound to each probe to 100% and graphed the relative amount bound by each mutant protein (). For example, the Exd/Hth51A heterodimer results in a 90% decrease in Abd-A complexes on DMXR2 versus a 40% decrease on RhoA. We also found that mutation of either the Hth (Hthm) or Hox (Hoxm) sites in DMXR2 and RhoA strongly compromised Hox complex formation in competition assays (). However, the Hth mutations did so to varying degrees as Hthm within DMXR2 nearly eliminated competition, whereas the same Hth mutation in RhoA still competes over 50% of the Hox complex. Altogether, these data demonstrate that while most cooperative Hox binding on DMXR2 and RhoA is mediated by Abd-A and Hth, the presence of a nearby Exd site greatly enhances complex formation on RhoA.
The role of Hth and the spacing between Exd and Hox binding sites
Currently, no other Abd-A targets contain Exd/Hox and Hth sites like DMXR1 for comparative purposes. However, over twenty-five cis
-regulatory elements for other Hox factors contain adjacent Exd(Pbx)/Hox sites (Mann et al., 2009
), and sequence comparisons reveal DMXR1 is the only element that contains a nucleotide (Adenine) inserted between its Hox and Exd sites (). In fact, a previous study using vertebrate proteins found that inserting an extra nucleotide within a Pbx/Hox binding site abolished DNA binding (Chang et al., 1996
). However, that study was performed in the absence of a Hth(Meis) protein and binding site. Hence, to test the dependence of Hox complex formation on Hth, we compared binding to wild type DMXR1 and a modified DMXR1 probe (DMXR1-Con) that removes the extra nucleotide and changes the Hox site from TAATTT to TT
(reverse complement is listed as most studies use this orientation) (, (Gebelein et al., 2002
)). Using Exd/Hth, Exd51A/Hth, and Exd/Hth51A with Abd-A, we found that both Exd and Hth binding are required for forming Abd-A/Exd/Hth complexes on DMXR1 (). In addition, DMXR1 probes containing Exd, Hth, or Hox1 site mutations all significantly decreased competition for Hox complexes compared to wild type DMXR1 (). In contrast, the same concentrations of Exd/Hth and Abd-A proteins on DMXR1-Con revealed that Hth and Exd binding are largely dispensable for complex binding (). These data indicate that either Exd or Hth is sufficient to mediate complex formation on DMXR1-Con. To better test this idea, competition assays using Exd, Hth, and Hox DMXR1-Con binding site mutations revealed that: 1) Hthm competed as well as wild type DMXR1-Con; 2) Exdm did not compete as well as wild type but did compete significantly better than Hoxm; and 3) ExdmHthm double mutations resulted in a significant decrease in competition when compared to either mutation alone (). These findings indicate that a Hth binding site is more critical for Abd-A complex formation on a suboptimal Exd/Hox site (DMXR1) than on an optimal binding site (DMXR1-Con). Surprisingly, we also found a significant amount of complex forms on DMXR1-Con but not DMXR1 in the absence of Exd binding, suggesting additional differences in Hox complex formation between these two probes.
DMXR1-Con alters both the spacing between Exd and Hox sites as well as the Hox binding sequence. To determine which of these changes permits Abd-A complex formation independent of Exd and/or Hth binding, we used two additional probes: DMXR1-ΔA deletes the extra Adenine between the Hox1 and Exd sites, and DMXR1-HoxC changes TAATTT to TTATGG but leaves the extra nucleotide (). We tested the relative strength of each probe in competition assays with DMXR1 and found each behaves similarly in competition assays except DMXR1-Con, which is a significantly better competitor (). We next used wild type versus 51A mutant Exd/Hth proteins to determine the relative dependence of Hox complex formation on Exd and Hth binding to each probe. For comparative purposes, the amount of wild type Abd-A/Exd/Hth bound to each probe was assigned to 100% and the relative amount bound by each mutant was determined (). On DMXR1, Exd51A/Hth results in an 80% decrease and Exd/Hth51A results in over a 70% decrease in Abd-A complex formation. In contrast, the same amount of Exd and Hth mutant proteins decrease binding to DMXR1-Con by only 15% and 10%, respectively. DMXR1-ΔA, on the other hand is highly dependent upon Exd (60% decrease) but not Hth (25% decrease), while DMXR1-HoxC is highly dependent upon Hth (80% decrease) and relatively independent of Exd (20% decrease). Altogether these findings indicate: 1) If the spacing between Hox and Exd sites is optimal, then Hox complexes form relatively independent of Hth (DMXR1-Con and DMXR1-ΔA). 2) If the Hox binding site is TTATGG as opposed to TAATTT, then a distant Hth site can mediate Hox complexes independent of Exd binding (DMXR1-Con and DMXR1-HoxC). Further studies revealed that merely changing the Hox site from TAATTT to TTATTT is sufficient to make DNA binding dependent upon distant Hth sites (data not shown).
Sequence preference for inserted nucleotide between the Exd and Hox sites
As mentioned above, previous studies using vertebrate proteins found that inserting an extra Cytosine nucleotide within a consensus Pbx/Hox binding site abolished DNA binding (Chang et al., 1996
). To determine if the identity of the nucleotide inserted between the Hox1 and Exd sites makes a significant difference in DNA binding activity, we used three additional probes that change the Adenine to either Thymine (DMXR1-A>T), Cytosine (DMXR1-A>C), or Guanine (DMXR1-A>G) as cold competitors for binding Abd-A and Exd/Hth. We found that changing Adenine to any other nucleotide significantly decreased Hox complex formation revealing that Adenine is the preferred nucleotide, then Thymine, Cytosine, and lastly Guanine (Supplemental Figure 1
Comparative analysis of Lab-Exd-Hth cis-regulatory elements
We next tested if the behavior of the abdominal Hox complex on DMXR1 and DMXR1-Con can be extrapolated to other Hox factors and their binding sites. For this analysis, we selected a set of six cis
-elements regulated by lab
Hox genes. As opposed to the posterior Abd-A Hox factor, lab
and its homologues are the most anterior Hox genes and regulate head structures in both vertebrates and invertebrates (Carpenter et al., 1993
; Dolle et al., 1993
; Mark et al., 1993
; Merrill et al., 1989
). Functional conservation between lab
and vertebrate genes has been demonstrated by showing Hoxb1
rescues a lab
null allele in Drosophila
, and a vertebrate cis
-regulatory element (Hoxb1 R3-PM2) drives gene expression in a lab
-dependent pattern in transgenic Drosophila
(Lutz et al., 1996
; Popperl et al., 1995
). Thus, we used purified Drosophila
Exd, Hth, and Lab proteins to analyze complex formation on six Drosophila
and vertebrate cis
-regulatory elements that contain adjacent Exd(Pbx)/Hox sites with variably spaced/oriented Hth(Meis) sites ().
Lab complex formation on all six probes was first analyzed using direct competition analysis. As shown in , a constant amount of Lab and Exd/Hth was bound to the labeled Lab48/95 probe in the absence and presence of different concentrations of each probe as cold competitors. Importantly, while each probe significantly competes for Lab/Exd/Hth complexes, they did so to differing degrees and in the following order: Hoxb2-PP2 > Lab48/95 ≥ Hoxb1 R3-PM2 > Hoxa2 PM-PH2 ≥ EVIII = Hoxa3 PHP1. Similar results were found using comparative gel shift analysis and calculating the percent probe bound under identical Exd/Hth and Lab protein conditions (data not shown). In , we list these cis-elements from strongest to weakest in terms of Lab complex formation and compare strength of binding with cis-element architecture and sequence. Importantly, we find that no one orientation of binding sites is favored as both the strongest and weakest sites share the same configuration. We also compared the Exd/Hox and Hth binding site sequences using position weight based matrices (PWMs).
Comparisons between Lab-Exd-Hth binding sites
Table 1 Comparisons of Hox cis-regulatory elements and Lab complex formation. Probes are listed from strongest (Top) to weakest (Bottom) in terms of Lab complex formation. The Exd (blue), Lab (red), and Hth (yellow) sites are listed in the same direction for (more ...)
For this purpose, we generated PWMs from two data sets. First, we used the recently published binding sequences for the individual Exd, Hth, and Lab proteins identified using a bacterial one-hybrid assay (Noyes et al., 2008
). Second, we used the published sequences identified using purified Meis1 and Pbx1/HoxB1 heterodimers bound to a random oligonucleotide library followed by reiterative purification/amplification (SELEX) (Chang et al., 1996
; Shen et al., 1997
). Sequences were imported into Target Explorer (http://luna.bioc.columbia.edu/Target_Explorer/
) and the program assigned the best matrix for each binding site (Supplemental Data
) (Sosinsky et al., 2003
). Analysis of the six Lab/HoxB1 regulatory elements using each PWM revealed the following: 1) scores for the six Hth sites are uniformly positive using the bacterial one-hybrid PWMs, but the Exd and Lab scores vary greatly and when summated the total scores do not correlate well with strength of Lab complex formation. 2) In contrast, all six probes scored positively using the in vitro
SELEX sites for Meis1 and Pbx1/HoxB1 and there is a strong correlation between strength of Lab complex formation and total PWM score. An additional interesting result that came from this analysis is that the reverse complement of the Exd/Lab site (TAATTGATCA
; Exd in italics, Hox in bold) in the EVIII probe scored significantly higher than the suggested sequence (TGATCAATTA
). This finding indicates that the orientation of these sites may differ from the original published report (Ebner et al., 2005
). However, since our data cannot discriminate between these two possibilities and structural studies would be required to determine the correct orientation, we left the orientation of binding sites as previously published.
We next determined the dependence of Lab complex formation on Exd and Hth binding using the wild type and mutant Exd/Hth proteins. As shown in , all six Lab complexes are heavily dependent upon Exd binding as Exd51A abolishes nearly all complex formation to each probe. The one exception is that some Lab/Exd51A/Hth complex forms on HoxB2-PP2, but when normalized, even this binding is 75% less than wild type (). The Hth51A protein also significantly disrupted Lab complexes but to a variable degree. For example, Lab/Exd/Hth51A disrupted over 90% of binding to Hoxa2 PM-PH2 but only 55% of binding to Lab48/95 and Hoxb1 R3-PM2. Interestingly, the Hoxa2 element has a poor Hox binding site (AGACCG) compared to Lab48/95 (GGATTG) and Hoxb1 (GGATGG), suggesting that like abdominal Hox complexes on DMXR1, Lab complex formation is more highly dependent upon Hth binding if the Exd/Hox site is suboptimal.
Anterior and posterior Hox factors differ in their Exd and Hth binding requirements for Hox complex formation
Unlike our findings using Abd-A on DMXR1-Con, all six of the Lab complexes required wild type Exd and Hth proteins for strong binding. This finding could be due to inherent differences between the Abd-A and Lab Hox factors or due to differences between the cis-regulatory elements tested. To distinguish between these possibilities, we performed gel shift analysis using Lab with wild type Exd/Hth on DMXR1-Con and found Lab readily forms complexes on this probe (, note: slightly more Lab protein was used than Abd-A to achieve a similar level of Hox complex formation, 75.3% vs 79.4% respectively). To determine if Lab can bind DMXR1-Con independent of either Exd or Hth binding, we performed comparative gel shifts using wild type and Exd/Hth mutant proteins and found that both Exd51A and Hth51A formed significantly less complex with Lab than wild type proteins (an 80% decrease with Exd51A and a 60% decrease with Hth51A, ). In contrast, the same amount of mutant Exd and Hth proteins disrupted less than 15% of binding with Abd-A (). As Lab is the most anterior Hox factor and Abd-A is one of the most posterior Hox factors, we also performed the same set of assays using the central Hox factor Sex Combs Reduced (Scr). As shown in , Scr behaved much like Abd-A in that it is able to form a significant amount of Hox complex on DMXR1-Con with either Exd51A or Hth51A.
Differences in complex formation between anterior and posterior Hox factors
We next determined if Abd-A or Scr could form Hox complexes with Exd51A and Hth51A on two of the Lab-regulated cis-elements. For this purpose, we first selected the Hoxa3-PHP1 site because it closely resembles the Exd/Hox site of DMXR1-Con (). Using the same amount of each respective Hox factor as for the DMXR1-Con gel shifts, we found that Scr and Abd-A bind significantly more Hoxa3-PHP1 than Lab (). Moreover, when tested with equal amounts of Exd51A/Hth and Exd/Hth51A, the Abd-A Hox factor was able to significantly bind Hoxa3-PHP1 in the presence of either protein mutant (). Scr, on the other hand, was intermediate between Lab and Abd-A in terms of its ability to form complexes on Hoxa3-PHP1 in the absence of Exd binding (Exd51A). Lastly, we selected the Lab48/95 probe, which contains an Exd/Hox site that is significantly different from DMXR1-Con. This probe was similarly bound by all three Hox factors with wild type Exd/Hth (). However, unlike on DMXR1-Con and Hoxa3-PHP1, Lab, Scr and Abd-A mediated Hox complex formation is heavily dependent upon Exd binding (Exd51A). Thus, Abd-A and to a lesser extent Scr forms complexes on Exd/Hox and Hth sites in the absence of Exd binding, but only on specific Hox sites (TTAT). Moreover, the anterior and posterior Hox factors significantly differ in their ability to form complexes independent of Exd binding. As discussed below, these data have implications for the mechanisms underlying the general ability of posterior Hox factors to phenotypically suppress anterior Hox factors during development.