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1.  Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms 
BMC Physiology  2011;11:9.
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.
PMCID: PMC3125234  PMID: 21627813
2.  The Truncated Hoxa1 Protein Interacts with Hoxa1 and Pbx1 in Stem Cells 
Journal of cellular biochemistry  2009;106(3):427-443.
Hox genes contain a homeobox encoding a 60-amino acid DNA binding sequence. The Hoxa-1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1-993), and another protein lacking this domain (Hoxa1-399). The functions of Hoxa1-399 are unknown. We detected Hoxa1-993 and Hoxa1-399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1-399 functions in cellular differentiation we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1-993 and its cofactor, Pbx1, bind to the Hoxb1 SOct-R3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1-399 (the F9-399 line) show that Hoxa1-399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1-399 reduces Hoxa1-993/Pbx1 binding to the Hoxb1 SOct-R3 region. GST-pull down experiments suggest that Hoxa1-399, Hoxa1-993, and Pbx1 form a trimer. However, the F9-399 line exhibits no differences in RA-induced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wild type.
PMCID: PMC3923656  PMID: 19115252
Homeobox; Transcription Factor; Splice Variant; cell differentiation; F9 cells; retinoids; Hoxb1; Pbx; teratocarcinoma; stem cell; vitamin A
3.  HOXA5-Induced Apoptosis in Breast Cancer Cells Is Mediated by Caspases 2 and 8 
Molecular and Cellular Biology  2004;24(2):924-935.
HOXA5 is a transcriptional factor whose expression is lost in more than 60% of breast carcinomas. Our previous work demonstrated that the overexpression of HOXA5 in MCF7 cells resulted in cell death through a p53-dependent apoptotic pathway. To determine whether p53-independent apoptotic pathways are involved in HOXA5-induced cell death, we engineered a p53-mutant breast cancer cell line, Hs578T, to inducibly express HOXA5. Induction of HOXA5 expression led to cell death with features typical of apoptosis within 24 h, and the expression levels of mutant p53 and its target genes either decreased or remained unchanged. To decipher apoptotic pathways, the HOXA5-expressing cells were treated with a variety of apoptotic inhibitors. Besides a general caspase inhibitor, caspase 2- and 8-specific inhibitors largely abolished HOXA5-induced apoptosis, whereas caspase 1-, 3-, 6-, and 9-specific inhibitors had no significant effects. Western blot analysis further confirmed that caspases 2 and 8 were activated after the induction of HOXA5 expression. Further, several small interfering RNAs which specifically silenced caspase 2 and caspase 8 expression significantly blocked HOXA5-induced apoptosis. HOXA5 expression could also sensitize cells to tumor necrosis factor alpha-induced apoptosis by at least 100-fold. These results indicate that expression of HOXA5 can induce apoptosis through an apoptotic mechanism mediated by caspases 2 and 8.
PMCID: PMC343791  PMID: 14701762
4.  Two isoforms of HOXA9 function differently but work synergistically in human MLL-rearranged leukemia 
Blood cells, molecules & diseases  2012;49(2):102-106.
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.
PMCID: PMC3399022  PMID: 22633751
HOXA9; HOXA9T; isoforms; leukemia
5.  Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells 
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.
PMCID: PMC2904782  PMID: 20540809
6.  HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes 
PLoS ONE  2009;4(9):e6845.
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.
PMCID: PMC2731163  PMID: 19727442
7.  Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis 
Oncogenesis  2014;3(12):e135-.
Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.
PMCID: PMC4275563  PMID: 25531430
8.  Cardiovascular defects in a mouse model of HOXA1 syndrome 
Human Molecular Genetics  2011;21(1):26-31.
Congenital heart disease is one of the most common human birth defects, yet many genes and pathways regulating heart development remain unknown. A recent study in humans revealed that mutations in a single Hox gene, HOXA1 (Athabascan Brainstem Dysgenesis Syndrome, Bosley-Salih-Alorainy Syndrome), can cause severe cardiovascular malformations, some of which are lethal without surgical intervention. Since the discovery of the human syndromes, there have been no reports of any Hox mouse mutants with cardiac defects, hampering studies to explore the developmental causes of the human disease. In this study, we identify severe cardiovascular malformations in a Hox mouse model, which mimic the congenital heart defects in HOXA1 syndrome patients. Hoxa1 null mice show defects such as interrupted aortic arch, aberrant subclavian artery and Tetralogy of Fallot, demonstrating that Hoxa1 is required for patterning of the great arteries and outflow tract of the heart. We show that during early embryogenesis, Hoxa1 is expressed in precursors of cardiac neural crest cells (NCCs), which populate the heart. We further demonstrate that Hoxa1 acts upstream of several genes, important for neural crest specification. Thus, our data allow us to suggest a model in which Hoxa1 regulates heart development through its influence on cardiac NCCs, providing insight into the mechanisms underlying the human disease.
PMCID: PMC3235008  PMID: 21940751
9.  Endothelin Type A Receptor (ETA) Expression Is Regulated by HOXA10 in Human Endometrial Stromal Cells 
Endothelin type A receptor (ETA) is a member of the superfamily of G protein-coupled receptors. Our laboratory conducted a microarray screen that identified ETA as target of HOXA10 transcriptional control in endometrium. Here, we confirm HOXA10-regulated ETA expression in endometrium. Endometrial biopsies were obtained from fertile reproductive-age individuals, and first trimester decidual samples were obtained at the time of elective termination. Immunohistochemistry (IHC) was used to identify ETA protein in endometrium as well as first trimester decidua. ETA was expressed in endometrial stromal cells throughout the menstrual cycle. ETA was also highly expressed in first trimester decidual cells. The regulatory relationship between HOXA10 and ETA was established by transient transfection analysis. The human endometrial stromal cell line (HESC) and the human endometrial epithelial cell line (Ishikawa) were transfected with pcDNA/HOXA10, HOXA10 small interfering RNA (siRNA), or respective controls. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to determine expression levels of HOXA10 and ETA in each group. ETA gene expression increased 9-fold (P < .05) after pcDNA/HOXA10 transfection of HESC. ETA was not regulated by HOXA10 in Ishikawa cells. We conclude that ETA is expressed in normal endometrium and decidua. Expression of this receptor is regulated by an essential mediator of endometrial receptivity, HOXA10. ETA may enhance the proliferative potential of endometrial cells in a manner similar to that seen in vascular smooth muscle cells. ETA likely acts as a molecular mechanism by which HOXA10 promotes stromal cell growth and prostaglandin production in both the implantation window and decidua.
PMCID: PMC3107845  PMID: 20371740
ETA; HOXA10; endometrium; decidua
10.  Changing homeodomain residues 2 and 3 of Hoxa1 alters its activity in a cell-type and enhancer dependent manner 
Nucleic Acids Research  2002;30(12):2663-2668.
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.
PMCID: PMC117285  PMID: 12060683
11.  Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of HoxA Genes in Developing Limbs 
PLoS Genetics  2013;9(12):e1004018.
HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.
Author Summary
Hox genes encode transcription factors with crucial roles during development. These genes are grouped in four different clusters names HoxA, B, C, and D. Mutations in genes of the HoxA and D clusters have been found in several human syndromes, affecting in some cases limb development. Despite their essential role and contrary to the genes of the HoxD cluster, little is known about how the HoxA genes are regulated. Here, we identified a large set of regulatory elements controlling HoxA genes during limb development. By studying spatial chromatin organization at the HoxA region, we found that the regulatory elements are spatially clustered regardless of their activity. Clustering of enhancers define tissue-specific chromatin domains that interact specifically with each other and with active genes in the limb. Our findings give support to the emerging concept that chromatin architecture defines the functional properties of genomes. Additionally, our study suggests a common constraint of the chromatin topology in the evolution of HoxA and HoxD regulation in the emergence of the hand/foot, which is one of the major morphological innovations in vertebrates.
PMCID: PMC3873244  PMID: 24385922
12.  MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5 
BMC Cancer  2012;12:348.
MicroRNAs (miRNAs) are short, non-coding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Although miR-196a has been implicated in several other cancers, its role in non-small cell lung cancer (NSCLC) is unknown. The aim of the present study was to examine the expression pattern of miR-196a in NSCLC and its clinical significance, as well as its biological role in tumor progression.
Expression of miR-196a was analyzed in 34 NSCLC tissues and five NSCLC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of DNA methylation on miR-196a expression was investigated by 5-aza-2-deoxy-cytidine treatment and bisulfite sequencing. The effect of miR-196a on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Analysis of target protein expression was determined by western blotting. Luciferase reporter plasmids were constructed to confirm the action of miR-196a on downstream target genes, including HOXA5. Differences between the results were tested for significance using Student’s t-test (two-tailed).
miR-196a was highly expressed both in NSCLC samples and cell lines compared with their corresponding normal counterparts, and the expression of miR-196a may be affected by DNA demethylation. Higher expression of miR-196a in NSCLC tissues was associated with a higher clinical stage, and also correlated with NSCLC lymph-node metastasis. In vitro functional assays demonstrated that modulation of miR-196a expression affected NSCLC cell proliferation, migration and invasion. Our analysis showed that miR-196a suppressed the expression of HOXA5 both at the mRNA and protein levels, and luciferase assays confirmed that miR-196a directly bound to the 3’untranslated region of HOXA5. Knockdown of HOXA5 expression in A549 cells using RNAi was shown to promote NSCLC cell proliferation, migration and invasion. Finally, we observed an inverse correlation between HOXA5 and miR-196a expression in NSCLC tissues.
Our findings indicate that miR-196a is significantly up-regulated in NSCLC tissues, and regulates NSCLC cell proliferation, migration and invasion, partially via the down-regulation of HOXA5. Thus, miR-196a may represent a potential therapeutic target for NSCLC intervention.
PMCID: PMC3503718  PMID: 22876840
Non-small cell lung cancer; miR-196a; Proliferation; Invasion; HOXA5
13.  HOXA5 Regulates hMLH1 Expression in Breast Cancer Cells1 
Neoplasia (New York, N.Y.)  2006;8(4):250-258.
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.
PMCID: PMC1600677  PMID: 16756717
Breast cancer; mismatch repair; homeotic gene; yeast; promoter analyses; HOX, homeotic; hMLH1, human MutL homolog 1; ChIP, chromatin immunoprecipitation; MMR, mismatch repair; Luc, luciferase
14.  Hoxa1 lineage-tracing indicates a direct role for Hoxa1 in development of the inner ear, the heart and the third rhombomere 
Developmental biology  2010;341(2):499-509.
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.
PMCID: PMC2862106  PMID: 20171203
Hoxa1; hindbrain; inner ear; heart
15.  Isolated Hoxa9 overexpression predisposes to the development of lymphoid but not myeloid leukemia 
Experimental hematology  2013;41(6):518-529.e5.
Hoxa9 is expressed in hematopoietic stem and progenitor cells although this expression is usually diminished as these cells undergo differentiation. In addition, aberrant expression of Hoxa9 is strongly associated with both T-cell and myeloid leukemia in mice and humans. Despite this strong association, enforced expression of Hoxa9 in murine bone marrow or thymus has only shown a modest ability to transform cells. To investigate this question, we used Vav regulatory elements to generate a transgenic mouse that targets Hoxa9 overexpression to all hematopoietic tissues. High level expression of the Hoxa9 transgene in the hematopoietic compartment was associated with embryonic lethality, as no pups from founders that expressed high levels of the transgene were born live. However, offspring of an additional founder line, which expressed lower levels of Hoxa9, developed a precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL), accompanied by spontaneous Notch1 mutations. In contrast to most murine models of leukemia associated with Hoxa9 overexpression, the Vav-Hoxa9 mice did not overexpress other Hoxa cluster genes, mir196b (a microRNA which is embedded in the Hoxa locus), Meis1, or Pbx3. The Hoxa9 transgenic mouse reported here provides a suitable system for the study of Hoxa9 collaborators that drive myeloid and lymphoid malignant transformation.
PMCID: PMC3718276  PMID: 23435313
Hoxa9; pre-T LBL; embryonic lethal; Notch1
16.  Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish 
The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.
Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.
We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.
PMCID: PMC4061917  PMID: 24902847
Zinc finger nuclease; TALEN nuclease; Retinoic acid signaling; Hindbrain; Nucleosome positioning; Gene expression
17.  NID2 and HOXA9 promoter hypermethylation as biomarkers for prevention and early detection in Oral Cavity Squamous Cell Carcinoma tissues and saliva 
Differentially methylated oral squamous cell carcinoma (OSCC) biomarkers, identified in-vitro and validated in well-characterized surgical specimens, have shown poor clinical correlation in cohorts with different risk profiles.
To overcome this lack of relevance we used the HumanMethylation27 BeadChip, publicly available methylation and expression array data, and Quantitative Methylation Specific PCR to uncover differential methylation in OSCC clinical samples with heterogeneous risk profiles.
A two stage-design consisting of Discovery and Prevalence screens was used to identify differential promoter methylation and deregulated pathways in patients diagnosed with OSCC and head and neck squamous cell carcinoma.
Promoter methylation of KIF1A (κ = 0.64), HOXA9 (κ = 0.60), NID2 (κ = 0.60), and EDNRB (κ = 0.60) had a moderate to substantial agreement with clinical diagnosis in the Discovery screen. HOXA9 had 68% sensitivity, 100% specificity and a 0.81 AUC. NID2 had 71% sensitivity, 100% specificity and a 0.79 AUC. In the Prevalence screen HOXA9 (κ = 0.82) and NID2 (κ = 0.80) had an almost perfect agreement with histologic diagnosis. HOXA9 had 85% sensitivity, 97% specificity and a 0.95 AUC. NID2 had 87% sensitivity, 95% specificity and a 0.91 AUC. A HOXA9 and NID2 gene panel had 94% sensitivity, 97% specificity and a 0.97 AUC. In saliva from OSCC cases and controls HOXA9 had 75% sensitivity, 53% specificity and a 0.75 AUC. NID2 had 87% sensitivity, 21% specificity and a 0.73 AUC.
This Phase I Biomarker Development Trial identified a panel of differentially methylated genes in normal and OSCC clinical samples from patients with heterogeneous risk profiles. This panel may be useful for early detection and cancer prevention studies.
PMCID: PMC3131432  PMID: 21558411
Genome-wide promoter methylation; Differential Methylation; Oral Squamous Cell Carcinoma; Biomarkers for Early Detection; Quantitative Methylation Specific PCR
18.  Allelic Variation within the Putative Autism Spectrum Disorder Risk Gene Homeobox-A-1 (HOXA1) and Cerebellar Maturation in Typically Developing Children and Adolescents 
Autism Research  2012;5(2):93-100.
Autism is known to be highly heritable, and has been associated with abnormalities in the development of several brain structures, including the cerebellum. Previous research has hinted that a gene controlling the development of posterior brain regions such as the cerebellum, may influence risk for autism. This gene is called Homeobox Domain A1 (HOXA1), and the variant within HOXA1 that has been most studied in relation to autism (A218G) falls within a gene region that is important for HOXA1 protein functioning. Although we know that autism appear to influence the dynamics of brain development, and that cerebellar anatomy continues to change over the lifespan – we do not know if A218G genotype influences cerebellar development over time. We studied this issue in typically developing controls who had a total of 296 repeat structural brain scans taken between ages 5 and 23 years of age. The volume of multiple cerebellar components was measures by hand in each scan, and we related developmental changes in these volumes to A218G genotype. We found that, in a part of the cerebellum implicated in autism, A218G genotype modified the rate of cerebellar growth. This suggests for the first time that the putative ASD risk gene HOXA1 has the capacity to modify the longitudinal development of cerebellar systems implicated in ASD neurobiology.
Homeobox-A-1 (HOXA1) has been proposed as a candidate gene for autism spectrum disorder (ASD) as it regulates embryological patterning of hind-brain structures implicated in autism neurobiology. In line with this notion, a non-synonymous single nucleotide polymorphism within a highly conserved domain of HOXA1 - A218G (rs10951154) - has been linked to both ASD risk, and cross-sectional differences in superior posterior lobar cerebellar anatomy in late adulthood. Despite evidence for early onset and developmentally dynamic cerebellar involvement in ASD, little is known of the relationship between A218G genotype and maturation of the cerebellum over early development. We addressed this issue using 296 longitudinally acquired structural magnetic resonance imaging brain scans from 116 healthy individuals between 5 and 23 years of age. Mixed models were used to compare the relationship between age and semi-automated measures of cerebellar volume in A-homozygotes (AA) and carriers of the G allele (Gcar). Total cerebellar volume increased between ages 5 and 23 years in both groups. However, this was accelerated in the Gcar relative to the AA group (Genotype-by-age interaction term, p=0.03), and driven by genotype-dependent differences in the rate of bilateral superior posterior lobar volume change with age (p=0.002). Resultantly, although superior posterior lobar volume did not differ significantly between genotype groups at age 5 (p=0.9), by age 23 it was 12% greater in Gcar than AA (p=0.002). Our results suggest that common genetic variation within this putative ASD risk gene has the capacity to modify the development of cerebellar systems implicated in ASD neurobiology.
PMCID: PMC3329569  PMID: 22359339
Autism; HOXA1; Cerebellum; Gene; Brain; MRI
19.  Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA. 
Journal of Bacteriology  1995;177(9):2373-2380.
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.
PMCID: PMC176894  PMID: 7730267
20.  Global analysis of genes regulated by HOXA10 in decidualization reveals a role in cell proliferation 
Molecular human reproduction  2008;14(6):357-366.
Homeobox (HOX) A10 is essential for fertility as demonstrated in transgenic mice, specifically affecting implantation and decidualization. Its role in human decidualization, however, remains unknown. In this study, we used gene silencing followed by microarray analysis to decipher the role of HOXA10 during decidualization of human endometrial stromal cells (HESCs). HOXA10 was knocked down using siRNA oligonucleotide transfection and cells were treated with estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + cAMP) to induce decidualization. Genes significantly regulated were identified using the Affymetrix microarray chip. With this method, 2361 transcripts were significantly altered by 1.5-fold or higher (P < 0.05) with H + cAMP treatment only. Of these genes, 258 were significantly up-regulated by HOXA10 knockdown and 236 transcripts were significantly down-regulated by more than 1.5-fold, totaling 494 genes that were regulated by HOXA10 during decidualization. Data analysis using the Ingenuity System revealed that many of the genes regulated by HOXA10 knockdown during H + cAMP treatment were associated with cell cycle. Real-time PCR was used to confirm that HOXA10 knockdown decreased expression of the cell cycle genes CDC2 and CCNB2. In addition, a higher percentage of cells were arrested in the G2/M phase. Next, we observed that cell proliferation as measured by BrdU incorporation was decreased upon HOXA10 knockdown and H + cAMP treatment. Apoptosis, on the other hand, as measured by Annexin V staining was not influenced by siHOXA10 in decidualizing cells. Together, these data demonstrate that during decidualization of HESC, HOXA10 is actively involved in promoting cell proliferation through the regulation of hundreds of genes.
PMCID: PMC2731304  PMID: 18456676
decidualization; HOXA10; endometrium; gene silencing; microarray analysis
21.  A SALL4/MLL/HOXA9 pathway in murine and human myeloid leukemogenesis 
The Journal of Clinical Investigation  2013;123(10):4195-4207.
The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.
PMCID: PMC3784519  PMID: 24051379
22.  Multiple Promoters and Alternative Splicing: Hoxa5 Transcriptional Complexity in the Mouse Embryo 
PLoS ONE  2010;5(5):e10600.
The genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.
Methodology/Principal Findings
We have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.
Our observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.
PMCID: PMC2868907  PMID: 20485555
23.  A Gal4/UAS system for conditional transgene expression in rhombomere 4 of the zebrafish hindbrain 
The zebrafish is well established as a model organism for the study of vertebrate embryogenesis, but transgenic lines enabling restricted gene expression are still lacking for many tissues.
We first generated the hoxb1a(β–globin):eGFPum8 line that expresses eGFP in hindbrain rhombomere 4 (r4), as well as in facial motor neurons migrating caudally from r4. Second, we generated the hoxb1a(β-globin):Gal4VP16um60 line to express the exogenous Gal4VP16 transcription factor in r4. Lastly, we prepared the UAS(β-actin):hoxa3aum61 line where the hoxa3a gene, which is normally expressed in r5 and r6, is under control of Gal4-regulated UAS elements. Crossing the hoxb1a(β-globin):Gal4VP16um60 line to the UAS(β-actin):hoxa3aum61 line drives robust hoxa3aexpression in r4. We find that transgenic expression of hoxa3a in r4 does not affect hoxb1a expression, but has variable effects on migration of facial motorneurons and formation of Mauthner neurons. While cases of somatic transgene silencing have been reported in zebrafish, we have not observed such silencing to date – possibly because of our efforts to minimize repetitive sequences in the transgenic constructs.
We have generated three transgenic lines that will be useful for future studies by permitting the labeling of r4-derived cells, as well as by enabling r4-specific expression of various transgenes.
PMCID: PMC3422023  PMID: 22499412
24.  The generation and expression of a Hoxa11eGFP targeted allele in mice 
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.
PMCID: PMC2855819  PMID: 18942146
Hoxa11; eGFP; targeted mutation
25.  The non-coding RNA Mistral activates Hoxa6 and Hoxa7 expression and stem cell differentiation by recruiting Mll1 to chromatin 
Molecular cell  2011;43(6):1040-1046.
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.
PMCID: PMC3176448  PMID: 21925392

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