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Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Hugh S. Taylor, M.D., Associate Professor, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520. E-mail: hugh.taylor{at}yale.edu.
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Introduction |
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The development of the paramesonephric duct in the embryo is dictated by the abdominal-B group of HOX genes (10, 11, 12, 13, 14, 15). Specifically, HOXA10 is responsible for proper development of the uterus (14, 16, 17). Whereas many HOX genes cease to function after completion of embryonic development, HOXA10 expression continues to be essential for reproduction in the adult female. We previously described the expression of HOXA10 mRNA in the adult endometrium and its importance in endometrial receptivity (14, 18, 19, 20, 21). HOXA10 is up-regulated in response to estrogen and progesterone during the window of implantation in the midsecretory phase of the menstrual cycle (20). In a murine model, repression of HOXA10 by a HOXA10 antisense expression vector leads to a decrease in litter size (19). We previously described several human conditions that result in diminished human embryo implantation in which HOXA10 expression is decreased (22, 23, 24).
HOX genes function as transcription factors and either activate or repress target genes. The pleiotropic effects of HOXA10 on endometrial differentiation and function are likely modulated through the regulation of multiple genes (25). Several target genes of HOXA10 have been characterized in human and primate endometrium; these include EMX2, IGF binding protein-1, and the ß-3 integrin subunit gene (26, 27, 28, 29). EMX2 is down-regulated by HOXA10 (29). ß-3 Integrin and IGF binding protein-1 are up-regulated by HOXA10 (27, 28).
Despite the numerous target genes and developmental functions of HOX proteins, their ability to discriminate between target genes based on DNA sequence alone is very poor (7, 19, 30, 31, 32, 33). Small preferences in target gene specificity exist in that the DNA binding sequence (TNAT) varies slightly from anterior to posterior. The more anteriorly expressed HOX genes tend to recognize TGAT or TAAT in target genes, as opposed to recognition of TTAT or TTAC by posteriorly expressed HOX genes (31, 32, 33, 34, 35, 36, 37). However, discrimination based on these binding sequences preferences is not sufficient to explain the distinct functions of individual HOX proteins. As a result, HOX proteins interact with cofactors that provide target gene specificity.
The PBC (Pbx, ceh-20, extra-denticle) family of TALE (three-amino acid loop extension) homeodomain proteins is a group of cofactors that provide target specificity to HOX transcription factors (38, 39, 40). This family includes Pbx and Meis, which have been shown to directly interact with HOX proteins in several systems. The majority of HOX proteins (paralogous groups 1–13) interact with Pbx (35), but only groups 9 and 10 have been shown to interact with Meis (34). The interaction of HOXA9, HOXB8, and HOXA10 with Pbx1 and Meis1 is essential for immortalization and differentiation of myeloid progenitors (41, 42, 43, 44). These DNA binding complexes may either activate or repress target genes depending on temporal or spatial expression (41, 42, 43, 45). They may have prodifferentiative or proproliferative properties in hematopoietic cells. As a result of the interaction between HOX proteins and PBC cofactors, the targeting of HOX transcription factors to their genes is specific and regulated.
Although the interaction of Pbx and Meis cofactors with HOX transcription factors has been demonstrated in other systems, the expression of these cofactors in the endometrium has not been determined. In the current study, we identify which of the Pbx and Meis family cofactors are expressed in the endometrium. We then determine the temporal and spatial expression pattern of the homeodomain protein HOXA10 and cofactors Pbx2 and Meis1 in the endometrium throughout the menstrual cycle. We also demonstrate the formation of HOXA10/TALE complexes on a HOXA10 binding site in the target gene EMX2.
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Materials and Methods |
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Endometrium samples were collected from 20 normally cycling women with no history of gynecologic disease by Pipelle endometrial biopsy under an approved human investigations committee protocol. Informed consent was obtained from all patients. Samples were representative of all stages of the menstrual cycle and consisted of at least five each from the early, mid-, and late secretory phases. Tissue was fixed in formalin, embedded in paraffin, cut into 5-µm sections, and mounted onto slides. Endometrial dating was confirmed based on the criteria of Noyes et al. (46) No discrepancies between menstrual history and histologic dating were identified.
Immunohistochemistry
Slides were deparaffinized and dehydrated through a serious of xylene and ethanol washes, followed by permeabilization in 95% cold ethanol. After a 5-min rinse in distilled water, an antigen-presenting step was performed by steaming the slides in 0.01 M sodium citrate buffer for 20 min, followed by removal of the staining jar from the steam chamber and cooling for 20 min. Slides were rinsed for 5 min in PBS with 0.1% Tween 20 (PBST), and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 min followed by a 5-min PBST wash. Nonspecific binding was blocked with 1.5% normal horse serum in PBST for 1 h at room temperature (RmT). Slides were incubated in the primary antibody overnight at 4 C. All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): HOXA10 (sc-17159), Meis1 (sc-10599), Meis2 (sc-10600), Pbx1 (sc-889), Pbx2 (sc-890), and Pbx3 (sc-891). Normal goat IgG (Santa Cruz Biotechnology) was used as a negative control for HOXA10 and Meis antibodies, and normal rabbit IgG (Vector Laboratories, Burlingame, CA) as a negative control for all Pbx antibodies. (Fig. 1
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-goat for HOXA10 and Meis and goat -rabbit for Pbx were applied for 1 h at 4 C. Slides were washed in 1x PBST, incubated in ABC Elite (Vector) for 15 min at RmT, washed in 1x PBST, and incubated for 5 min in diaminobenzidine (Vector). A 20-sec exposure to hematoxylin was used as a counterstain. For each individual primary antibody, all slides were processed simultaneously. Slides were rehydrated through 3-min ethanol and xylene washes and mounted with Permount. Cell culture
Ishikawa cells were grown in 150-mm tissue culture dishes with MEM (Sigma, St. Louis, MO) containing 10% fetal bovine serum, 1% antibiotic/antimycotic, 1% sodium pyruvate, and 1.2% sodium bicarbonate. Burkitt-like lymphoma cells were grown in RPMI 1640 with supplements identical with those used for the Ishikawa cells.
Western blot
To obtain whole-cell extracts, cells were rinsed once with RmT 1x PBS. Radioimmunoprecipitation buffer (1x PBS, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin) was added, and culture dishes were rocked for 15 min at 4 C. Cells were then scraped and lysate was incubated on ice for 45 min. The lysate was centrifuged at 10,000 x g for 10 min at 4 C and supernatant was collected (47).
BJAB cell lysate was used as a positive control for Pbx2 and megakaryoblastic cell lysate (Santa Cruz Biotechnology) was used as a positive control for Meis1. Cell lysates were boiled in Laemmli sample buffer for 5 min and loaded onto a 4–15% precast gradient gel (Bio-Rad Laboratories, Hercules, CA). The gel was run at 70 V for 3.5 h. Protein was transferred overnight at 4 C onto a nitrocellulose membrane. The membrane was blocked in 5% milk PBST for 1 h at RmT. Primary antibody was applied (Meis1 at 1:200 and Pbx2 at 1:1000) in 1.5% milk PBST for 2 h at RmT. Peroxidase-conjugated secondary antibody was added in 5% milk PBST for 1 h at RmT. Peroxidase goat -rabbit (Pierce, Rockford, IL) and peroxidase donkey -goat (Santa Cruz Biotechnology) were used at 1:10,000 for Pbx2 and Meis1, respectively. Finally, Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA) was applied for 1 min. The membrane was exposed on X-OMAT film (Kodak, Rochester, NY) and developed using the 3000RA processor (Kodak).
Probe labeling and purification
Single-stranded oligonucleotides and their complementary sequences were synthesized by the Department of Pathology at Yale University. The probes contained the HOXA10 and Pbx binding sites and the flanking sequences found in the EMXC portion of the EMX2 gene (29). The sequences were as follows:
EMXC wild-type 5'-AGGAAGCTGTTTATGTGATCCCCG-3'
EMXC Hox-mut 5'-AGGAAGCTGTGCATGTGATCCCCG-3'
EMXC Pbx-mut 5'-AGGAAGCTGTTTATGCAATCCCCG-3'
EMXC double-mut 5'-AGGAAGCTGTGCATGCAATCCCCG-3'
The complementary probes were annealed and end labeled in the following reaction: 20 pmol DNA, 1x kinase buffer, 10 U/µl T4 polynucleotide kinase (New England Biolabs, Beverly, MA), 50 µCi 32P-ATP, and 2 µl distilled water. The reaction was incubated for 1 h at 37 C followed by the addition of 70 µl of binding buffer [10 mM Tris-HCl (pH 7.5), 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 12% glycerol]. The probes were purified using MicroSpin G-25 columns (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions.
EMSA
Cells were washed in PBS and trypsinized. The cell pellet was washed with PBS and resuspended in 200 µl lysis buffer (25 mM HEPES, 12.5 mM MgCl2, 0.1 mM EDTA, 100 mM KCl, 1 mM dithiothreitol, 10% glycerol, and 1 µg/ml leupeptin and pepstatin). The cells were then centrifuged at 90,000 rpm for 15 min at 4 C. The supernatant was aliquoted and flash frozen. The binding reaction was performed using the binding buffer described above (see Probe preparation). For lanes 2–4 (see Fig. 6), 5 µg protein was incubated with 9 µl binding buffer and 25 ng/µl salmon sperm for 30 min on ice. Then 80,000 cpm probe were added and incubated another 30 min on ice. To supershift the complexes for lanes 5–7 (Fig. 5), 5 µg protein was incubated with 10 µg antibody in 9 µl binding buffer overnight at 4 C, followed by a 30-min incubation on ice with 25 ng/µl salmon sperm, and 80,000 cpm probe were added and incubated on ice for 30 min. Samples were loaded onto a 5% 29:1 acrylamide-bisacrylamide gel and run at 300 V for 3 h at 4 C. The gel was dried (Bio-Rad Laboratories), exposed overnight on X-OMAT film (Kodak), and developed with the Kodak 3000RA processor.
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Results |
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We previously demonstrated an increase in endometrial HOXA10 mRNA levels in both glandular and stromal cells during the midsecretory phase of the menstrual cycle (20). To analyze the expression pattern of HOXA10 protein, immunohistochemical analysis was performed on endometrial biopsies throughout the menstrual cycle. HOXA10 protein was found in both stromal and glandular cells (Fig. 1
, A–D). Expression in the stromal cells was localized to the nucleus and appeared constant at high unvarying levels through all stages of the cycle. Throughout the proliferative phase and until d 16 of the early secretory phase, HOXA10 expression was nearly absent in glandular epithelium (Fig. 1A). Low levels of expression became apparent in the nucleus of most of the glandular cells on d 17 (Fig. 1B). The signal intensity continued to increase in all glandular cells until cycle d 27, suggesting an increase in HOXA10 protein expression (Fig. 1, C and D). The relative intensity of stromal HOXA10 expression was approximately equal to that seen in the glands on d 27.
Meis1 and Pbx2 are expressed in the endometrium
To determine the expression pattern of TALE family cofactors throughout the menstrual cycle, we used immunohistochemical analysis on the same endometrial biopsy specimens as used for the analysis of HOXA10 expression. Meis1 and Pbx2, but not Pbx1, Pbx3, or Meis2, expression was clearly identified in the endometrium. Pbx2 protein was localized to the nucleus of glandular and stromal cells and appeared constant at high unvarying levels through all stages of the cycle (Fig. 2, A–D). The expression of Meis1 differed between the stromal and glandular cells. In the stromal cells, Meis1 was nuclear and relatively high, at apparently unvarying levels (Fig. 3, A–D). Within the glandular cells, Meis1 was also nuclear and expressed in the early proliferative phase (Fig. 3A). Glandular Meis1 expression was minimal or absent through d 17 (Fig. 3B) and undetectable from d 24 through d 27 (Fig. 3, C and D).
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Ishikawa cells are a well-differentiated endometrial epithelial adenocarcinoma cell line that is commonly used as a model of endometrial glandular cells (48, 49, 50). To determine the expression of Meis1 and Pbx2 in Ishikawa cells, Western blot analysis was performed using whole-cell protein extract. The MEG-01 whole-cell extract was used a positive control for Meis1 and BJAB whole-cell extract as a positive control for Pbx2. Results demonstrated the absence of Meis1 expression (Fig. 4A) and the presence of Pbx2 expression (Fig. 4B) in Ishikawa cells. This finding is consistent with results observed in vivo in that Pbx2, but not Meis1, was expressed in secretory glandular cells.
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The EMX2 gene is a known direct target of HOXA10 regulation in the adult endometrium. Sequence analysis revealed a putative Pbx2 binding site 3' of the HOXA10 binding site in the EMXC region of the EMX2 gene (29). To investigate the dimerization of Meis1 and Pbx2 with HOXA10, a probe containing a portion of the EMXC sequence was created and used with Ishikawa whole-cell extract in an EMSA, as demonstrated in Fig. 5. Lane 1 contains radiolabeled probe without protein extract. Lane 2 reveals a HOXA10-Pbx2 heterodimer binding to the wild-type EMX2 probe. A nonspecific band is also observed below the dimer. To confirm the presence of HOXA10 and Pbx2 in the complex, antibodies against each of the two proteins were used to supershift the complexes (lanes 6 and 7 containing the HOXA10 and Pbx2 proteins, respectively). The disappearance of the shift and the appearance of a supershift in lanes 6 and 7 reveal that HOXA10 and Pbx2, respectively, are components of this protein complex. Meis1 did not form a heterodimeric complex with HOXA10 or the HOXA10/Pbx2 complex on this regulatory element (data not shown).
A mutated Pbx binding site in the EMX2 probe prevents HOXA10-Pbx2 binding
To determine the importance of the individual HOXA10 and Pbx2 sites in the ability of the heterodimer to bind, three EMX2 probes were created containing mutations in either or both of the binding sites. The probes were used with Ishikawa whole-cell extract in an EMSA to determine heterodimer binding (Fig. 5). In lane 3, use of the probe with the mutated HOXA10 binding site revealed that the heterodimer was able to bind to the probe despite the absence of the HOXA10 site. In lane 4, whole-cell extract was incubated with the probe with the mutated Pbx binding site. The formation of the heterodimer is decreased or absent, suggesting that the Pbx2 site is necessary for the HOXA10-Pbx2 heterodimer to bind the EMX2 probe. Heterodimer formation is similarly decreased or absent in lane 5 using the EMX2 probe in which both the HOXA10 and Pbx binding sites have been mutated.
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Discussion |
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Although the function of HOXA10 in the uterus has been defined, the molecular mechanisms underlying its actions are poorly understood. It is necessary for HOXA10 to interact with cofactors such as those of the Pbx and Meis families to attain target gene specificity (38, 39, 40, 52, 53). Here we determined the presence of cofactors Pbx2 and Meis1 in the endometrium and their expression pattern throughout the menstrual cycle. We also demonstrate the importance of Pbx2 and the Pbx2 binding site in HOXA10 target genes, which allows high affinity binding of HOXA10 to its target genes.
We previously demonstrated that HOXA10 mRNA is expressed in the adult endometrium from the early proliferative to the early secretory phase and is up-regulated during the mid- to late secretory phase (20). Here we demonstrate the expression pattern of the HOXA10 protein. Immunohistochemical analysis revealed that the HOXA10 protein is constitutively expressed in the stroma throughout the menstrual cycle but is absent in the glandular epithelium until d 17. After this time point, protein expression increases in the glands throughout the remainder of the cycle. The increase in HOXA10 mRNA at the midsecretory phase likely reflects the increase in expression in the glandular epithelium.
We compared the spatial and temporal expression of Pbx2 and Meis1 to that of HOXA10. Meis2, Pbx1, and Pbx3 were not identified in the endometrium and so are unlikely to interact with HOXA10. Pbx2 and Meis1 expression was observed, and each had a specific pattern of expression throughout the cycle. Both were constitutively expressed in the stroma. Meis1 was apparent in early proliferative glandular epithelium and was absent throughout the rest of the cycle. The expression of Pbx2 in the glandular epithelium was similar to its expression in the stroma.
HOXA10, Meis1, and Pbx2 are all expressed in the stroma throughout the menstrual cycle. This coexpression makes it likely that these three proteins may form heterotrimeric complexes that bind HOXA10 target genes in these cells. Because Meis1 and HOXA10 are not cotemporally expressed in the glands, it is unlikely that Meis1 interacts with HOXA10 in this cell type. Pbx2 interacts with HOXA10 in the glands during the secretory phase of the menstrual cycle, and both proteins are expressed at this time point.
To confirm that this potential protein-protein interaction does occur in endometrial cells, we determined their interaction on a known HOXA10 target gene, EMX2. In a previous study, we identified a HOXA10 binding site between base pairs –700 to –550 of the EMX2 gene (29). Here we identified a Pbx binding site, although its location with respect to the HOXA10 binding site is not that which is typically observed for HOX-Pbx dimeric complexes. The common HOX-Pbx consensus site is an octomer of 5'-ATGATTNATNN-3' in which the Pbx half-site (TGAT) is 5' and directly adjacent to the HOX half-site (TNAT) (9, 35). In the –700 to –550 region of the EMX2 gene, the TGAT site is 3' of the TTAT site, and the two are separated by one nucleotide (TTATGTGAT). Here we demonstrated that this sequence is a nonconsensus binding site to which HOXA10-Pbx2 heterodimers bind. Ablation of the Pbx half-site results in a loss of HOXA10-Pbx2 binding to the EMX2 probe, suggesting the importance of this sequence in HOXA10 target gene specificity and the importance of the Pbx2/HOXA10 protein-protein interaction for high-affinity HOXA10 binding to its target genes.
A similar interaction of HOXA10, Pbx1, and Meis1 occurs at the HOXA10 site in the p21 promoter in myeloid differentiation. p21 reporter expression was not enhanced after cotransfection with HOXA10 and Meis1, compared with a significant increase in expression after treatment with Pbx1 and HOXA10. The greatest increase in expression was observed when all three transcription factors were included (41). It is likely that similar regulation occurs with HOXA10, Pbx2, and Meis1 in the endometrium.
The observation that Pbx and Meis cofactors are involved in HOXA10 target gene recognition in the endometrial cells suggests that these proteins may be essential for endometrial receptivity. It is likely that EMX2 is regulated by not only HOXA10 but also a transcription complex involving Pbx2. We have demonstrated that Pbx2 and Meis1 are present in the adult endometrium. The temporal and spatial expression pattern of Meis1 and Pbx2 as well as that of HOXA10 makes it likely that HOX-Pbx2 dimers in the glands and HOXA10-Pbx2-Meis1 trimers in the stroma activate or repress target gene expression. These transcription factor complexes likely regulate endometrial receptivity.
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Footnotes |
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First Published Online October 19, 2004
Abbreviations: HOX, Homeo box; PBST, PBS with 0.1% Tween 20; RmT, room temperature; TALE, three amino-acid loop extension.
Received May 3, 2004.
Accepted October 10, 2004.
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-induced differentiation of myeloid leukemia cell lines. J Biol Chem 275:20117–201261 integrin. J Steroid Biochem Mol Biol 59:31–39[CrossRef][Medline]
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