The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.
We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).
Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.
Homeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited.
To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay.
Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect.
Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.
In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3.
Hoxa3; branchial nerve; neural crest; placode; axon guidance; hindbrain; cell migration
Hox genes are crucial for body axis specification during embryonic development. Hoxa11 has been previously reported to play a role in anterioposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximal-distal patterning of the limbs. Hoxa11 expression has also been observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock-in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in-frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis.
Hoxa11; eGFP; targeted mutation
Transcription factor Cdx4 and transcriptional coregulator menin are essential for Hoxa9 expression and normal hematopoiesis. However, the precise mechanism underlying Hoxa9 regulation is not clear.
Methods and Findings
Here, we show that the expression level of Hoxa9 is correlated with the location of increased trimethylated histone 3 lysine 4 (H3K4M3). The active and repressive histone modifications co-exist along the Hoxa9 regulatory region. We further demonstrate that both Cdx4 and menin bind to the same regulatory region at the Hoxa9 locus in vivo, and co-activate the reporter gene driven by the Hoxa9 cis-elements that contain Cdx4 binding sites. Ablation of menin abrogates Cdx4 access to the chromatin target and significantly reduces both active and repressive histone H3 modifications in the Hoxa9 locus.
These results suggest a functional link among Cdx4, menin and histone modifications in Hoxa9 regulation in hematopoietic cells.
Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.
Homeobox genes encode transcription factors that dictate developmental identity, including that of the Mullerian tract. These genes also direct differential Mullerian transformation of the ovarian cancer cells. The homeobox gene HOXA10 controls uterine organogenesis during embryonic development and similarly is expressed in endometroid epithelial ovarian cancer. Here we confirmed aberrant regulation of HOXA10 expression in epithelial uterine and ovarian carcinomas. We identified a HOXA10 epithelial regulatory element containing an enhancer that drove HOXA10 expression specifically in gynaecologic epithelium. We further identified an adjoining dominant repressor element that restricted regulation by the epithelial enhancer to a subset of epithelial cell types. The repressor contained two functional WT1 binding sites. We identified a strong inverse correlation between HOXA10 expression and that of the Wilms’ Tumour 1 (WT1) gene in multiple benign and malignant gynaecologic tissues, suggesting functionality of the WT1 sites in the repressor. Mutation of the two WT1 binding sites abolished WT1 binding to the element as well as the ability to affect epithelial enhancer activity in reporter assays. Similarly, decreased expression of WT1 using siRNA prevented repressor activity. The Mullerian phenotype seen in ovarian cancer is dependent on gain of HOX gene expression secondary to the loss of WT1-mediated HOX repression. This suggests that Gynaecologic epithelial histologic type is regulated by WT1 expression through its selective repression of HOX genes.
WT1; HOXA10; uterine cancer; ovarian cancer
During limb development, posterior Hox genes of the Hoxa- and Hoxd cluster provide positional information along the limb axis. Here we report a new function for Hoxa11 and Hoxd11 in regulating the early steps of chondrocyte differentiation. We analyzed forelimbs of Hoxa11−/−;d11−/− and Ulnaless mice, which are characterized by specifically shortened zeugopods. By detailed morphological and molecular analyses, we show that loss of Hoxa11 and Hoxd11 in the ulna of both mutants leads to an arrest of chondrocyte differentiation at a step before the separation into round and columnar cells takes place. Furthermore, we demonstrate that Hoxa11 and Hoxd11 act upstream of Runx2 and Shox2, two key regulators of chondrocyte differentiation. We hypothesize that Runx2 activates Shox2 in early chondrocytes, which at later stages induces Runx2 expression to regulate hypertrophic differentiation. These results give insight into mechanisms by which positional information might be translated into a specific bone pattern.
The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-α)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp −210 to −221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-α-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-α-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.
The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints.
Homeobox protein HOXA5 functions as a transcriptional factor for genes that are not only involved in segmentation identity but also in cell differentiation. Although HOXA5 has been shown to regulate the expression of the tumor-suppressor protein p53, its role in breast tumorigenesis is not well understood. Using yeast as a model system, we now demonstrate that overexpression of HOXA5 in yeast can be used to identify downstream target genes that are homologous in humans. One such identified gene was that of the mismatch repair pathway component MutL homolog 1. Analysis of the promoter region of the gene for human MutL homolog 1 (hMLH1) displayed several putative HOXA5-binding sites. In transient transfection experiments, the overexpression of HOXA5 transactivated the hMLH1 promoter-reporter construct. In addition, chromatin immunoprecipitation assay using a human breast cancer cell line MCF-7 demonstrated that HOXA5 binds to the hMLH1 promoter in vivo. Furthermore, we demonstrate that, in the presence of HOXA5, there is an increase in in vivo repair activity in MCF-7 cells. Taken together, our results indicate that HOXA5 is a transcriptional regulator of hMLH1 in breast cancer cells.
Breast cancer; mismatch repair; homeotic gene; yeast; promoter analyses; HOX, homeotic; hMLH1, human MutL homolog 1; ChIP, chromatin immunoprecipitation; MMR, mismatch repair; Luc, luciferase
HOXA9 plays a critical role in both normal hematopoiesis and leukemogenesis, particularly in the development and maintenance of mixed lineage leukemia (MLL)-rearranged leukemia. Through reverse transcription polymerase chain reaction (RT-PCR) analysis of HOXA9 transcripts in human leukemia and normal bone marrow samples, we identified a truncated isoform of HOXA9, namely HOXA9T, and found that both HOXA9T and canonical HOXA9 were highly expressed in leukemia cell lines bearing MLL rearrangements, relative to human normal bone marrow cells or other subtypes of leukemia cells. A frameshift in HOXA9T in exon I causes a premature stop codon upstream of the PBX binding domain and the homeodomain, which leads to the generation of a non-homeodomain-containing protein. Unlike the canonical HOXA9, HOXA9T alone cannot transform normal bone marrow progenitor cells. Moreover, HOXA9T cannot cooperate with MEIS1 to transform cells, despite the presence of a MEIS1-binding domain. Remarkably, although the truncated isoforms of many proteins function as dominant-negative competitors or inhibitors of their full-length counterparts, this is not the case for HOXA9T; instead, HOXA9T synergized with HOXA9 in transforming mouse normal bone marrow progenitor cells through promoting self-renewal and proliferation of the cells. Collectively, our data indicate that both truncated and full-length forms of HOXA9 are highly expressed in human MLL-rearranged leukemia, and the truncated isoform of HOXA9 might also play an oncogenic role by cooperating with canonical HOXA9 in cell transformation and leukemogenesis.
HOXA9; HOXA9T; isoforms; leukemia
The N-terminal region of human HOXA13 has seven discrete polyalanine tracts. Our previous analysis of these tracts in multiple major vertebrate clades suggested that three are mammal-specific. We now report the N-terminal HOXA13 repetitive tract structures in the monotreme Tachyglossus aculeatus (echidna). Contrary to our expectations, echidna HOXA13 possesses a unique set of polyalanine tracts and an unprecedented polyglycine tract. The data support the conclusion that the emergence of expanded polyalanine tracts in proteins occurred very early in the stem lineage that gave rise to mammals, between 162 and 315 MYA.
HOXA13; polyalanine; monotreme; trinucleotide repeat expansion; echidna
While investigating the mechanism of action of the HOXA9 protein, we serendipitously identified Meis1 as a HOXA9 regulatory target. Since HOXA9 and MEIS1 play key developmental roles, are cooperating DNA binding proteins and leukemic oncoproteins, and are important for normal hematopoiesis, the regulation of Meis1 by its partner protein is of interest. Loss of Hoxa9 caused downregulation of the Meis1 mRNA and protein, while forced HOXA9 expression upregulated Meis1. Hoxa9 and Meis1 expression was correlated in hematopoietic progenitors and acute leukemias. Meis1+/− Hoxa9−/− deficient mice, generated to test HOXA9 regulation of endogenous Meis1, were small and had reduced bone marrow Meis1 mRNA and significant defects in fluorescence-activated cell sorting-enumerated monocytes, mature and pre/pro-B cells, and functional B-cell progenitors. These data indicate that HOXA9 modulates Meis1 during normal murine hematopoiesis. Chromatin immunoprecipitation analysis did not reveal direct binding of HOXA9 to Meis1 promoter/enhancer regions. However, Creb1 and Pknox1, whose protein products have previously been reported to induce Meis1, were shown to be direct targets of HOXA9. Loss of Hoxa9 resulted in a decrease in Creb1 and Pknox1 mRNA, and forced expression of CREB1 in Hoxa9−/− bone marrow cells increased Meis1 mRNA almost as well as HOXA9, suggesting that CREB1 may mediate HOXA9 modulation of Meis1 expression.
Loss of Hoxa1 function results in severe defects of the brainstem, inner ear and cranial ganglia in humans and mice as well as cardiovascular abnormalities in humans. Since Hoxa1 is expressed very transiently during an early embryonic stage, it has been difficult to determine whether Hoxa1 plays a direct role in the precursors of the affected organs or if all defects result from indirect effects due to mispatterning of the hindbrain. In this study we use a Hoxa1-IRES-Cre mouse to genetically label the early Hoxa1-expressing cells and determine their contribution to each of the affected organs, allowing us to conclude in which precursor tissue Hoxa1 is expressed. We found Hoxa1 lineage-labeled cells in all tissues expected to be derived from the Hoxa1 domain, such as the facial and abducens nuclei and nerves as well as r4 neural crest cells. Additionally, we detected the lineage in derivatives that were not thought to have expressed Hoxa1 during development. In the brainstem the anterior border of the lineage was found to be in r3, which is more anterior than previously reported. We also observed an interesting pattern of the lineage in the inner ear, namely a strong contribution to the otic epithelium with the exception of sensory patches. Moreover, lineage-labeled cells were detected in the atria and outflow tract of the developing heart. In conclusion, Hoxa1 lineage-tracing uncovered new domains of Hoxa1 expression in rhombomere 3, the otic epithelium and cardiac precursors, suggesting a more direct role for Hoxa1 in development of these tissues than previously believed.
Hoxa1; hindbrain; inner ear; heart
The epigenetic activator Mixed lineage leukemia 1 (Mll1) is paramount for embryonic development and hematopoiesis. Here we demonstrate that the long, non-coding RNA (lncRNA) Mistral (Mira) activates transcription of the homeotic genes Hoxa6 and Hoxa7 in mouse embryonic stem cells (mESC) by recruiting Mll1 to chromatin. The Mira gene is located in the spacer DNA region (SDR) separating Hoxa6 and Hoxa7, transcriptionally silent in mESCs, and activated by retinoic acid. Mira-mediated recruitment of Mll1 to the Mira gene triggers dynamic changes in chromosome conformation, culminating in activation of Hoxa6 and Hoxa7 transcription. Hoxa6 and Hoxa7 activate the expression of genes involved in germ layer specification during mESC differentiation in a cooperative and redundant fashion. Our results connect the lncRNA Mira with the recruitment of Mll1 to target genes and implicate lncRNAs in epigenetic activation of gene expression during vertebrate cell fate determination.
Hoxa13 is expressed early in the caudal mesoderm and endoderm of the developing hindgut. The tissue specific roles of Hoxa13 function have not been described. Hand-Foot-Genital syndrome, a rare dominantly inherited human malformation syndrome characterized by distal extremity and genitourinary anomalies, is caused by mutations in the Hoxa13 gene (HFGa13). We show evidence that one specific HFGa13 mutation likely acts as a dominant negative in vivo. When chick HFGa13 is overexpressed in the chick’s caudal endoderm early in development, caudal structural malformations occur. The phenotype is specific to HFGa13 expression in the posterior endoderm and includes taillessness and severe gut/genitounrinary (GGU) malformations. Finally, we show that chick HFGa13 negatively regulates expression of Hoxd13 and antagonizes functions of both endogenous Hoxa13 and Hoxd13 proteins. We suggest a fundamental role for epithelial specific expression of Hoxa13 in the epithelial-mesenchymal interaction necessary for tail growth and posterior GGU patterning.
Abnormalities; Multiple; etiology; genetics; Animals; Body Patterning; Chick Embryo; Endoderm; physiology; Extremities; embryology; Fibroblast Growth Factor 8; Fibroblast Growth Factors; metabolism; Homeodomain Proteins; genetics; metabolism; Humans; Intestines; embryology; Limb Deformities; Congenital; etiology; genetics; Molecular Sequence Data; Mutation; Sequence Deletion; Syndrome; Tail; abnormalities; embryology; Transcription Factors; Urogenital Abnormalities; etiology; genetics; Urogenital System; embryology; cloaca; tail; colon; hindgut; HOX; HOXA13; chick
Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 54. Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified beta-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33 degrees C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment.
The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx–Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt–β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.
HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using RT-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrantly activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a PI3K inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with TRAIL resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets, and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.
glioblastoma; prognosis; PI3K; HOXA; MGMT
HOX genes, encoding homeodomain transcription factors, are dynamically expressed in endometrium, where they are necessary for endometrial growth, differentiation, and implantation. In human endometrium, the expression of HOXA10 and HOXA11 is driven by sex steroids, with peak expression occurring at time of implantation in response to rising progesterone levels. However, the maximal HOXA10 and HOXA11 expression fails to occur in women with endometriosis. In endometriosis, altered progesterone receptor expression or diminished activity may lead to attenuated or dysregulated progesterone response and decreased expression of progesterone-responsive genes including HOX genes in the eutopic endometrium. In turn, other mediators of endometrial receptivity that are regulated by HOX genes, such as pinopodes, αvβ3 integrin, and IGFBP-1, are downregulated in endometriosis. HOXA10 hypermethylation has recently been demonstrated to silence HOXA10 gene expression and account for decreased HOXA10 in the endometrium of women with endometriosis. Silencing of progesterone target genes by methylation is an epigenetic mechanism that mediates progesterone resistance. The relatively permanent nature of methylation may explain the widespread failure of treatments for endometriosis-related infertility.
HOX genes; implantation; endometrium; endometriosis
Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.
Homeobox genes of the Abdominal B (AbdB) family constitute a distinct subset of vertebrate Hox genes. Analysis of the murine Hoxa-10 gene, one member of this family, revealed several properties specific to this class. Two transcripts of Hoxa-10, a10-1 and a10-2, encode homeodomain proteins of 55 kDa (399 amino acids) and 16 kDa (96 amino acids), respectively. These proteins have identical homeodomains and C-terminal regions encoded by a common 3' exon but differ significantly in the sizes of their N-terminal regions because of the usage of alternative 5' exons. The 5' exon of the a10-2 form is also present in transcripts of Hoxa-9, the next 3' gene, indicating that splicing can occur between adjacent AbdB Hox genes within a cluster. Both Hoxa-10 transcripts demonstrated identical patterns of expression in the posterior body and proximal limb bud, differentiating them from AbdB morphogenetic and regulatory transcripts and suggesting a role with other AbdB Hox genes in the patterning of these structures. Finally, a binding site selection identified the sequence AA(A/T)TTTTATTAC as the Hoxa-10 homeodomain consensus binding site, with a TTAT core sequence. Preferential recognition of a TTAT core therefore differentiates the AbdB class from Antennapedia (Antp) class gene products which bind a TAAT core. Thus, in vertebrates, structural similarities, coordinate transcriptional regulation, sites of expression, and binding site preferences all serve to distinguish AbdB from Antp Hox genes.
During the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.
The second and third amino acid residues of the N-terminal arm of most Hox protein homeodomains are basic (lysine or arginine), whereas they are asparagine and alanine, respectively, in the Hoxa1 homeodomain. Previous reports pinpointed these residues as specificity determinants in the function of Hoxa1 when it is acting as a monomer. However, in vitro data supported that these residues do not influence the target specificity of Hoxa1 in Pbx1a–Hoxa1 heterodimers. Here, we have analysed the transcriptional activity of a Hoxa1(NA-KR) mutant for which the asparagine and alanine residues of the homeodomain have been replaced by lysine and arginine, respectively. Comparison between the wild-type and mutant Hoxa1 reveals that they show distinct activity on the TSEII enhancer of the somatostatin gene, but that they are equally active in the presence of Pbx and Prep cofactors. This therefore corroborates the biochemical evidence having shown that the second and third residues of the homeodomain do not contribute to the DNA binding of Hoxa1–Pbx dimers. However, on the hoxb1 autoregulatory enhancer, Hoxa1 and Hoxa1(NA-KR) may display distinct activity despite the presence of Pbx, in a cell-type dependent manner. Therefore, our data suggest that, depending on the enhancer, these residues may contribute to the functional specificity of Hoxa1 and that this contribution may not be abrogated by the interaction with Pbx.