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Cell patterning during embryogenesis is essential for establishing the identity of the developing body plan. Hox genes are fundamental regulators of tissue organisation along the anterior-posterior body axis of the developing embryo. These homeodomain-containing proteins act as transcription factors during normal development. The function of the homeodomain is to bind sequence-specific DNAmotifs which allows either activation or repression of downstream effector genes, which consequently results in the control of tissue-specific determination and differentiation. Aberrant expression of such Hox genes, including Hoxa-9 can result in homeotic transformations leading to phenotypic malformations and oncogenesis. However the normal function of Hoxa-9 is poorly understood. This study explored the potential role for Hoxa-9 in normal development and differentiation. An in situ hybridisation approach was taken to define the expression of Hoxa-9 in the developing mouse. Hoxa-9 was found to expressed in a temporarily and spatially regulated manner, in particular being detected in the developing cardiac atria, ventricles and cardiac vessels during E9.5-E12 stages of development. The expression of this homeotic gene during in vitro differentiation of embryonic stem cells into cardiomyocytes and haematopoietic cells demonstrated a profile that correlated with the emergence of these cell types. The functioning relationship between Hoxa-9 expression and lineage commitment was Airther explored using over-expression in embryonic stem cells. A potential role for Hoxa-9 in normal development is discussed.

Whole genome transcriptome analysis has revealed that a large proportion of the genome in higher metazoa is transcribed, yet only a small proportion of this transcription is protein-coding. One possible function of non-coding transcription is that it enables complex and diverse body plans to evolve through variation in deployment of a relatively common set of protein-coding genes. Functional studies suggest that long non-coding RNAs (lncRNAs) regulate gene expression via diverse mechanisms, operating in both cis and trans to activate or repress target genes. An emerging theme common to lncRNA function is interaction with proteins that modify chromatin and mediate epigenetic regulation. The Hox gene complexes are particularly rich in lncRNAs and require precise and fine-tuned expression to deploy Hox transcription factors throughout development. Here we identify and functionally characterize two novel lncRNAs within the D. melanogaster Hox complex, in the interval between Scr and Antp. We use nascent transcript fluorescent in-situ hybridization (ntFISH) to characterize the embryonic expression patterns of each lncRNA with respect to flanking Hox genes, and to analyze co-transcription within individual nuclei. We find that the transcription of one lncRNA, ncX, is an initial response to early transcription factors and may activate Scr expression, while transcription of the other lncRNA, ncPRE is consistent with activation and/or maintenance of Scr expression. ntFISH performed in D.virilis embryos revealed the presence of a lncRNA ortholog with highly similar expression to ncX, indicating functional conservation of lncRNA transcription across ~60 million years of evolution. We identify the ncPRE lncRNA locus as a binding site for multiple proteins associated with Polycomb/Trithorax response elements (PREs/TREs) and show that DNA encoding the ncPRE lncRNA functions as a bona fide PRE, mediating trans-interactions between chromosomes and silencing of nearby genes. We find that transcription through the ncPRE DNA relieves silencing, suggesting a role for endogenous transcription of the ncPRE lncRNA in relieving Polycomb-silencing and enabling Scr activation. We demonstrate that both lncRNA transcripts are required for proper Scr expression, and over-expression of either lncRNAs from ectopic genomic loci has no effect on Scr expression, but ectopic expression at the endogenous locus is associated with ectopic Scr activation, indicating that the lncRNA-mediated regulation functions locally at the site of transcription on the chromosome. ncX may mediate transvection effects previously observed at the Scr locus, independent of the protein Zeste. Together our results support a model of competing mechanisms in the regulation of Scr expression - a background of Polycomb repression acting from the ncPRE locus, which in the first thoracic segment is counteracted by lncRNA transcription and Trithorax binding to ncPRE, enabling activation and maintenance of Scr expression. This work provides a functional insight into the complex regulatory interactions between lncRNAs and epigenetic mechanisms, essential to establish and maintain the precise expression pattern of Hox genes through development.

Xenoturbella bocki has recently been identified as one of the most basal deuterostomes, although an even more basal phylogenetic position cannot be ruled out. Here we report on a polymerase chain reaction survey of partial Hox homeobox sequences of X. bocki. Surprisingly, we did not find evidence for more than five Hox genes, one clear labial/PG1 ortholog, one posterior gene most similar to the PG9/10 genes of Ambulacraria, and three central group genes whose precise assignment to a specific paralog group remains open. We furthermore report on a re‐evaluation of the available published evidence of Hox genes in other basal deuterostomes.

To understand the gene regulatory network (GRN) governing caudal hindbrain formation in embryonic zebrafish, several early expressed factors have been manipulated, and multiple genetic mutants have been characterized. Such analyses have identified morphogens such as Retinoic Acid (RA) and Fibroblast growth factors (FGFs), as well as transcription factors like hoxb1b, hoxb1a, hnf1ba, and valentino as being required for rhombomere (r) r4-r6 formation in zebrafish. Considering that the caudal hindbrain is relatively complex – for instance, unique sets of neurons are formed in each rhombomere segment – it is likely that additional essential genes remain to be identified and integrated into the caudal hindbrain GRN. Our results reveal that r4 gene expression is unaffected by the individual loss of hoxb1b, hoxb1a or RA, but is under the combinatorial regulation of RA together with hoxb1b. In contrast, r5/r6 gene expression is dependent on RA, FGF, hnf1ba and valentino – as individual loss of these factors abolishes r5/r6 gene expression. Analysis of six mutant lines (gas6, gbx1, sall4, eglf6, celf2, and greb1l) did not reveal rhombomere or neuronal defects, but transcriptome analysis of one line (gas6 mutant) identified expression changes for genes involved in several developmental processes – suggesting that these genes may have subtle roles in hindbrain development. We conclude that r4-r6 formation is relatively robust, such that very few genes are absolutely required for this process. However, there are mechanistic differences in r4 versus r5/r6, such that no single factor is required for r4 development while several genes are individually required for r5/r6 formation.

Yes / Background The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development and which are dys-regulated in some cancers. In this study we examined the expression and oncogenic function of HOX genes in mesothelioma, a cancer arising from the pleura or peritoneum which is associated with exposure to asbestos. Methods We tested the sensitivity of the mesothelioma-derived lines MSTO-211H, NCI-H28, NCI-H2052, and NCI-H226 to HXR9, a peptide antagonist of HOX protein binding to its PBX co-factor. Apoptosis was measured using a FACS-based assay with Annexin, and HOX gene expression profiles were established using RT-QPCR on RNA extracted from cell lines and primary mesotheliomas. The in vivo efficacy of HXR9 was tested in a mouse MSTO-211H flank tumor xenograft model. Results We show that HOX genes are significantly dysregulated in malignant mesothelioma. Targeting HOX genes with HXR9 caused apoptotic cell death in all of the mesothelioma-derived cell lines, and prevented the growth of mesothelioma tumors in a mouse xenograft model. Furthermore, the sensitivity of these lines to HXR9 correlated with the relative expression of HOX genes that have either an oncogenic or tumor suppressive function in cancer. The analysis of HOX expression in primary mesothelioma tumors indicated that these cells could also be sensitive to the disruption of HOX activity by HXR9, and that the expression of HOXB4 is strongly associated with overall survival. Conclusion HOX genes are a potential therapeutic target in mesothelioma, and HOXB4 expression correlates with overall survival. / The authors gratefully acknowledge the support of the British Lung Foundation, grant number ICAPPG10-1. KJH acknowledges support from the ICR/RM NIHR Biomedical Research Centre.

A família de genes HOX compreende um conjunto de fatores de transcrição altamente conservados evolutivamente. Em mamíferos, os genes HOX se subdividem em 4 clusters: HOXA, HOXB, HOXC e HOXD, atuando no desenvolvimento embrionário com a regulação de processos biológicos como proliferação, diferenciação, migração, angiogênese e apoptose que são reativados durante a carcinogênese. Estudos recentes apontam que os genes HOX podem exercer papel relevante na formação de diversos tumores sólidos, todavia ainda não foi possível caracterizar sistematicamente a expressão dos genes HOX em tumores bem como determinar seus alvos em tumores. Desta forma, o objetivo geral deste trabalho consistiu na caracterização in silico do modelo de atuação genes HOX na carcinogênese. Para cumprir este objetivo foi identificado o perfil diferencial dos genes HOX entre amostras normais e tumorais. Alvos de genes HOX foram identificados e, quando diferencialmente expressos, foram associados com os genes HOX, independentemente dos índices de metilação e CNA. Por fim, as associações finais entre os genes HOX e seus alvos foram enriquecidas com os bancos de dados KEGG e GO. Identificou-se diferentes assinaturas de expressão de genes HOX em diferentes tumores, associadas com o eixo ântero-posterior do corpo humano, bem como os folhetos embrionários originários aos tecidos tumorais, compatível com o padrão de expressão no desenvolvimento embrionário. Um número considerável de genes HOX atuam preferencialmente via enhancers na regulação de seus alvos. Como exemplo, os genes HOXB7 e HOXC11, que funcionam como moduladores anti tumorais. Finalmente, o estudo mostra que diante do número crescente de dados genômicos públicos, é possível viabilizar projetos de grande valor científico. / The HOX gene family comprises a set of evolutionarily highly conserved transcription factors. In mammals, HOX genes are subdivided into four clusters: HOXA, HOXB, HOXC and HOXD, acting on the embryonic development with regulation of biological processes such as proliferation, differentiation, migration, angiogenesis and apoptosis that are reactivated during carcinogenesis. Recent studies indicate that HOX genes may play a relevant role in the formation of several solid tumors, but it has not been possible to systematically characterize the expression of HOX genes in tumors as well as to determine their targets in tumors. Thus, the general aim of this project was to characterize the in vivo model of HOX genes in carcinogenesis. To accomplish this goal the differential profile of HOX genes was identified between normal and tumor samples. HOX gene targets were identified and, when differentially expressed, were associated with HOX genes regardless of methylation and CNA indices. Finally, the final associations between the HOX genes and their targets were enriched with the KEGG and GO databases. Different signatures of HOX gene expression were identified in different tumors, associated with the anteroposterior axis of the human body, as well as the embryonic leaflets originating from the tumor tissues, compatible with the expression pattern in the embryonic development. A considerable number of HOX genes preferentially act via enhancers in the regulation of their targets. As an example, the HOXB7 and HOXC11 genes, which function as pro-tumor modulators. Finally, the study shows that in view of the growing number of public genomic data, it is possible to make feasible projects of great scientific value.

Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.