An autoinhibitory effect of the homothorax domain of Meis2

Authors


D. Wotton, Center for Cell Signaling, University of Virginia, Box 800577, HSC, Charlottesville, VA 22908, USA
Fax: +1 434 924 1236
Tel: +1 434 243 6752
E-mail: dw2p@virginia.edu

Abstract

Myeloid ecotropic insertion site (Meis)2 is a homeodomain protein containing a conserved homothorax (Hth) domain that is present in all Meis and Prep family proteins and in the Drosophila Hth protein. The Hth domain mediates interaction with Pbx homeodomain proteins, allowing for efficient DNA binding. Here we show that, like Meis1, Meis2 has a strong C-terminal transcriptional activation domain, which is required for full activation of transcription by homeodomain protein complexes composed of Meis2 and Pbx1. We also show that the activity of the activation domain is inhibited by the Hth domain, and that this autoinhibition can be partially relieved by the interaction of Pbx1 with the Hth domain of Meis2. Targeting of the Hth domain to DNA suggests that it is not a portable trans-acting repression domain. However, the Hth domain can inhibit a linked activation domain, and this inhibition is not limited to the Meis2 activation domain. Database searching reveals that the Meis3.2 splice variant, which is found in several vertebrate species, disrupts the Hth domain by removing 17 codons from the 5′-end of exon 6. We show that the equivalent deletion in Meis2 derepresses the C-terminal activation domain and weakens interaction with Pbx1. This work suggests that the transcriptional activity of all members of the Meis/Prep Hth protein family is subject to autoinhibition by their Hth domains, and that the Meis3.2 splice variant encodes a protein that bypasses this autoinhibitory effect.

Structured digital abstract

Abbreviations
AD

activation domain

EST

expressed sequence tag

GBD

Gal4 DNA-binding domain

HD

homeodomain

hr1

homology region 1

hr2

homology region 2

Hth

homothorax

HTH

Hth protein

Meis

myeloid ecotropic insertion site

SV40

simian virus 40

Introduction

Homeodomain (HD) proteins were first identified in flies, and are conserved across diverse species from yeasts to mammals [1,2]. The characteristic DNA-binding HD is ∼ 60 amino acids in length and consists of three α-helices [3]. It is the third α-helix within the HD that is the primary DNA-binding region, although there are other DNA contacts outside helix 3 [4–7]. In addition to binding DNA, the HD is a protein interaction module that mediates interactions with other DNA-binding proteins and non-DNA-binding transcriptional regulators. HD proteins can be recruited to DNA by direct DNA binding, and indirectly via interaction with other transcription factors [8,9]. However, even when HD proteins bind their cognate DNA-binding site, they generally bind to other DNA-binding cofactors [10–12]. Meis2 is a member of the TALE superfamily of HD proteins, which are characterized by the presence of a three amino acid loop insertion between helices 1 and 2 of the HD [13–15]. The presence of this loop between helices 1 and 2 is unlikely to affect DNA binding directly, but plays a role in protein–protein interactions [6,7]. TALE superfamily HD proteins participate in both activating and repressing transcription factor complexes. For example, proteins such as Tgif1 and Tgif2 are obligate transcriptional repressors that are primarily recruited to DNA by interactions with other DNA-binding proteins [16–18]. In contrast, Meis–Pbx complexes appear to be primarily involved in transcriptional activation [9,19,20].

In humans and mice, there are three myeloid ecotropic insertion site (Meis) paralogs and two Prep genes, which are closely related to the Meis group. Mammalian Meis1 was identified initially as a common site of viral integration in mouse myeloid leukemia cells [21], and the related Meis2 and Meis3 genes were identified by sequence similarity [22,23]. Meis1 plays a key role in the progression of acute myeloid leukemia and mixed lineage leukemia, and fusion proteins generated by chromosomal rearrangements in mixed lineage leukemia can induce increased expression of Meis1 [24–26]. Prep1 plays a role in hematopoietic stem cell function, and in early T-cell development [27–29]. Pbx proteins, which are common partners of Meis family members, have also been implicated in tumorigenesis. Pbx1 can be fused to the transcription factor E2A as a result of the t(1;19) translocation in pre-B-cell leukemia [30,31]. This fusion prevents interaction with Meis proteins and converts Pbx1 to a strong transcriptional activator.

In addition to the HD, Meis and Prep proteins share a second region of high sequence conservation, termed the homothorax (Hth) domain [15,32,33]. This domain is named for the Drosophila Hth protein (HTH). The Hth domain interacts with Pbx proteins, thereby promoting cooperative binding of Meis–Pbx dimers to a composite DNA element [34,35]. The interaction of the Meis and Pbx partners also facilitates binding of the Pbx partner to DNA [34]. Interestingly, this requirement for a Meis partner is lost in oncogenic Pbx fusion proteins, such as the E2a–Pbx protein. Additionally, the interaction of Meis family proteins with a Pbx protein allows for recruitment of the Meis protein to a DNA-bound Pbx–Hox complex, without the need for direct binding of the Meis protein to a consensus Meis site [8,9]. A conformational change in Pbx1a and interaction with a Meis protein are required for nuclear localization of Pbx1, suggesting that the Meis and Pbx partners are regulated by mutual interaction [36]. Recent evidence has suggested that the p160 Myb-binding protein interacts with the Hth domain of Prep1 and is a negative regulator of Prep1–Pbx complexes [37]. Thus, the Hth region of Meis family proteins is clearly a key regulatory domain within these proteins that can mediate both positive and negative influences on transcriptional activity. Interestingly, splice variants of mammalian Meis1 and Meis2, and Drosophila HTH, that encode proteins lacking the HD have been identified [38,39]. The Meis2e variant, which is truncated prior to the end of the first α-helix of the HD, has been suggested to act as a dominant negative form of the Meis protein that may be able to interfere with the formation of fully functional Meis–Pbx complexes [39]. HTH that lacks the HD can carry out many of the developmental functions of full-length HTH, but cannot substitute for it in all cases [38].

Here, we demonstrate that the Meis2 and Prep1 Hth domains inhibit the ability of the full-length proteins to activate transcription. In the case of Meis2, the C-terminus contains a strong transcriptional activation domain (AD), the activity of which is inhibited by the Hth domain. This autoinhibition can be relieved, in part, by interaction with Pbx1, and maps to a region of the Hth domain that also contributes to Pbx interaction. Finally, we show that the Meis3.2 splice variant generates a protein lacking 17 amino acids from the Hth domain. Removal of the equivalent region from Meis2 results in both decreased interaction with Pbx1 and weakened autoinhibition.

Results

Meis2 contains a C-terminal AD

Several splice variants of Meis2 have been described, most of which affect the region C-terminal to the HD, whereas Meis2e lacks most of the HD and everything C-terminal to it [39]. To test whether Meis2 could activate transcription, we targeted both Meis2d and Meis2e to DNA by fusing them to the Gal4 DNA-binding domain (GBD; Fig. 1E). When they were targeted to a minimal TATA element containing a promoter via multiple Gal4 sites, we observed several-fold activation by Meis2d, but no activation by Meis2e (Fig. 1A). However, this activation by Meis2d was relatively weak, particularly in light of the recent identification of a strong AD in the C-terminal region of the related Meis1 protein [40]. Interestingly, when we deleted the Hth domain from Meis2d in the context of the GBD fusion protein, we observed a dramatic increase in the level of transcriptional activation as compared with the wild-type Meis2d fusion protein (Fig. 1A). The GBD fusion protein lacking the Hth domain also significantly increased transcription from the more active simian virus 40 (SV40) promoter, although the wild-type Meis2d and Meis2e fusion proteins were unable to do so (Fig. 1B). No repression of SV40 promoter activity was observed by either Meis2d or Meis2e, whereas a GBD–TGIF repressor fusion protein decreased the activity of this reporter (Fig. 1B). To test whether derepression of transcriptional activity by removal of the Hth domain might be a more general feature of Meis family proteins, we tested the effects of GBD fusion proteins on Prep1 and a version of Prep1 lacking its Hth domain. Prep1 did not activate the TATA-containing reporter, whereas the Hth deletion mutant increased transcription at least 10-fold (Fig. 1C). Importantly, the higher levels of transcriptional activation by the Hth deletion mutants did not appear to result simply from increased expression of these constructs as compared with the wild-type Meis2d or Prep1 fusion proteins (Fig. 1D). To further define the Meis2d transcriptional AD, we tested two other GBD fusion proteins, which contained either the Meis2 HD and C-terminal region, or just the region C-terminal to the HD. As shown in Fig. 1C, both fusion proteins activated gene expression to a similar degree as the Hth deletion mutant, suggesting that the approximately 150 amino acids C-terminal to the HD of Meis2d contain a transcriptional AD.

Figure 1.

 Meis2 contains a C-terminal AD. HepG2 cells were transfected with the indicated GBD fusion proteins, and the (Gal)5-TATA luciferase reporter (A), or the (Gal)5-SV40 reporter (B). Luciferase activity was assayed after 48 h, and is presented as the mean + standard deviation of duplicate transfections (arbitrary units). (C) A series of Meis2 and Prep1 deletion constructs fused to the GBD were assayed as in (A). (D) The relative expression of the indicated GBD fusion proteins was analyzed by western blot (WB) with a GBD antibody. The specific full-length bands are indicated by arrows. The numbers below the lanes correspond to the numbered constructs in (E) and Figure 4 (F). The positions of molecular mass markers (95, 72, 55 and 43 kDa) are shown to the left. (E) GBD expression constructs are shown schematically. The scale below shows amino acid numbers.

Both the Meis2 AD and the Hth domain are required for transcriptional activation by Meis–Pbx

To test whether the Meis2 AD is required in the context of transcriptional regulation in complex with Pbx1, we tested two reporters, one in which luciferase activity is under the control of two copies of a canonical Meis–Pbx-binding site and a minimal TATA element, and one with two copies of the Hoxb1 auto-regulatory element (ARE) r3 element [9]. Coexpression of Meis2d and Pbx1 together with the Pbx–Meis reporter resulted in > 10-fold activation as compared with the control, or with expression of either protein alone (Fig. 2A). Meis2e did not activate this reporter with Pbx1, and activation was clearly impaired by deletion of the Meis2d AD, or by a point mutation (R332M) that decreases binding to a consensus Meis site. To confirm that these constructs were able to interact with Pbx1a, we performed coimmunoprecipitaion assays from COS1 cells transfected with T7-tagged Pbx1a and Flag-tagged Meis2d or Meis2d mutants. As shown in Fig. 2B, removal of the Hth domain abolished interaction with Pbx1a. We also tested the Meis2 mutant that lacks the AD (Meis2dΔAD, encoding amino acids 2–345 of Meis2), and one that binds DNA poorly (R332M; this contains a point mutation in helix 3 of the HD, which alters a critical DNA contact residue), and both retained the ability to interact with Pbx1a. Importantly, the expression levels of both the R332M mutant and the AD deletion mutant were similar to those of wild-type Meis2d.

Figure 2.

 The Meis2 AD is required for Pbx-dependent transcriptional activation. (A) HepG2 cells were transfected with the indicated expression constructs and a luciferase reporter in which luciferase expression is driven by two copies of a Meis–Pbx consensus binding site and a minimal TATA element. Meis2d(ΔAD) encodes amino acids 2–345 of Meis2, and so lacks the AD, and the R332M mutant has a point mutation in the HD that prevents binding to a consensus Meis site. (B) COS1 cells were transfected with T7-tagged Pbx1a and the indicated Flag-tagged Meis2 expression constructs. Complexes were isolated on Flag agarose, and analyzed for coprecipitating T7-Pbx1a. Expression in the lysates is shown below. (C) Cells were transfected and analyzed as in (A), with increasing amounts of coexpressed Meis2e. (D) HepG2 cells were transfected with the indicated Meis2 expression constructs and HoxB1 or Pbx1 expression constructs as indicated, together with a luciferase reporter containing two copies of the Hox ARE r3 element, which binds Hox and Pbx proteins. (E) The effect of expressing increasing amounts of either the Meis2e splice variant or the AD deletion mutant of Meis2 on Hox ARE luciferase reporter activity was assayed as in (C). Triangles in (C) and (E) represent ratios of 1 : 1, 1 : 2, 1 : 4 and 1 : 6 of Meis2d to Meis2e or Meis2dΔAD. (F) HepG2 cells were assayed as in (E), with the indicated ratios of transfected Meis2d and Meis2e. Expression of the Meis2 proteins was assayed by Flag western blot (right). Numbers 1–6 above the luciferase data correspond to lanes 1–6 of the blot. IP, immunoprecipitation; WB, western blot.

We next tested the possibility that Meis2e might interfere with activation by Meis2d and Pbx1. However, as shown in Fig. 2C, even when Meis2e was cotransfected at a five-fold excess relative to Meis2d, we observed minimal inhibition of the Pbx–Meis reporter by Meis2e. Meis family proteins can also be recruited to DNA without the requirement for DNA binding, by interactions with other HD proteins, such as Pbx1 and Hox proteins. To test the importance of the Meis2d AD for this mode of transcriptional regulation, we used a reporter based on the Hoxb1 ARE, which contains a composite binding site for Pbx1 and Hoxb1, but lacks a Meis2 consensus site. Transfection of Meis2d, Pbx1a or Hoxb1 expression constructs individually did not dramatically activate this reporter (Fig. 2D). However, coexpression of either Meis2d or Hoxb1 with Pbx1a resulted in 15-fold to 20-fold activation, and coexpression of all three proteins together resulted in even greater activation. In contrast, Meis2e or the AD deletion mutant of Meis2d failed to increase activity over that seen with Pbx1a and Hoxb1 alone (Fig. 2D). As expected, because this reporter does not contain a Meis2-binding site, the R332M mutation did not affect activity. As with the Pbx–Meis reporter, we did not observe interference by overexpression of Meis2e in the presence of Meis2d, Pbx1a, and Hoxb1 (Fig. 2E). However, at high levels of overexpression, the Meis2d mutant lacking the AD was able to inhibit activation of this reporter (Fig. 2E). We next tested whether further increasing Meis2e levels, with a relatively low level of Meis2d, would allow Meis2e to interfere with Meis2d function. When Meis2e was cotransfected at a ratio of up to 10 : 1 with Meis2d, we did observe some interference (Fig. 2F). However, it should be noted that the level of Meis2d in this experiment resulted in only modest reporter activation over that seen with HoxB1 and Pbx1a alone.

To test whether the Hth domain was required for activation of Pbx-dependent reporters by Meis2d, we expressed wild-type or the Hth deletion mutant of Meis2d alone or with Pbx1a, and tested activation of the Meis–Pbx reporter and the Hoxb1 ARE. As shown in Fig. 3A, we observed a small increase in activity from the Meis–Pbx reporter with the Hth deletion mutant as compared with wild-type Meis2d, but this mutant was unable to cooperate with Pbx1a to activate the reporter. With the Hoxb1 ARE, Meis2 lacking the Hth domain was completely nonfunctional, consistent with an absolute requirement for recruitment via Pbx1 (Fig. 3B). Together, these results suggest that the Meis2d AD is required for transcriptional activation, whether Meis2d binds directly to DNA or is recruited by other HD proteins. Additionally, it appears that the protein encoded by the Meis2e splice variant has a limited ability to act as an effective dominant negative.

Figure 3.

 The Hth domain is required for Pbx1-dependent transcription. HepG2 cells were cotransfected with the indicated expression constructs and either the Meis–Pbx-TATA luc reporter (A) or the Hoxb1 ARE reporter (B). Luciferase activity was measured after 48 h, and is presented as the average of duplicate transfections.

The Hth domain inhibits the activity of a linked AD

To further delineate the region required for the inhibitory effect of the Hth domain, we created a series of GDB fusion proteins (Fig. 4F). Deletion of either the N-terminal 65 or the N-terminal 97 amino acids did not derepress the Meis2d AD, whereas a smaller internal deletion (removing amino acids 150–193), which encompasses homology region 2 (hr2) of the Hth domain, derepressed it to a similar degree as the full Hth deletion (Fig. 4A). To test whether the inhibitory activity of the Hth domain was specific to the Meis2 AD, we next created an AD swap construct, in which the relatively proline-rich Meis2d AD was replaced with the acidic AD from the Drosophila TGIFa protein [41]. As shown in Fig. 4B, this chimeric construct did not activate the Gal4 reporter, but was significantly derepressed by deletion of the Hth domain, suggesting that the inhibitory effect of this domain is not specific to the Meis2d AD. Comparison of the relative expression levels of these GBD fusion proteins (see Fig. 1E) suggests that the increased transcriptional activation seen with Hth deletion does not correlate with expression level. To test the possibility that the Hth domain was a portable transcriptional repression domain, we targeted increasing amounts of GBD–Meis2d or GBD–Meis2e to the SV40 promoter, which has a high basal level of activity. As shown in Fig. 4C, we observed a little more than two-fold activation of this promoter by Meis2d, and little repression (1.3-fold) by Meis2e, which lacks the AD, but retains the Hth domain. We next compared the effects of targeting either Meis2e or TGIF to two promoters with lower basal activity than the SV40 promoter. As shown in Fig. 4D,E, GBD–TGIF resulted in maximal repression of at least 2.5-fold for both reporters, whereas we observed much lower-level repression by GBD–Meis2e. However, on the Gal-TK reporter, GBD–Meis2e resulted in repression by up to 1.7-fold (a 42% reduction in activity), suggesting that it may have weak repressive activity (Fig. 4E). Thus, it appears that the Hth domain is able to effectively inhibit the activity of at least two different linked ADs, but does not act as a potent general transcriptional repression domain.

Figure 4.

 The Hth domain inhibits a linked AD. HepG2 cells were cotransfected with the Gal-TATA luciferase reporter (A, B) or the Gal-SV40 reporter (C) and the indicated GBD–Meis2 fusion proteins. The effects of increasing amounts of GBD or GBD fusions with TGIF and Meis2e were tested on the Gal-TATA luciferase (D) or Gal-TK-luciferase (E) reporters. (F) The GBD–Meis2 fusion proteins are shown schematically. The AD from Drosophila TGIFa is indicated as dTA.

Mutational analysis of the Hth domain

Previous work has identified point mutations within the Hth domain that weaken interaction with Pbx1 [35]. An interaction between Prep1 and the transcriptional repressor p160Mybbp1 has been mapped to the Prep1 Hth domain, and specifically to a leucine-rich motif in homology region 1 (hr1) [37]. To test whether Pbx1 or p160Mybbp1 interaction might contribute to the inhibitory effect of the Hth domain, we created three GBD–Meis2d mutants, which should affect either Pbx1 interaction (NNGT and IL-AA; Fig. 5A) or interaction with both Pbx1 and p160Mybbp1 (LL-AA). In addition, we noticed a relatively close match to the consensus interaction motif for CtBP [PxDL(R/S/T) [42]; PIDLV in Meis2], which is missing from our hr2 and Hth deletion constructs. As this sequence is conserved in most Meis relatives, except for the Prep subfamily, we also created a mutant lacking the PIDLV. We first tested the effects of targeting the GBD fusion proteins to the TATA-containing luciferase reporter. As shown in Fig. 5B, none of these mutations resulted in significant derepression of GDB–Meis2d. When we tested the effects of the NNGT and IL-AA mutations on transcription, using the Pbx–Meis and Hox ARE reporters, we observed some decrease in activity in the presence of Pbx1a relative to that seen with wild-type Meis2d and Pbx1a, consistent with a weakened Pbx1 interaction (Fig. 5C,D). In contrast, we did not see any effect of either the LL-AA or ΔPIDLV mutations, and none of these mutations resulted in increased Meis2d transcriptional activity, as would be expected if they affected the inhibitory function of the Hth domain. To verify that the Pbx1 interaction mutants (NNGT and IL-AA) did indeed affect interaction with Pbx1, we performed coimmunoprecipitation experiments from transfected COS1 cells. As shown in Fig. 4E, significantly less Pbx1a coprecipitated with the NNGT and IL-AA mutant forms of Meis2d than with the wild type, whereas the LL-AA mutant had little effect in this assay.

Figure 5.

 Pbx1 derepresses GBD–Meis2d. (A) The Meis2d Hth domain is shown schematically, together with the sequence of four mutant forms of Meis2d. (B) HepG2 cells were transfected with GBD–Meis2 expression constructs and the (Gal)5-TATA luciferase reporter, and luciferase activity was measured after 48 h. The indicated Meis2 expression constructs were coexpressed with Pbx1a and HoxB1, as indicated, and luciferase activity from the Meis–Pbx reporter (C) or Hox ARE reporter (D) was assayed after 48 h. (E) The indicated Flag-tagged Meis2 mutants, Meis2d or Meis2e, were coexpressed with T7-tagged Pbx1a in COS1 cells. Protein complexes were isolated on Flag agarose, and analyzed for coprecipitating T7-Pbx1a. Expression in the lysates is shown below. (F) HepG2 cells were transfected with GBD–Meis2 expression constructs and the (Gal)5-TATA luciferase reporter, together with T7-tagged Pbx1a or a truncation mutant that encodes the N-terminal 233 amino acids (including the Meis2 interaction domains). Luciferase activity was measured after 48 h. IP, immunoprecipitation; WB, western blot.

As the Pbx interaction mutants in hr2 of Meis2 failed to derepress Meis2d transcriptional activity, we tested the alternative possibility, that interaction with Pbx might help to alleviate the inhibitory effect of hr2. To do this, we used GBD fusions with Meis2d and the Hth deletion mutant, and coexpressed either full-length Pbx1a, or the N-terminal 233 amino acids of Pbx1a, which contain the Meis interaction domains. As shown in Fig. 5F, we observed a 3.3-fold increase in the activity of GBD–Meis2d with full-length Pbx1a, and an almost eight-fold increase in the presence of the N-terminal fragment of Pbx1a. In contrast, there was relatively little effect on the Hth deletion mutant of Meis2d, even when a low level of GBD–Meis2d(ΔHth) was used, such that an increase in activity on this reporter would be easily detectable. These data suggest that interaction of Pbx1a with the Hth region can, to some degree, relieve the inhibitory effect of hr2 on transcriptional activation.

Pbx interaction is separable from autoinhibition

The Hth domain of Meis2 contains two regions, termed hr1 and hr2, which are highly conserved from flies to mammals, and are present in multiple Meis paralogs (Fig. 6A). As hr2 appeared to be most important for inhibition of transcriptional activity, we generated a series of mutant forms of Meis2d in which we changed charged and hydrophobic residues to alanines (Fig. 6A). We also noticed that hr2 contains three highly conserved cysteines, which we also converted to alanines. We first tested whether these four Meis2d mutants were expressed at similar levels as the wild type, and whether they were able to interact with Pbx1a. As shown in Fig. 6B, all four mutants were expressed at similar levels as wild-type Meis2d, and all appeared to interact with Pbx1a to some degree. However, the interaction of the L3-A mutant with Pbx1a was reduced by at least as much as that of the previously described LL-AA mutant. Additionally, the EEK-A mutant was somewhat impaired for Pbx1a interaction. Next, we used the Gal4 system to test the effects of these mutations on transcriptional activity. Two amounts of each GBD–Meis2 fusion protein were transfected, together with the Gal-TATA luciferase reporter. Among the four mutant forms of Meis2, we observed around two-fold derepression with two of them, the L3-A and YIL-A mutants, whereas the others showed similar activity in this assay as the wild type (Fig. 6C). We next tested the effect of these mutants on activation of the Pbx–Meis and Hox ARE reporters. As shown in Fig. 6D,E, only the YIL-A mutant resulted in any increase in activity over that seen with wild-type Meis2d. The L3-A mutant, which caused derepression in the GBD fusion assay, failed to do so with these reporters, presumably because of its decreased interaction with Pbx1a. These data suggest that interaction with Pbx1a and the autoinhibitory activity are separable functions.

Figure 6.

 Mutational analysis of hr2. (A) An alignment of the Hth domains from Meis relatives is shown. Amino acids that are identical or similar between all sequences shown are shaded black and gray respectively. The sequences shown are human Meis1, Meis2, Meis3, Prep1, and Prep2, Xenopus laevis Meis1, Meis3, and Prep (XlMs1, XlMs3, and XlPrep), Drosophila melanogaster HTH (DmHth), and a Meis-like protein from Caenorhabditis elegans (Unc-62). Brackets above the sequences indicate hr1 and hr2. Mutations within Meis2 hr2 are shown below. Dots indicate no change. (B) COS1 cells were transfected with the indicated Flag-tagged Meis2 expression constructs and T7-Pbx1a. Proteins were isolated on Flag agarose, and the presence of coprecipitating Pbx1a was analyzed by T7 western blot. Expression in the lysates is shown below. (C) Two amounts of each of the indicated GBD–Meis2d fusion proteins were cotransfected into HepG2 cells with the (Gal)5-TATA luciferase reporter, and luciferase activity was assayed after 48 h. The dashed line indicates the maximum activation level achieved by Meis2d. HepG2 cells were transfected with the indicated Meis2d, Pbx1a and HoxB1 expression constructs, together with the Meis–Pbx reporter (D) or Hox ARE reporter (E), and luciferase activity was determined after 48 h. The dashed lines indicate activity with wild-type Meis2d. IP, immunoprecipitation; WB, western blot.

Alternative splicing of Meis3 affects the Meis autoinhibitory domain

Several Meis2 splice variants have been identified that primarily affect the region C-terminal to the HD [39]. However, we were interested in whether alternative splicing of Meis2 or other Meis family members might affect the autoinhibitory function of the Hth domain. Database searching revealed the presence of two isoforms of human Meis3 (termed Meis3.1 and Meis3.2), which were also found in the expressed sequence tag (EST) database. Although only a single mouse Meis3 isoform is listed in GenBank, two forms that are equivalent to human Meis3.1 and Meis3.2 can be found in the mouse EST database. Interestingly, Meis3.1 encodes a protein with the full Hth domain, whereas the Meis3.2 splice variant lacks 17 codons from the 5′-end of exon 6 (Fig. 7A). The region missing in Meis3.2 encodes the equivalent of amino acids 164–180 in Meis2, which form about half of hr2 (see Fig. 6A). To confirm that the two isoforms of Meis3 were indeed expressed, we performed RT-PCR analysis on RNA from HepG2 cells, using primers that span intron 5 and exon 6 of Meis2 or Meis3, and would be expected to generate two products if both isoforms were expressed. As shown in Fig. 7B, we amplified PCR products of the expected size for both Meis3.1 and Meis3.2, whereas only a single longer isoform of Meis2 was detected, suggesting that the alternative splicing event is specific to Meis3. Comparison of the genomic structures of Meis1, Meis2, Meis3 and Prep1 reveals that the three Meis genes, in both mice and humans, have a similar overall structure at least up to exon 6, whereas in Prep1 a single exon encompasses the equivalent of exons 5 and 6 from Meis3. Among the three Meis genes, intron 5 is considerably smaller (< 200 bp) in human and mouse Meis3 than in either of the other genes. Examination of the 5′ and 3′ splice sites surrounding intron 5 provides some clues as to why Meis3 may undergo this alternative splicing event. Position 5 of the 5′ splice site in Meis3 is a guanosine (Fig. 7A), which is characteristic of genes that undergo alternative splicing, whereas, in Meis1 and Meis2, this residue is an adenosine, which correlates with constitutive splicing [43]. Although the 3′ splice site in Meis3 is actually a better match to the consensus than in Meis1 or Meis2, the region upstream of this, within intron 5 of Meis3, is almost completely devoid of adenosines (only three of the first 74 bases, excluding the 3′ splice site, are adenosines). In Meis3, no good match to the branchpoint consensus is present, whereas the Meis1 and Meis2 introns have better branchpoint consensus sequences [44]. Additionally, Meis1 is unlikely to undergo a similar alternative splicing event, as a match to the consensus 3′ splice site is not found at the same internal position within exon 6. To determine how widely the Meis3.2 isoform was expressed, we performed RT-PCR on RNA isolated from several human cell lines and mouse tissues, using PCR primers that span the alternative splice junction in mouse or human Meis3. The relative intensities of the bands corresponding to the Meis3.1 and Meis3.2 splice variants were then quantified. As shown in Fig. 7C, the Meis3.2 variant represented ∼ 25% of the total Meis3 message in most human cell lines tested. In the prostate cancer metastasis-derived cell line LNCaP, the majority of the Meis3 was Meis3.2, suggesting that some variation is possible. Analysis of a panel of mouse tissues, taken from wild-type C57BL/6J mice, revealed that the Meis3.2 variant represented between 20% and 50% of the total (Fig. 7D). Thus, it appears that this alternative splice form of Meis3 represents a significant proportion of the total Meis3 in both mouse tissues and human cell lines, at least at the mRNA level.

Figure 7.

 A Meis3 splice variant disrupts the Hth domain. (A) Meis3.1 and Meis3.2 splice variants are shown schematically. The first few amino acids encoded at each splice junction are shown. The sequences at the splice junctions, together with exon and intron lengths, are shown below for mouse and human Meis1, Meis2, and Meis3. The consensus splice sequences are shown below, with identical bases shaded black. The asterisk indicates the base that correlates with alternative or constitutive splicing. (B) The presence of alternative splicing around the 5′-end of exon 6 of Meis2 and Meis3 was tested by RT-PCR. The positions of molecular mass markers are shown to the left, and the size in base pairs of the products to the right (the Meis2 equivalent of Meis3.2 would be expected at 149 bp). (C, D) RNA from a series of human cell lines (C) or mouse tissues (D) was analyzed by RT-PCR, using primers that span the alternative splice site in Meis3, such that both the Meis3.1 and Meis3.2 isoforms were amplified. The relative amount of each splice form as a percentage of the total Meis3 is plotted in the upper panels. Representative RT-PCR reactions are shown below. (E) The indicated Flag-tagged Meis2 constructs were coexpressed with T7-tagged Pbx1b, or a deletion mutant lacking the HD (amino acids 2–233) in HeLa cells. Protein complexes were isolated on Flag agarose, and analyzed for coprecipitating T7-Pbx1b. Expression in the lysates is shown below. (F) Each of the indicated GBD–Meis2d fusion proteins, or GBD alone, was cotransfected into HepG2 cells with the (Gal)5-TATA luciferase reporter, and luciferase activity was assayed after 48 h. IP, immunoprecipitation; WB, western blot.

To test whether removal of the sequence encoded by the first 17 codons of exon 6 might affect Meis function, we created a version of Meis2d in which amino acids 164–180 were deleted. This generates the Meis2d equivalent of Meis3.2, to allow for comparison with our previous mutational analysis. We first tested the effects of this deletion on Pbx-dependent transcriptional reporters, and observed no increase in activity over that seen with Meis2d (data not shown). To test the possibility that the lack of effect on Pbx-dependent reporters was due to changes in the ability of the deletion mutant to interact with Pbx1, we performed coimmunoprecipitation experiments from transfected HeLa cells. As shown in Fig. 7E, the mutants of Meis2d lacking either amino acids 164–180 or the entire hr2 were both dramatically reduced in their ability to interact with Pbx1. Although there was still some residual interaction of Meis2d lacking amino acids 164–180 with full-length Pbx1, this was lost when we used a deletion mutant of Pbx1 [Pbx1(2–233)] that lacks the HD but not the Meis interaction domains (Fig. 7E). To test the effects on Pbx-independent transcriptional activation, we created a fusion protein comprising GBD and the Meis2d mutant lacking amino acids 164–180. As shown in Fig. 7F, deletion of amino acids 164–180 from GBD–Meis2d resulted in a 3.3-fold increase in transcriptional activity over that seen with wild-type Meis2d. Together, these data suggest that the Meis3.2 splice variant produces a protein that is unable to interact with Pbx1, but is also relieved of the autoinhibitory effect of the Hth domain.

Discussion

We have shown that Meis2d, like Meis1, contains a C-terminal transcriptional AD. The activity of the AD is inhibited by the conserved Hth domain, and this autoinhibitory activity appears to be a general feature of Meis family proteins.

Previous work has identified a transcriptional AD C-terminal to the HD of Meis1a [40]. When assayed as a GBD fusion protein, the C-terminal half of Meis1a (amino acids 232–390, lacking the Hth domain) had robust transcriptional activity, as shown here for Meis2d. However, the activity of the full-length Meis1a was not tested, and on the basis of our work we expect that its activity would be inhibited by the conserved Hth domain. The Meis1a isoform, in which the C-terminal AD was mapped, is equivalent to the Meis2a splice variant, and these two proteins share 74% identity and 80% similarity over their C-terminal domains. Comparison of the Meis2d isoform analyzed here with the public databases reveals a predicted splice variant of Meis1 (Meis1e, gb accession: EAW99896), which shares 75% identity (86% similarity) over the 132 amino acid domain C-terminal to the HD in Meis2d. We therefore suggest that the autoinhibitory function of the Hth domain in Meis2d is likely to be a common feature of Meis family proteins. Switching the AD of Meis2d for that of an unrelated protein still allowed for autoinhibition, suggesting that this function is not dependent on a specific AD, and supporting the notion that it may function for all Meis paralogs. Coexpression of Pbx1a was able to partially relieve the inhibitory effect of the Hth domain on Meis2d, at least in the GBD fusion protein assay. However, this derepression by Pbx1 was not very robust, perhaps suggesting that another factor or other signals are required to fully derepress Meis2d.

The Meis2e splice variant retains the Hth domain, but lacks both the HD and the AD. It could therefore interact with Pbx, but would be unable to bind to DNA or contribute a transcriptional AD, if recruited to DNA. One possibility is that Meis2e represents a naturally occurring dominant negative form of Meis2 that might be able to interfere by competing with other Meis isoforms for binding to Pbx1, for example. Our attempts to test this possibility met with limited success; we observed an interfering effect of Meis2e only when it was expressed at very high levels relative to Meis2d. This may not be surprising when both the Pbx and Meis partners bind DNA, as a Meis2d–Pbx1 complex formed on DNA would probably be more stable than one in which Meis2e is unable to contact DNA. Where Meis2 is recruited without the need for it to bind to DNA, such as via Hox–Pbx complexes, Meis2e might be expected to be better able to interfere. Even with the Hox ARE reporter, we observed relatively little inhibition by even high levels of Meis2e, perhaps suggesting that it is less well incorporated into a DNA-bound Pbx–Hox complex. However, it remains possible that this may represent a normal function for Meis2e and similar Meis isoforms created by alternative splicing.

Although the Hth domain was effective at limiting the activity of a linked AD, Meis2e had relatively little repression activity when targeted to DNA via a heterologous DNA-binding domain. An alternative possibility for the function of Meis2e-like proteins is provided by work on the gene encoding Drosophila HTH, which has been shown to code for a full-length isoform and one lacking the HD [38]. In Drosophila, most HTH functions could be performed by both isoforms, although for antenna development only full-length HTH was sufficient. It may therefore be that Meis2e-like proteins are functional for some activities, but that some processes can only be carried out by full-length Meis paralogs. The lack of a dramatic dominant negative or repressive effect of Meis2e in our assays is consistent with this interpretation, although we show that the Meis2d AD contributes to transcriptional activation by Meis–Pbx–Hox complexes. As Meis2e lacks both a DNA-binding domain and an AD, it is not clear what positive functions such a protein might have. One possibility is that if it is recruited to DNA via interaction with other proteins, it might act to prime specific genes for later activation by Meis2d. However, in the case of the Drosophila HTH variant that lacks the HD, the full-length protein was unable to substitute completely during fly development, suggesting that there may be functions specific to the versions of Meis-related proteins lacking HDs [38].

Recent work has shown an interaction between Prep1 and the repressor p160Mybbp1 mediated by hr1 of Prep1 [37]. However, our data suggest that p160Mybbp1 recruitment is not responsible for the autoinhibitory function of the Meis2 Hth domain. Subcellular localization of Meis2d might also be expected to affect its ability to activate transcription. If the Hth domain was responsible for maintaining cytoplasmic localization of Meis2d, then its deletion might be expected to derepress activity, and the autoinhibition could be relieved by binding to Pbx1, if this allowed for nuclear entry. Although the localization of Prep1 to the nucleus has been shown to be dependent on interaction with Pbx1, a deletion mutant of Prep1 lacking the Hth domain was cytoplasmic in the absence or presence of Pbx1 [45]. Thus the nuclear/cytoplasmic localization of Prep1, and possibly of other Meis paralogs, may play a role in regulating transcriptional activity, but it appears that the Hth domain does not maintain the cytoplasmic localization of Prep1. Additionally, the greatest derepression that we observed was in the context of the GBD fusion proteins, which contain an nuclear localization signal within the GBD part of the protein. Another possible explanation for the observed autoinhibitory activity is that the Hth domain mediates some intramolecular interaction, or affects the conformation of Meis2d. The ability of Pbx1 to somewhat derepress GBD–Meis2d would fit with this model if interaction of Pbx1 with the Hth domain altered the conformation or intramolecular interactions, allowing access to the AD. As the autoinhibition affected an unrelated AD when this was put in place of the native Meis2d AD, it appears that any intramolecular interactions with the Hth domain are likely to be with regions of Meis2 other than its AD.

Among the three Meis and two Prep genes present in humans, alternative splicing appears to affect the Hth region of only Meis3. This alternative splicing event removes 51 nucleotides from exon 6, creating Meis3.2 in humans. The intron–exon structure of Meis1, Meis2 and Meis3 is relatively well conserved in this region of the genes – in both mouse and human, the 17 codons removed in human Meis3.2 are present at the 5′-end of exon 6 of all three genes. In contrast, in the Prep1 gene, the equivalents of exons 5 and 6 in the Meis genes are present in a single exon. Database searching reveals the presence of multiple ESTs from both mouse and human Meis3, which represent the 3.2 isoform, and we show that a similar Meis3 isoform is also present in multiple mouse tissues. Semiquantitative RT-PCR suggests that the Meis3.2 splice variant represents 20–50% of the total Meis3 mRNA expressed in most mouse tissues and human cell lines. It may therefore represent a significant proportion of the functional Meis3 protein. However, further work will be required to determine the relative levels of the proteins encoded by these two splice variants. Some ESTs that probably encode a similar Meis3 isoform are present in pig, cow, and zebrafish. Despite the overall conservation between Meis paralogs, there is no evidence for alternative splicing of Meis1 and Meis2 creating a similar isoform. It has been suggested that Pbx proteins are the major DNA-binding partners for Meis proteins, consistent with the presence of an intact Hth domain in the majority of Meis isoforms [34]. However, we suggest that the Meis3.2 splice variant encodes a Pbx-independent Meis protein, which will bind DNA independently of Pbx, and does not possess the autoinhibitory function of the Hth domain.

In summary, our data suggest that one function of the conserved Hth domain is to inhibit the activity of the transcriptional AD of Meis family proteins. This autoinhibition can be relieved by interaction with Pbx, suggesting that this may provide a mechanism for better control of the transcriptional activity of Meis proteins.

Experimental procedures

Plasmids

Flag-tagged and T7 epitope-tagged expression constructs were generated in a modified pCMV5 by PCR. GBD fusion proteins were created within pM (Clontech, Mountain View, CA, USA). Gal4 luciferase reporters were as previously described [18]. The Pbx–Meis site and Hox ARE reporters were created in pGL2 basic (Promega, Madison, WI, USA). Briefly, a double-stranded oligonucleotide containing the adenovirus major late TATA element was inserted into the BglII and HindIII sites, as previously described [18]. Double-stranded oligonucleotides containing either a consensus Meis2-binding and Pbx1-binding site or the Pbx–Hox-binding site from the Hox B1 ARE were phosphorylated with polynucleotide kinase (NE Biolabs, Ipswich, MA, USA) and ligated into the TATA-luc vector. Oligonucleotide sequences for reporters were as follows (upper strand only): Pbx–Meis, 5′-GATCGTTGATTGACAGA-3′; and Hox ARE, 5′-GATCGGGTGATGGATGGGCC-3′.

Luciferase assays

HepG2 cells were transfected with firefly luciferase reporters and a phCMVRLuc control (Promega), together with appropriate expression constructs, using Exgen 500 (MBI Fermentas, Hanover, MD, USA). After 48 h, promoter activity was assayed with luciferase assay reagent (Biotium, Hatward, CA, USA), using a Berthold LB953 luminometer. Results were standardized using Renilla luciferase activity, assayed with 0.09 μm colenterazine (Biosynth, Naperville, IL, USA).

Immunoprecipitation and Western blotting

COS1 and HeLa cells were maintained in DMEM with 10% bovine growth serum (Hyclone, Logan, UT, USA), and were transfected using LipofectAmine (Invitrogen, Carlsbad, CA, USA). Thirty-six hours after transfection, cells were lysed by sonication in 75 mm NaCl, 50 mm Hepes (pH 7.8), 20% glycerol, 0.1% Tween-20 and 0.5% NP40 with protease and phosphatase inhibitors. Immunocomplexes were precipitated with Flag M2–agarose (Sigma, St Louis, MO, USA). Following SDS/PAGE, proteins were electroblotted to Immobilon-P (Millipore, Billerica, MA, USA) and incubated with antisera specific for Flag tags (Sigma) or T7 epitope tags (EMD Chemicals, Gibbstown, NJ, USA). The GBD antibody was from Cell Signaling.

RT-PCR

RNA was isolated and purified using an Absolutely RNA kit (Agilent, Santa Clara, CA, USA). For quantitative RT-PCR, cDNA was generated using Superscript III (Invitrogen), and analyzed by PCR using a DNA engine cycler and Promega Taq. Intron-spanning primer pairs were selected using primer3 (http://frodo.wi.mit.edu/). Oligonucleotides for RT-PCR were as follows: Meis2-F, 5′-AGGACATCGCGGTCTTCG-3′; Meis2-R, 5′-GAGGTCGATGGGCATTTTC-3′; Meis3-F, 5′-GATGATCCAGCCATCCA-3′; Meis3-R, 5′-GGCTGGGTAGTCCTCGAAGT-3′; mMeis3-F, 5′-GTCCAGGCCATCCAGGTACT-3′; and mMeis3-R, 5′-TCCTCCCTGCAACTACCATC-3′. The relative intensities of the Meis3.1 and Meis3.2 bands were quantified using imagej software, from PCR reactions that had not left the linear range.

Ancillary