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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2674-2680
Copyright © 2002 by The Endocrine Society

The Impact of the Human Genome on Endocrinology: Original Articles

HOXA11 Silencing and Endogenous HOXA11 Antisense Ribonucleic Acid in the Uterine Endometrium

Y. Michael Chau, Susan Pando and Hugh S. Taylor

Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Dr. Hugh S. Taylor, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: . hugh.taylor{at}yale.edu

Abstract

Hoxa11 is an essential regulator of embryonic uterine development and the cyclic development of the adult uterine endometrium. Hoxa11 is required for female fertility, as evidenced by targeted mutation. Here we demonstrate a naturally occurring Hoxa11 (mouse)/HOXA11 (human) antisense transcript present in the adult mouse and human endometrium. HOXA11 antisense transcript levels varied during the menstrual cycle, with peak antisense RNA levels occurring in the midproliferative phase, varying inversely with mRNA expression levels. HOXA11 protein levels correlated temporally with peak mRNA levels. In primary stromal cell culture, progesterone down-regulated HOXA11 antisense transcription, and this was followed by up-regulation of HOXA11 mRNA, suggesting a possible role for the antisense transcript in regulating mRNA expression. Attempts to block Hoxa11 function by transfection of the murine uterus with Hoxa11 antisense oligonucleotides failed to interrupt normal uterine function, suggesting that Hoxa11 antisense does not regulate Hoxa11 mRNA by formulation of sense/antisense duplexes. We propose that HOXA11 antisense functions by transcriptional interference, repressing HOXA11 expression by competing for transcription of the common gene, rather than by sense/antisense interaction.

HOX (HUMAN)/HOX (mouse) transcription factors act as regulators of embryonic development (1, 2). The mammalian Hox genes are organized into four clusters. Within each cluster the order of genes on the chromosome parallels the order of expression along the anterior-posterior axis of the developing embryo. Those Hox genes at the 5'-end of the cluster are involved in the development of posterior structures, including the reproductive tract. Hox D and A cluster gene expression has been identified in the developing reproductive tract (3, 4, 5). Differential expression of Hoxa genes along the axis of the undifferentiated paramesonephric duct provides developmental identity resulting in unique adult structures (3, 4). Specifically, Hoxa9 is expressed in the developing oviducts, Hoxa10 in the developing uterus, Hoxa11 in the developing uterus and cervix, and finally Hoxa13 in the primordia of the upper vagina.

Hox gene expression is unusual in the reproductive tract in that it persists in the adult, probably providing developmental plasticity to the adult reproductive tract (3, 6, 7). This plasticity allows the reproductive tract to undergo the significant developmental remodeling involved in the establishment and maintenance of pregnancy. Two Hox genes are known to be necessary for the establishment of pregnancy. Targeted mutation of either the Hoxa10 or the Hoxa11 gene in mice results in sexually dimorphic female infertility (8, 9, 10). The defect results from uterine failure to support the preimplantation embryo or allow implantation. Although subtle developmental defects exist in the uteri of these mice, the sterility is a result of absent adult maternal Hox expression; blocking adult uterine Hoxa10 expression in a wild-type mouse with normal embryonic Hoxa10 expression results in a decrease in the number of embryos that implant (11). Human uterine endometrium similarly expresses HOXA10 and HOXA11 in a menstrual cycle-dependent fashion (6, 7). HOXA10 and HOXA11 expression dramatically increase in the midsecretory phase, the time of blastocyst implantation (12). Women with defective implantation have been demonstrated to have altered levels of HOXA10 and HOXA11 expression, further demonstrating the role of these genes in human pregnancy (13).

Hoxa10 expression can be blocked with synthetic antisense oligonucleotides. Hoxa10 mRNA levels are not significantly changed after antisense treatment, whereas protein levels are decreased, and Hoxa10-mediated reproductive function is severely compromised (11). This suggests that Hoxa10 antisense interferes with mRNA function at the level of RNA processing, transport, or translation, rather than transcription. Due to targeting to the translation start site and the apparently unaltered transcript size, disruption of translation most likely accounts for the altered protein levels.

Although there is no naturally occurring antisense Hoxa10 transcript, Hoxa1l antisense is transcribed, resulting in an abundant, naturally occurring antisense RNA in the mouse embryo. The prevalence of eukaryotic genes that transcribe both sense and antisense RNA has only recently been recognized (14, 15, 16, 17, 18). Few have been ascribed a well defined regulatory role. In the developing mouse embryo, there is a distinct temporal complementary pattern of Hoxa11 sense and antisense transcription (9); the increasing antisense RNA abundance in regions of decreasing sense RNA is consistent with an antisense role in regulating Hoxa11 expression.

In this report we identify HOXA11 antisense RNA in the adult mouse and human uterus. The antisense abundance varies through the menstrual cycle and is regulated by progesterone. Synthetic Hoxa11 antisense oligos did not block Hoxa11 expression or function in mice. Progesterone-mediated down-regulation of Hoxa11 antisense transcription results in increased Hoxa11 sense transcription. These data suggest transcriptional interference as a mechanism of regulating Hoxa11 expression in both the embryo and the adult uterus.

Materials and Methods

Tissue collection

Endometrial tissue was collected by Pipelle biopsy from 30 normal fertile women at the time of tubal ligation under and approved human investigation committee protocol. Tissue was either immediately frozen in liquid nitrogen for Northern analysis or was fixed in 4% paraformaldehyde, cryoprotected in sucrose, and frozen for in situ hybridization. A portion of tissue was sent for histologic analysis and menstrual cycle dating as determined by the criteria reported by Noyes et al. (19). CD-1 mouse uteri were collected in proestrus immediately after cervical dislocation and were frozen in liquid nitrogen for Northern analysis.

Immunohistochemistry

Cellular expression of HOXA11 was evaluated by immunohistochemistry using a mouse polyclonal antibody (Berkley Antibody Co., Richmond, CA). Four random biopsies were obtained from each uterus. The specimens were embedded in paraffin and sectioned into serial 5-µm sections. The sections were deparaffinized in xylene and ethanol. Blocking of endogenous peroxidase was performed by incubation with 0.6% H2O2. The sections were incubated with 1.5% normal goat blocking serum for 45 min and then incubated overnight at 4 C with the primary antibody. In control sections, the primary antibody was substituted with an equimolar concentration of mouse IgG (Vector Laboratories, Inc., Burlingame, CA).

The slides were then incubated with biotinylated mouse secondary IgM antiserum for 30 min at room temperature, followed by a 45-min incubation with avidin and biotinylated peroxidase (Vectastain, Vector Laboratories, Inc.). The slides were then incubated in diaminobenzidine (400 µg/ml) for 5 min. Counterstaining was performed with hematoxylin and Li2CO3.

RNA isolation and Northern analysis

Total RNA was isolated from human endometrium or whole mouse uterine by separately homogenizing the tissue in 1 ml TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD)/100 mg or tissue wet wt. RNA was extracted with chloroform and precipitated with isopropyl alcohol according to the manufacturer’s recommendations. Total RNA (20 µg/lane) was electrophoresed through 1.0% agarose/0.66 M formaldehyde gels and transferred to nylon membrane. Hybridization was performed overnight at 60 C in 50% formaldehyde, 1x SSC, 5x Denhardt’s reagent, 0.2% tRNA, and 32P-labeled riboprobe at 2 x 106 cpm/ml. The membrane was washed twice at 68 C for 30 min each time in 1x SSC and 0.1% SDS and exposed to Kodak-XAR film at -80 C (Eastman Kodak Co., Rochester, NY).

Statistical analysis

Densitometry was performed on nonsaturated autoradiograms using laser densitometry (Molecular Dynamics, Inc., Sunnyvale, CA). ANOVA was used to distinguish between phases of the menstrual cycle. A t test was used to determine differences in implantation rates between control and experimentally treated mice and to compare RNA expression between steroid-treated cells and controls. Statistical significance was defined as P value less than 0.05.

In situ hybridization

Ten-micron sections of endometrial tissue were cut at -20 C and stored at -80 C on Vectabond-coated slides. 33P-Labeled probes complimentary to either the HOXA11 sense or antisense transcripts, as described below, were transcribed from linearized templates using in vitro transcription (Promega Corp., Madison, WI). Frozen sections were allowed to come to room temperature and then placed in 4% paraformaldehyde in PBS, followed by 2x SSC. Acetylation was performed in 0.026 M acetic anhydride in 0.1% triethanolamine (pH 8.0), followed by a 2x SSC wash. Dehydration was performed in serially increasing ethanol baths, and sections were dried at room temperature. Hybridization was performed by adding 100 µl probe diluted in 1x Denhardt’s solution, 50% formamide, 0.1 M NaCl, 10 mM Tris-HCl, pH 8.0), 1 mM EDTA, 50 mM dithiothreitol, 500 mg/ml yeast tRNA, and 10% dextran sulfate. Hybridization was performed using 3 x 106 cpm/slide at 50 C for 16 h. After hybridization, sections were washed twice in 2x SSC at 55 C, followed by treatment with 50 mg/ml ribonuclease A (Sigma, St. Louis, MO) for 1 h at 37 C. Slides were washed overnight at 50 C in 2x SSC containing 0.1% ß-mercaptoethanol.

After washing, the sections were dehydrated in serially increasing concentrations of ethanol, air-dried, then dipped in Kodak NTB-2 emulsion (Eastman Kodak Co.) and exposed for 2 wk at 4 C. The slides were developed with Kodak D10 developer, counterstained with hematoxylin and eosin. For each probe five sections were hybridized with the antisense probe, and two sections were hybridized with the sense probe.

Primary stromal cell culture

Endometrial samples were obtained from four different normal cycling women in the proliferative phase. Endometrial epithelium and stromal cells were separated as described previously. Briefly, the tissue was finely minced, and cells were dispersed by incubation in the HBSS containing HEPES (25 mM), penicillin (200 U/ml), streptomycin (200 µg/ml), collagenase (1 mg/ml, 15 U/mg), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg) for 20–30 min at 37 C with agitation. The cells were separated by filtration through a wire sieve with 73-µm diameter pores. The stromal cells were found in the filtrate, whereas the endometrial glands were retained by the sieve. The stromal cells were pelleted, washed, and suspended in phenol red-free Ham’s F-12/DMEM (1:1) containing antibiotics and 10% charcoal-stripped FCS. The cells were passaged once and grown to confluence. Confluent monolayers were maintained in phenol red-free, serum-free medium for 48 h and subsequently treated with 17ß-estradiol (5 x 10-8 M) or medroxyprogesterone acetate (10-7 M) for 4 h. Immunocytochemical analysis of endometrial cells was conducted after the first passage. Factor VIII (20), cytokeratin (21), 3C10 (22, 23), and vimentin (24, 25) were used as markers of endothelial cells, epithelial cells, macrophages, and stromal cells. Epithelial cells and macrophages accounted for approximately 3% and 0.2% of the cells, respectively; endothelial cells were absent.

Probes

The HOXA11 probe consists of 103 bp of the 3'-untranslated region of the HOXA11 gene. This probe has been well characterized by ourselves and others. The Hoxa11 antisense probe was a gift from S. S. Potter (9). Plasmids containing each probe were linearized and used as a template for the generation of riboprobes. 32P-Labeled HOXA11 sense, HOXA11 antisense, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes were generated by in vitro transcription.

Western analysis

Mice uteri or human endometrial biopsies were lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM NaSO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride) and centrifuged at 12,000 x g for 2 min at 4 C, and the supernatant was collected. The amount of protein was quantified by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA). Thirty-microgram aliquots were loaded on to a 6% SDS-polyacrylamide gel, size fractionated, and transferred to a nitrocellulose membrane using a Transblot apparatus (Bio-Rad Laboratories, Inc.) at 100 V for 2 h at 4 C. The membrane was immersed in a 3% gelatin-Tris-buffered saline (TBS; 20 mM Tris and 500 mM NaCl) blocking solution for 30 min at room temperature, washed for 10 min in TBS (20 mM Tris 500 mM NaCl, and 0.05% Tween 20, pH 7.5), and then incubated for 1 h with a 1:1000 dilution of HOXA11 polyclonal antibody (BabCo). The membrane was washed with TBS for 5 min at room temperature and incubated for 1 h with a 1:200 dilution of goat antimouse IgG-horseradish peroxidase (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was then washed twice in TBS for 5 min each time at room temperature and immersed in a horseradish peroxidase color developer buffer (Bio-Rad Laboratories, Inc.) for 30 min. Photographs were taken immediately after color development.

Gene transfection

Nulliparous reproductive age female and male CD1 mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Female mice were mated and examined every 8 h until detection of a vaginal plug. The presence of a vaginal plug was designated d 1 of pregnancy. The mice were anesthetized 30–60 h after plug detection, with 250 µl of a 5% xylazine/10% ketamine mixture given by ip injection. In accordance with our previously reported protocol, laparotomy was performed to expose the uterus, and 25 µl of the DNA/liposome complex were injected into the base of each uterine horn using a 27-gauge needle (11). The incision was closed in two layers (peritoneal and cutaneous) with 4-0 vicryl suture. Ten individual antisense oligonucleotides were used complementary to the start of Hoxa11 translation, a strategy we have previously used to block HOXA10 (11). Each was a 30-bp phosphothiorate-modified deoxyribonucleotide. Controls consisted of a 30-bp phosphothiorate-modified deoxyribonucleotide with the same nucleotide composition as the experimental oligo, but a random sequence order. These experiments were conducted in accordance with an approved protocol issued by the Yale animal care and use committee.

Results

Hoxa11/HOXA11 antisense RNA is expressed in adult mouse and human uterus

To determine whether HOXA11 antisense RNA is present in mouse and human uterus, endometrial tissue was obtained from each, and RNA was extracted. Northern analysis using a 32P-labeled HOXA11 sense riboprobe identified abundant HOXA11 antisense RNA expression in both the mouse and human uterus (Fig. 1Go). To localize the transcript within the human endometrium, in situ hybridization was performed with 33P-labeled HOXA11 sense and antisense riboprobes. Figure 2Go demonstrates abundant antisense RNA and sense RNA in the same tissue and cell types. High levels of expression of both sense and antisense transcript were seen in the uterine stroma, with diminished levels in the epithelium. Myometrial expression was present at low levels (data not shown). Similar spatial and cellular localization of Hoxa11 sense and antisense transcripts was observed in the mouse uterus (data not shown).



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  		Figure 1. HOXA11 antisense expression in mouse uterine and human endometria. Northern analysis demonstrates mouse uterine and human endometrial RNA hybridized to a probe specific to Hoxa11/HOXA11 antisense transcript. Abundant antisense RNA is present in both species. Hybridization to a probe specific for G3PDH is shown as a control. M, Mouse uterine RNA; H, human endometrial RNA.

 


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  		Figure 2. In situ hybridization demonstrating HOXA11 and HOXA11 antisense transcripts in human endometrium. In situ hybridization demonstrates HOXA11 mRNA in human endometrial glands and stroma (top panel). A control specimen, hybridized to a nonsense oligonucleotide revealed minimal signal (center panel). HOXA11 antisense transcript was abundantly expressed in both glands and stroma (lower panel). Both sense and antisense transcripts were more abundant in the stroma and had similar cellular distributions.

 
HOXA11 antisense shows a distinct temporal expression pattern complementary to that of HOXA11 sense

We have previously shown that during the human menstrual cycle, the level of HOXA11 sense transcript rises in the midsecretory phase, corresponding to the time of endometrial receptivity to blastocyst implantation (7). To determine the temporal pattern of HOXA11 antisense abundance, endometrium was collected from 30 cycling women throughout the menstrual cycle. RNA was extracted, processed for Northern analysis, and hybridized with a 32P-labeled HOXA11 sense riboprobe to detect the HOXA11 antisense transcript. Results were normalized G3PDH. As demonstrated in Fig. 3Go, the level of HOXA11 antisense RNA abundance was elevated during the proliferative phase of the menstrual cycle. Levels dropped significantly in the luteal phase at the time of a significant rise in HOXA11 sense transcription. Figure 3BGo demonstrates the menstrual cycle-specific pattern of expression. HOXA11 antisense RNA reached peak abundance in the late proliferative phase.



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  		Figure 3. HOXA11 antisense transcript abundance varies during the menstrual cycle. A, Endometrial RNA was extracted separately from the proliferative and secretory phases of the menstrual cycle. Northern analysis revealed a decrease in HOXA11 antisense abundance in the secretory phase compared with the proliferative phase. Sense transcript abundance increased in the secretory phase. G3PDH is shown as a control. B, Five samples of endometrium were obtained from each of the early, mid, and late proliferative phases (EP, MP, and LP, respectively) and from the early, mid, and late luteal phases of the cycle (EL, ML, and LL, respectively), totaling 30 specimens. Northern analysis and densitometry was performed, and each was normalized to G3PDH expression for the same sample. The relative abundance of the HOXA11 antisense transcript is shown. HOXA11 antisense abundance increased through the proliferative phase and decreased in the secretory phase (P < 0.01, proliferative vs. secretory phase).

 
HOXA11 protein expression corresponds to sense mRNA abundance

Previous reports have described HOXA11 mRNA levels only. To determine whether HOXA11 protein abundance corresponded to mRNA abundance, immunohistochemistry was performed on sections of human uterine tissue, and Western analysis was performed on tissue homogenates. Figure 4Go demonstrates HOXA11 protein expression in human endometrium. Western analysis demonstrated high levels of protein expression in the secretory phase, corresponding to the time of maximum mRNA abundance and HOXA11 functional importance (Fig. 4AGo). Diminished levels were noted in proliferative phase endometrium. Immunohistochemistry showed abundant HOXA11 expression in luteal phase endometrium. HOXA11 protein expression was noted in epithelial and stromal cells (Fig. 4BGo).



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  		Figure 4. HOXA11 protein expression increased in the secretory phase. A, Western analysis revealed that endometrial HOXA11 protein expression correlated with increased mRNA expression in the secretory phase. Lanes A and B contain proteins from proliferative and secretory endometrium, respectively. B, Immunohistochemistry revealed HOXA11 protein expression in the endometrial glands and stroma. The inset shows the corresponding control.

 
Progesterone regulates HOXA11 antisense RNA abundance

We have previously demonstrated roles for sex steroids in the regulation of HOX gene expression (4, 6, 7). Here we investigated the role of progesterone in the regulation of HOXA11 antisense transcription. Primary human uterine stromal cell cultures were treated with progesterone at concentrations up to the maximum physiological range (10-6 M). A dose-responsive decrease in HOXA11 antisense RNA abundance was seen with progesterone treatment (Fig. 5Go). This corresponds to the decrease in HOXA11 antisense abundance observed in the secretory phase, when progesterone is the predominant hormonal influence on the endometrium. Treatment with 17ß-estradiol did not result in significantly reduced HOXA11 antisense transcription at physiological concentrations. Supraphysiological 17ß-estradiol did reduce HOXA11 antisense. Each experiment was repeated in triplicate.



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  		Figure 5. Sex steroid regulation of HOXA11 antisense RNA. Primary stromal cell cultures were treated with increasing concentrations or progesterone or estradiol. HOXA11 antisense RNA abundance was measured by Northern analysis 16 h after treatment. Each data point is the average of three independent experiments. A, Progesterone treatment produced a dose-responsive decrease in HOXA11 antisense transcript. The maximal effect occurred over the physiological range of progesterone concentrations. All points were statistically different from controls (P < 0.05). B, 17ß-estradiol repressed HOXA11 antisense transcript only at supraphysiological concentrations. HOXA11 antisense transcript levels were statistically different from controls at an E2 concentration of 10-7.5 M or more.

 
Synthetic Hoxa11 antisense oligonucleotide failed to block Hoxa11 protein expression or function

Taken together, the data presented above suggested a role for HOXA11 antisense in down-regulating HOXA11 sense transcription in the proliferative phase of the menstrual cycle. Previously we have demonstrated the ability to block Hoxa10 expression and function with a synthetic Hoxa10 antisense oligonucleotide (11). Transfection of Hoxa10 antisense oligonucleotide resulted in decreased Hoxa10 protein, but not mRNA, suggesting a role for antisense in directly binding sense mRNA and blocking translation, mRNA transport, or inducing a subtle defect in splicing. To determine whether naturally occurring Hoxa11 antisense has a similar role, Hoxa11 antisense oligonucleotides were tested for the ability to decrease Hoxa11 protein expression or Hoxa11 function in the mouse. Following our previous protocol, Hoxa11 antisense oligonucleotides were transfected into the uterus of the female mouse on d 2 after vaginal plug detection (11). This corresponds to the time of decreased naturally occurring Hoxa11 antisense RNA abundance. Western analysis of Hoxa11 expression in mice treated with Hoxa11 antisense oligonucleotide demonstrated that Hoxa11 protein expression was not diminished by Hoxa11 antisense RNA (data not shown). As predicted by the lack of effect on protein expression, Hoxa11 antisense had no effect on Hoxa11 function. Mice transfected with Hoxa11 antisense oligonucleotide (n = 42) had litter sizes similar to those of mice transfected with a missense oligonucleotide of the same length and nucleotide composition (n = 19; Fig. 6Go). In contrast to the effect of Hoxa10 antisense on Hoxa10 translation and function, Hoxa11 antisense appeared to have no such direct affect on Hoxa11 translation or function.



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  		Figure 6. Transfection with HOXA11 antisense oligonucleotide does not block implantation. Transfection of the mouse uterus 2 d after vaginal plug detection with a synthetic 30-mer phosphothiorate-modified deoxyoligonucleotide complimentary to the HOXA11 translation start site resulted in an average implantation rate of 11.4 pups/litter on d 9 of pregnancy. Transfection with a control nucleotide of the same length and composition, but in random order, resulted in an average of 9.9 pups/liter. HOXA11 antisense did not block implantation. n = 42 antisense-treated mice and 19 controls. P = 0.45 (not significant), by t test.

 
Increased transcription of Hoxa11 antisense decreased Hoxa11 sense RNA abundance

To determine whether competition for transcription of a common gene regulates the relative abundance of Hoxa11 sense and antisense RNA, Hoxa11 antisense transcription was inhibited with progesterone in human stromal cell culture. Progesterone treatment led to decreased Hoxa11 antisense transcription and increased Hoxa11 sense mRNA abundance. Figure 7Go demonstrates an initial decrease in Hoxa11 antisense abundance, followed by an increase in sense mRNA abundance. This temporal relationship suggests that the process of Hoxa11 antisense transcription may interfere with Hoxa11 sense transcription from the same gene.



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  		Figure 7. Temporal relationship of HOXA11 transcript in response to progesterone. At time zero, human endometrial stromal cells were treated with 10-6 M progesterone. HOXA11 antisense transcript abundance rapidly decreased. This was followed by an increase in HOXA11 sense transcript. Increased HOXA11 mRNA was transcribed only after antisense inhibition was released. Each value is the mean of three independent experiments. Sense expression was different from antisense expression at all time points except for 4 h (P < 0.05).

 
Discussion

Abundant HOXA11 antisense RNA in the uterine endometrium and its regulation by progesterone in the menstrual cycle

Hoxa11 is essential for normal development of the embryonic mouse and is necessary for uterine receptivity to implantation (9). In the human, HOXA11 expression rises in the midsecretory phase, the time of implantation, suggesting a role similar to that described in mice (7). The aberrant expression noted in some disorders of the endometrium results in decreased implantation, consistent with a role for maternal HOXA11 expression in human fertility (13). HOXA11 levels are low in the proliferative phase of the menstrual cycle, when the endometrium is unreceptive to implantation of the blastocyst. The mechanism by which HOXA11 expression is repressed in the proliferative phase was previously unknown. Here we report the presence of HOXA11 antisense RNA in the human uterus. Naturally occurring antisense RNA transcripts have been established as playing a role in gene regulation (14, 15, 16, 17, 18). The observation of HOXA11 mRNA and antisense RNA in the same cell type is consistent with a role for HOXA11 antisense in regulating HOXA11 function. HOXA11 antisense is abundantly expressed in the proliferative phase of the menstrual cycle, when HOXA11 mRNA transcripts are expressed at low levels. Repression of HOXA11 antisense transcription in the secretory phase correlates temporally with increased HOXA11 mRNA. This secretory phase expression is probably mediated by progesterone, which down-regulates HOXA11 antisense RNA. The spatial and temporal expression patterns of HOXA11 antisense RNA in vivo suggest a role in regulating HOXA11 expression and in endometrial receptivity. Progesterone decreases HOXA11 antisense transcription, which probably allows up-regulation of HOXA11 mRNA.

Interestingly the same reciprocal pattern between HOXA11 antisense and HOXA11 mRNA is observed in the developing mouse limb (9). It is unclear what regulates the expression of HOXA11 antisense in the murine embryo, but it is likely under differential transcriptional regulation. Similarly, Hoxd3, a homeobox gene involved in specification of the first cervical vertebrate, has an antisense transcript (26). This transcript shows spatial-temporal regulation different from that of Hoxd3 mRNA. Differential regulation of sense and antisense transcripts may be a common mechanism to regulate Hox gene expression. Here we demonstrate that this mechanism of regulation is also present in the adult uterus and probably plays a role in the regulation of endometrial receptivity. The sex steroid progesterone is a novel regulator of antisense transcription, decreasing HOXA11 antisense abundance in the secretory phase.

Mechanism of Hoxa11 antisense action

Hoxa11 antisense RNAs were originally found in a murine embryonic cDNA library screen using probe from the 5'-region of the sense Hoxa11 cDNA sequence (9). These antisense cDNA are polyadenylated and alternately processed. The sense and antisense cDNAs showed considerable sequence overlap of more than 500 bp. Complementary distributions of sense and antisense RNA transcripts are seen in the developing murine limb, suggesting a possible regulatory function. To distinguish between spurious and functional transcripts, this group further characterized the evolutionary conservation of the antisense transcripts (27). A 99% nucleotide sequence identity between mouse and human was observed in the approximately 500-bp region of overlap between the mouse sense and antisense transcripts. This conservation is strongly suggestive of a functional significance for the Hoxa11 antisense transcript.

Synthetic antisense oligonucleotides are routinely used to inhibit gene expression (27, 28, 29). The mechanisms by which they exert their effects are complex and are not completely characterized. Mechanisms by which oligonucleotides affect protein expression include effects on transcription, nuclear processing, nuclear transport, mRNA stability, and translation. It has recently become apparent that naturally occurring antisense molecules exist in eukaryotes (14, 15, 16, 17, 18, 31, 32, 33, 34, 35, 36). These molecules may regulate gene expression of their complementary mRNAs by directly annealing to them (37, 38, 39). An alternative mechanism of action, possible for naturally occurring antisense RNAs, but not synthetic oligonucleotides, is the regulation of gene expression by competition for transcription of the same gene (40, 41, 42, 43). An antisense molecule transcribed at a high rate will interfere with the ability of the sense strand to be transcribed, a mechanism termed transcriptional interference.

HOXA11 antisense is abundantly transcribed in the proliferative phase, whereas HOXA11 mRNA abundance is relatively low. Synthetic Hoxa11 antisense oligonucleotides are unable to block Hoxa11 protein expression or Hoxa11 function, as was previously demonstrated for Hoxa10. Hoxa10 does not have a known naturally occurring antisense transcript. These data suggest that Hoxa11 antisense does not function by binding Hoxa11 sense, but by another mechanism. The repression of Hoxa11 antisense transcription by progesterone is followed by an increase in Hoxa11 sense mRNA transcription, suggesting that there is competition for the transcription of these two transcripts. Taken together, these data suggest that the mechanism by which Hoxa11 antisense represses Hoxa11 mRNA is by transcriptional interference.

Acknowledgments

Footnotes

This work was supported by NIH Grant HD36887 (to H.S.T.).

Abbreviations: G3PDH, Glyceraldehyde-3-phosphate dehydrogenase; TBS, Tris-buffered saline.

Received October 31, 2001.

Accepted February 11, 2002.

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