Hox Gene Expression Research Articles

1917P - Luminal B breast cancer prognosis prediction by comprehensive analysis of Homeobox genes

BackgroundHomeobox (HOX) family consists of 39 genes which act as master regulators in embryonic development. Each of the genes is also known to play key roles in progression of breast cancer, including epithelial to mesenchymal transition, tumor angiogenesis and endocrine therapy resistance. Although there are numerous reports on individual HOX genes and cancer, none of them have comprehensively analyzed the whole gene family. Since HOX genes strongly coordinate within the family during the embryonic period, we considered that the analysis of the whole HOX family is also indispensable in breast cancer. MethodsWe collected 702 breast cancer data from four publicly available array datasets (GSE11121, GSE7390, GSE3494, GSE2990) and performed unsupervised hierarchal clustering into two clusters by the expression of HOX genes. We constructed model formulas for cluster prediction by dividing the samples into learning and validation groups. We used three machine learning methods: support-vector machine (SVM), neural network and Bayes. The model formulas were validated by validation samples. We also used 512 TCGA breast cancer data to calculate covariations of the genes in breast cancer. ResultsBy the clustering of four array datasets, the DFS of the two clusters in PAM50-classified luminal B patients were statistically different (p=0.016), and the gene ontology analysis revealed that the Wnt pathway was activated in the poor prognostic cluster. All cluster prediction models for luminal B sample achieved accuracies of over 90%. From TCGA breast cancer data, we found that HOX genes covariate the most with other HOX genes, especially within the chromosomally proximal groups. ConclusionsComprehensive analysis of the whole HOX family lead to the prediction of luminal B breast cancer prognosis. Considering that Wnt signaling controls HOX genes during the embryonic stage, we suppose a Wnt pathway activated, poor prognostic subgroup in luminal B breast cancer which can be identified by the expression of HOX genes. The cluster prediction model by machine learning was acceptable for its future adaptation in clinical settings. We also proved that HOX genes strongly covariate within the gene family in cancer, not only during the embryonic stage. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Evolutionary lability in Hox cluster structure and gene expression in Anolis lizards.

Hox genes orchestrate development by patterning the embryonic axis. Vertebrate Hox genes are arranged in four compact clusters, and the spacing between genes is assumed to be crucial for their function. The genomes of squamate reptiles are unusually rich and variable in transposable elements (TEs), and it has been suggested that TE invasion is responsible for the Hox cluster expansion seen in snakes and lizards. Using de novo TE prediction on 17 genomes of lizards and snakes, I show that TE content of Hox clusters are generally 50% lower than genome‐wide TE levels. However, two distantly related lizards of the species‐rich genus Anolis have Hox clusters with a TE content that approaches genomic levels. The age distribution of TEs in Anolis lizards revealed that peaks of TE activity broadly coincide with speciation events. In accordance with theoretical models of Hox cluster regulation, I find that Anolis species with many TEs in their Hox clusters show aberrant Hox gene expression patterns, suggesting a functional link between TE accumulation and embryonic development. These results are consistent with the hypothesis that TEs play a role in developmental processes as well as in evolutionary diversifications.

The Role of MicroRNAs in Early Chondrogenesis of Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (hiPSCs) play an important role in research regarding regenerative medicine. Particularly, chondrocytes differentiated from hiPSCs seems to be a promising solution for patients suffering from osteoarthritis. We decided to perform chondrogenesis in a three-week monolayer culture. Based on transcriptome analysis, hiPSC-derived chondrocytes (ChiPS) demonstrate the gene expression profile of cells from early chondrogenesis. Chondrogenic progenitors obtained by our group are characterized by significantly high expression of Hox genes, strongly upregulated during limb formation and morphogenesis. There are scanty literature data concerning the role of microRNAs in early chondrogenesis, especially in chondrogenic differentiation of hiPSCs. The main aim of this study was to investigate the microRNA expression profile and to select microRNAs (miRNAs) taking part in early chondrogenesis. Our findings allowed for selection crucial miRNAs engaged in both diminishing pluripotency state and chondrogenic process (inter alia hsa-miR-525-5p, hsa-miR-520c-3p, hsa-miR-628-3p, hsa-miR-196b-star, hsa-miR-629-star, hsa-miR-517b, has-miR-187). These miRNAs regulate early chondrogenic genes such as: HOXD10, HOXA11, RARB, SEMA3C. These results were confirmed by RT-qPCR analysis. This work contributes to a better understanding of the role of miRNAs directly involved in chondrogenic differentiation of hiPSCs. These data may result in the establishment of a more efficient protocol of obtaining chondrocyte-like cells from hiPSCs.

Homeobox B9 integrates bone morphogenic protein 4 with inflammation at atheroprone sites.

AimsAtherosclerosis develops near branches and bends of arteries that are exposed to disturbed blood flow which exerts low wall shear stress (WSS). These mechanical conditions alter endothelial cells (EC) by priming them for inflammation and by inducing turnover. Homeobox (Hox) genes are developmental genes involved in the patterning of embryos along their anterior–posterior and proximal–distal axes. Here we identified Hox genes that are regulated by WSS and investigated their functions in adult arteries.Methods and resultsEC were isolated from inner (low WSS) and outer (high WSS) regions of the porcine aorta and the expression of Hox genes was analysed by quantitative real-time PCR. Several Hox genes (HoxA10, HoxB4, HoxB7, HoxB9, HoxD8, HoxD9) were significantly enriched at the low WSS compared to the high WSS region. Similarly, studies of cultured human umbilical vein EC (HUVEC) or porcine aortic EC revealed that the expression of multiple Hox genes (HoxA10, HoxB9, HoxD8, HoxD9) was enhanced under low (4 dyn/cm2) compared to high (13 dyn/cm2) WSS conditions. Gene silencing studies identified Hox genes (HoxB9, HoxD8, HoxD9) that are positive regulators of inflammatory molecule expression in EC exposed to low WSS, and others (HoxB9, HoxB7, HoxB4) that regulated EC turnover. We subsequently focused on HoxB9 because it was strongly up-regulated by low WSS and, uniquely, was a driver of both inflammation and proliferation. At a mechanistic level, we demonstrate using cultured EC and murine models that bone morphogenic protein 4 (BMP4) is an upstream regulator of HoxB9 which elicits inflammation via induction of numerous inflammatory mediators including TNF and downstream NF-κB activation. Moreover, the BMP4-HoxB9-TNF pathway was potentiated by hypercholesterolaemic conditions.ConclusionsLow WSS induces multiple Hox genes that control the activation state and turnover of EC. Notably, low WSS activates a BMP4-HoxB9-TNF signalling pathway to initiate focal arterial inflammation, thereby demonstrating integration of the BMP and Hox systems in vascular pathophysiology.

Lissamphibian limbs and the origins of tetrapod hox domains

The expression and function of hox genes have played a key role in the debate on the evolution of limbs from fins. As an early branching tetrapod lineage, lissamphibians may provide information on the origin of the limb’s hox domains and particularly how the plesiomorphic tetrapod pattern compares to the hox pattern present in fish fins. Here, we comparatively investigated the expression of hox genes in the developing limbs of axolotl and Xenopus laevis as well as in the fins of the direct developing cichlid Astatotilapia burtoni. In contrast to axolotl, which has only very low digital expression of hoxd11, Xenopus limbs recapitulate the reverse collinear hoxd expression pattern known from amniotes with clearly defined proximal and distal hoxd11 expression domains. For hoxa genes, we observe that in Xenopus limbs, as in axolotl, a clear distal domain of hoxa11 expression is present, although in the presence of a hoxa11 antisense transcript. Investigation of fins reveals the presence of hoxa11 antisense transcription in the developing fin rays in a domain similar to that of hoxa13 and overlapping with hoxa11 sense transcription. Our results indicate that full exclusion of hoxa11 from the autopod only became firmly established in amniotes. The distal antisense transcription of hoxa11, however, appears to predate the evolution of the limb, but likely originated without the concurrent implementation of the transcriptional suppression mechanism that causes mutually exclusive hoxa11 and hoxa13 domains in amniotes.

THE ROLE OF VENTX HOMEOBOX GENE DURING HUMAN HAEMATOPOIETIC DEVELOPMENT

The Ventx genes are non-clustered homeobox transcription factors that confer a ventral phenotype on mesodermal cells in the developing embryo. Ventx genes are conserved in vertebrates but have been lost in rodents. In the human haematopoietic system, VENTX promotes myeloid differentiation and is expressed in some acute myeloid leukaemias (AML). Using a DOX (Doxycycline) inducible VENTX expression system, we found that VENTX overexpression in hPSC impaired mesoderm formation, but VENTX enforced expression after mesoderm commitment resulted in the emergence of an increased percentage of immature blood cells that co-expressed CD90 and CD34. These cells displayed high clonogenic capacity in methylcellulose, but only after DOX was removed from the media. This suggested that VENTX expression held cells in a non-proliferative state. Transcriptional profiling revealed increased expression of HOXA genes in haematopoietic cells following VENTX induction, consistent with their immature phenotype. Conversely, genes involved in myeloid differentiation were downregulated during VENTX overexpression, as were genes involved in proliferation, such as MYC and MYB. ATAC-sequencing demonstrated that VENTX closes selected chromatin loci, in particular areas targeted by HOXB13 and CDX transcription factors. We hypothesise that VENTX might act as a transcriptional suppressor during haematopoietic differentiation and we are currently investigating the genomic targets of VENTX, combining ATAC-seq and ChIP-seq with the transcriptional profiling. In summary, VENTX overexpression generated immature blood cells in a quiescent state, preventing their proliferation and differentiation into myeloid lineages.

1010 - CHARACTERIZING HOXA-EXPRESSING ENDOTHELIAL AND HAEMATOPOIETIC POPULATIONS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS

Haematopoietic stem cell (HSC) transplantation is a key therapy for patients with leukaemia or bone marrow failure. Repopulating HSCs derived from human pluripotent stem cells (hPSCs) would provide a source of blood stem cells for many patients who might benefit from this therapy but lack a suitable donor.Success is contingent on the development of hPSC differentiation protocols that generate and support the developmental continuum from correctly patterned haemogenic endothelium to the emerging preHSCs, and their subsequent maturation to repopulating (r) HSCs. Whilst many hPSC differentiation protocols generate blood cells similar to those arising from the human yolk sac, our recent work has demonstrated that we can differentiate blood cells from hPSCs that phenotypically and transcriptionally resemble the preHSCs found in the aorta-gonad-mesonephros (AGM) of the human embryo. An important finding from these studies was that the expression of HOXA genes distinguished yolk sac like (HOXA-) from AGM-like (HOXA+) endothelium and blood cells. In response to these observations, we developed a hPSC reporter line in which tdTOMATO is linked to expression of HOXA5, a gene that is expressed in intra-embryonic haemogenic endothelium, preHSCs and rHSCs. Validation of the reporter demonstrated that HOXA5-expressing cells were also enriched for expression of the whole HOXA cluster. We have shown that this line enables us to enumerate the proportion of endothelia and blood cells that express HOXA5 during differentiation, and that this faithfully reflects the bias towards a yolk sac (TOMATO-) or AGM-like (TOMATO+) fate imposed by the protocol used. This line will enable fine-tuning of the differentiation process to identify conditions that support development and maintenance of cells that carry a stem cell 'signature' and also express HOXA genes – a hypothesized requirement for rHSC.

A Single MicroRNA-Hox Gene Module Controls Equivalent Movements in Biomechanically Distinct Forms of Drosophila

SummaryMovement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. Here we explore this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which we previously showed to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). Our study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, we also discover that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, we provide evidence that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. Our results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. Our work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.

Long non-coding RNA HOXB-AS3 promotes myeloid cell proliferation and its higher expression is an adverse prognostic marker in patients with acute myeloid leukemia and myelodysplastic syndrome

BackgroundLong non-coding RNAs (lncRNAs) represent the majority of cellular transcripts and play pivotal roles in hematopoiesis. However, their clinical relevance in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains largely unknown. Here, we investigated the functions of HOXB-AS3, a lncRNA located at human HOXB cluster, in the myeloid cells, and analyzed the prognostic significances in patients with AML and MDS.MethodsshRNAs were used to downregulate HOXB-AS3 in the cell lines and the effect was evaluated by quantitative polymerase chain reaction. The proliferation of the cell lines was illustrated by proliferation and BrdU flow assays. Further, we retrospectively analyzed the HOXB-AS3 expression in 193 patients with AML and 157 with MDS by microarray analysis, and evaluated its clinical importance.ResultsDownregulation of HOXB-AS3 suppressed cell proliferation. Mechanistically, HOXB-AS3 potentiated the expressions of several key factors in cell cycle progression and DNA replication without affecting the expressions of HOX genes. In AML, patients with higher HOXB-AS3 expression had shorter survival than those with lower HOXB-AS3 expression (median overall survival (OS), 17.7 months versus not reached, P < 0.0001; median relapse-free survival, 12.9 months versus not reached, P = 0.0070). In MDS, patients with higher HOXB-AS3 expression also had adverse prognosis compared with those with lower HOXB-AS3 expression (median OS, 14.6 months versus 42.4 months, P = 0.0018). The prognostic significance of HOXB-AS3 expression was validated in the TCGA AML cohort and another MDS cohort from our institute. The subgroup analyses in MDS patients showed that higher HOXB-AS3 expressions could predict poor prognosis only in lower-risk (median OS, 29.2 months versus 77.3 months, P = 0.0194), but not higher-risk group.ConclusionsThis study uncovers a promoting role of HOXB-AS3 in myeloid malignancies and identifies the prognostic value of HOXB-AS3 expression in AML and MDS patients, particularly in the lower-risk group.

PRC2-Associated Chromatin Contacts in the Developing Limb Reveal a Possible Mechanism for the Atypical Role of PRC2 in HoxA Gene Expression

Preventing inappropriate gene expression in time and space is as fundamental as triggering the activation of tissue- or cell-type-specific factors at the correct developmental stage and in the correct cells. Here, we study the impact of Polycomb repressive complex 2 (PRC2) function on HoxA gene regulation. We analyze chromatin conformation of the HoxA cluster and its regulatory regions and show that in addition to the well-known role of PRC2 in silencing Hox genes via direct binding, its function is required for the changes in HoxA long-range interactions distinguishing proximal limbs from distal limbs. This effect stems from the differential PRC2 occupancy over the HoxA cluster and, at least in part, from the ability of PRC2-bound loci to engage in long-range contacts. Unexpectedly, PRC2 also impacts chromatin conformation in a way that promotes enhancer-promoter contacts required for proper HoxA expression, pointing to a dual role of PRC2 in gene regulation.

Elevated HOX gene expression in acute myeloid leukemia is associated with NPM1 mutations and poor survival

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cells and the most common malignant myeloid disorder in adults. Several gene mutations such as in NPM1 (nucleophosmin 1) are involved in the pathogenesis and progression of AML. The aim of this study was to identify genes whose expression is associated with driver mutations and survival outcome. Genotype data (somatic mutations) and gene expression data including RNA-seq, microarray, and qPCR data were used for the analysis. Multiple datasets were utilized as training sets (GSE6891, TCGA, and GSE1159). A new clinical sample cohort (Semmelweis set) was established for in vitro validation. Wilcoxon analysis was used to identify genes with expression alterations between the mutant and wild type samples. Cox regression analysis was performed to examine the association between gene expression and survival outcome. Data analysis was performed in the R statistical environment. Eighty-five genes were identified with significantly altered expression when comparing NPM1 mutant and wild type patient groups in the GSE6891 set. Additional training sets were used as a filter to condense the six most significant genes associated with NPM1 mutations. Then, the expression changes of these six genes were confirmed in the Semmelweis set: HOXA5 (P = 3.06E−12, FC = 8.3), HOXA10 (P = 2.44E−09, FC = 3.3), HOXB5 (P = 1.86E−13, FC = 37), MEIS1 (P = 9.82E−10, FC = 4.4), PBX3 (P = 1.03E−13, FC = 5.4) and ITM2A (P = 0.004, FC = 0.4). Cox regression analysis showed that higher expression of these genes – with the exception of ITM2A – was associated with worse overall survival. Higher expression of the HOX genes was identified in tumors harboring NPM1 gene mutations by computationally linking genotype and gene expression. In vitro validation of these genes supports their potential therapeutic application in AML.

PS990 INTEGRATED ANALYSIS OF NTAL RELATED GENES IMPROVES OUTCOMES PREDICTION OF EUROPEAN LEUKEMIANET 2008 RISK STRATIFICATION IN ACUTE MYELOID LEUKEMIA

Background: In the context of myelodysplasias (MDS) and acute mydeloid leukemias (AML), the discovery in hematopoietic cells of aberrant DNA methylation profiles and mutations in genes associated with DNA methylation regulation has allowed for the development of demethylating agents. However, such treatments affect the whole bone marrow (BM), including mesenchymal stromal cells (MSCs), the epigenetic landscape of which is poorly known. Aims: Considering the importance of BM-MSCs in the pathophysiology of MDS and AML, the aim of this study was to compare the methylation profiles of MSCs from MDS and AML patients to those of healthy donors (HD). Methods: The AML-FILOtheque (#BB-0033–00073, Cochin hospital, Paris, France) provided samples from AML patients. BM from MDS patients was collected in the course of the MILESYM clinical study (authorized by the French Ministry of Research, #DC-2016–2739). BM samples were collected after informed consent from age-matched HD during hip surgery (Tours University Hospital). All BM samples were collected prior to treatment and patients with a history of chemotherapy were excluded. The study cohort comprised 32 HD, 19 patients newly diagnosed for MDS and 21 de novo AML. Targeted next-generation sequencing (NGS) was used to determine the genetic landscape of the AML samples. MSCs were isolated from whole BM samples during a single culture passage and cryopreserved until use; thawed cells were cultured for two further passages (P1 and P2) and all experiments were performed with P2-MSCs. Global DNA methylation analysis was performed via 5-methylcytosine quantification (dot-plots); regarding methylome beadchip analysis, bisulfite-converted DNA samples were analyzed with an Infinium® MethylationEPIC BeadChip array (over 850,000 methylation sites, Illumina); initial quality control was performed with the GenomeStudio software (Illumina); the Bioconductor's ChAMP package was used with R software to conduct bio-informatics analyses; after filtering, beta values for the remaining sites were calculated and normalized using the Beta Mixture Quantile dilatation (BMIQ) algorithm; comparisons were performed to identify differentially methylated probes (DMPs) and regions (DMRs); DMRs were detected at a threshold of 7 sites per region using the DMRcate algorithm; gene-set enrichment analysis (GSEA) was computed with DMRs-coupled genes. Specific gene expression (key regulators of DNA methylation, HOX gene family) was quantified by RT-qPCR (LightCycler® 480) on total mRNA previously tested on a 2100 Bioanalyzer (Agilent Technologies). Results: MDS and AML-MSCs displayed a global DNA hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed an aberrant methylation profile in all MDS and in a subgroup of AML-MSCs. The latter was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4 and HOXC6), and impacted their expression. Summary/Conclusion: This study highlight the fact that modifications of DNA methylation occur in MDS/AML-MSCs, both globally and at focal levels, specifically dysregulating the expression of HOX genes known for their involvement in leukemogenesis. Such DNA methylation alterations in MSCs could be the consequence of the malignant disease or could participate in its development.

PS989 ABNORMAL DNA METHYLATION MODIFIES HOX GENES EXPRESSION IN BONE MARROW MESENCHYMAL STROMAL CELLS OF MYELODYSPLASIAS AND DE NOVO ACUTE MYELOID LEUKEMIAS

Background:In the context of myelodysplasias (MDS) and acute mydeloid leukemias (AML), the discovery in hematopoietic cells of aberrant DNA methylation profiles and mutations in genes associated with DNA methylation regulation has allowed for the development of demethylating agents. However, such treatments affect the whole bone marrow (BM), including mesenchymal stromal cells (MSCs), the epigenetic landscape of which is poorly known.Aims:Considering the importance of BM‐MSCs in the pathophysiology of MDS and AML, the aim of this study was to compare the methylation profiles of MSCs from MDS and AML patients to those of healthy donors (HD).Methods:The AML‐FILOtheque (#BB‐0033–00073, Cochin hospital, Paris, France) provided samples from AML patients. BM from MDS patients was collected in the course of the MILESYM clinical study (authorized by the French Ministry of Research, #DC‐2016–2739). BM samples were collected after informed consent from age‐matched HD during hip surgery (Tours University Hospital). All BM samples were collected prior to treatment and patients with a history of chemotherapy were excluded. The study cohort comprised 32 HD, 19 patients newly diagnosed for MDS and 21 de novo AML. Targeted next‐generation sequencing (NGS) was used to determine the genetic landscape of the AML samples. MSCs were isolated from whole BM samples during a single culture passage and cryopreserved until use; thawed cells were cultured for two further passages (P1 and P2) and all experiments were performed with P2‐MSCs. Global DNA methylation analysis was performed via 5‐methylcytosine quantification (dot‐plots); regarding methylome beadchip analysis, bisulfite‐converted DNA samples were analyzed with an Infinium® MethylationEPIC BeadChip array (over 850,000 methylation sites, Illumina); initial quality control was performed with the GenomeStudio software (Illumina); the Bioconductor's ChAMP package was used with R software to conduct bio‐informatics analyses; after filtering, beta values for the remaining sites were calculated and normalized using the Beta Mixture Quantile dilatation (BMIQ) algorithm; comparisons were performed to identify differentially methylated probes (DMPs) and regions (DMRs); DMRs were detected at a threshold of 7 sites per region using the DMRcate algorithm; gene‐set enrichment analysis (GSEA) was computed with DMRs‐coupled genes. Specific gene expression (key regulators of DNA methylation, HOX gene family) was quantified by RT‐qPCR (LightCycler® 480) on total mRNA previously tested on a 2100 Bioanalyzer (Agilent Technologies).Results:MDS and AML‐MSCs displayed a global DNA hypomethylation and under‐expression of DNMT1 and UHRF1. Methylome analysis revealed an aberrant methylation profile in all MDS and in a subgroup of AML‐MSCs. The latter was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4 and HOXC6), and impacted their expression.Summary/Conclusion:This study highlight the fact that modifications of DNA methylation occur in MDS/AML‐MSCs, both globally and at focal levels, specifically dysregulating the expression of HOX genes known for their involvement in leukemogenesis. Such DNA methylation alterations in MSCs could be the consequence of the malignant disease or could participate in its development.

Deciphering The Potential Role of Hox Genes in Pancreatic Cancer.

The Hox gene family plays an important role in organogenesis and animal development. Currently, 39 Hox genes that are clustered in four chromosome regions have been identified in humans. Emerging evidence suggests that Hox genes are involved in the development of the pancreas. However, the expression of Hox genes in pancreatic tumor tissues has been investigated in only a few studies. In addition, whether specific Hox genes can promote or suppress cancer metastasis is not clear. In this article, we first review the recent progress in studies on the role of Hox genes in pancreatic cancer. By comparing the expression profiles of pancreatic cancer cells isolated from genetically engineered mice established in our laboratory with three different proliferative and metastatic abilities, we identified novel Hox genes that exhibited tumor-promoting activity in pancreatic cancer. Finally, a potential oncogenic mechanism of the Hox genes was hypothesized.

Hox genes: Downstream "effectors" of retinoic acid signaling in vertebrate embryogenesis.

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.

An atlas of anterior hox gene expression in the embryonic sea lamprey head: Hox-code evolution in vertebrates

In the hindbrain and the adjacent cranial neural crest (NC) cells of jawed vertebrates (gnathostomes), nested and segmentally-restricted domains of Hox gene expression provide a combinatorial Hox-code for specifying regional properties during head development. Extant jawless vertebrates, such as the sea lamprey (Petromyzon marinus), can provide insights into the evolution and diversification of this Hox-code in vertebrates. There is evidence for gnathostome-like spatial patterns of Hox expression in lamprey; however, the expression domains of the majority of lamprey hox genes from paralogy groups (PG) 1-4 are yet to be characterized, so it is unknown whether they are coupled to hindbrain segments (rhombomeres) and NC. In this study, we systematically describe the spatiotemporal expression of all 14 sea lamprey hox genes from PG1-PG4 in the developing hindbrain and pharynx to investigate the extent to which their expression conforms to the archetypal gnathostome hindbrain and pharyngeal hox-codes. We find many similarities in Hox expression between lamprey and gnathostome species, particularly in rhombomeric domains during hindbrain segmentation and in the cranial neural crest, enabling inference of aspects of Hox expression in the ancestral vertebrate embryonic head. These data are consistent with the idea that a Hox regulatory network underlying hindbrain segmentation is a pan vertebrate trait. We also reveal differences in hindbrain domains at later stages, as well as expression in the endostyle and in pharyngeal arch (PA) 1 mesoderm. Our analysis suggests that many Hox expression domains that are observed in extant gnathostomes were present in ancestral vertebrates but have been partitioned differently across Hox clusters in gnathostome and cyclostome lineages after duplication.

A homeotic shift late in development drives mimetic color variation in a bumble bee

Natural phenotypic radiations, with their high diversity and convergence, are well-suited for informing how genomic changes translate to natural phenotypic variation. New genomic tools enable discovery in such traditionally nonmodel systems. Here, we characterize the genomic basis of color pattern variation in bumble bees (Hymenoptera, Apidae, Bombus), a group that has undergone extensive convergence of setal color patterns as a result of Müllerian mimicry. In western North America, multiple species converge on local mimicry patterns through parallel shifts of midabdominal segments from red to black. Using genome-wide association, we establish that a cis-regulatory locus between the abdominal fate-determining Hox genes, abd-A and Abd-B, controls the red-black color switch in a western species, Bombus melanopygus Gene expression analysis reveals distinct shifts in Abd-B aligned with the duration of setal pigmentation at the pupal-adult transition. This results in atypical anterior Abd-B expression, a late developmental homeotic shift. Changing expression of Hox genes can have widespread effects, given their important role across segmental phenotypes; however, the late timing reduces this pleiotropy, making Hox genes suitable targets. Analysis of this locus across mimics and relatives reveals that other species follow independent genetic routes to obtain the same phenotypes.

365 Multivalent chromatin looping orchestrates cellular reprogramming for advanced gene therapy

The discovery of cellular plasticity, an epigenetic phenomenon by which differentiated cells alter their identity through reprogramming, dedifferentiation or transdifferentiation has led to a paradigm shift in regenerative medicine for treatment of genetic diseases and injuries. Current surgical methods pose high mortality rates making stem cell-based therapy an attractive option. HOX genes, a set of transcription factor genes, play key roles during stem cell renewal, multilineage differentiation and reprogramming of differentiated cells. Delineating the molecular mechanisms of HOX genes regulation in diverse cellular identities will provide clues to modulate cell fate for development of novel, more effective stem cell-based treatments. Recent studies highlight that a cell identity is determined not only by specific gene expression profile and posttranslational modifications but largely by its characteristic three-dimensional (3D) genomic organization. Chromatin maintains a high-order topology organizing genes into structural domains through long-range chromatin looping that is essential for controlled gene regulation. Existing techniques to study the link between chromatin looping and gene regulation require either large alterations of the linear DNA sequence or prior knowledge of loop-mediating factors. We have pioneered a technology to form multivalent chromosomal contacts and loops in 3D space at will. Previous studies revealed that primary adult fibroblasts retain several HOX gene expression patterns similar to that in embryonic cells. We will affect chromosomal remodeling using our new multiplexed chromatin looping system to examine how 3D architectural reorganization of chromatin at the Hox loci in human dermal fibroblasts can alter cellular identities (via reprogramming and transdifferentiation) with the potential for reproducible and efficient clinical-grade cell production. This study will provide insights for linking chromatin architecture to cutaneous gene expression that affects cellular plasticity in both adult and embryonic cells that may have transformative outcomes for regenerative medicine.

A Case of Identity: HOX Genes in Normal and Cancer Stem Cells.

Stem cells are undifferentiated cells that have the unique ability to self-renew and differentiate into many different cell types. Their function is controlled by core gene networks whose misregulation can result in aberrant stem cell function and defects of regeneration or neoplasia. HOX genes are master regulators of cell identity and cell fate during embryonic development. They play a crucial role in embryonic stem cell differentiation into specific lineages and their expression is maintained in adult stem cells along differentiation hierarchies. Aberrant HOX gene expression is found in several cancers where they can function as either oncogenes by sustaining cell proliferation or tumor-suppressor genes by controlling cell differentiation. Emerging evidence shows that abnormal expression of HOX genes is involved in the transformation of adult stem cells into cancer stem cells. Cancer stem cells have been identified in most malignancies and proved to be responsible for cancer initiation, recurrence, and metastasis. In this review, we consider the role of HOX genes in normal and cancer stem cells and discuss how the modulation of HOX gene function could lead to the development of novel therapeutic strategies that target cancer stem cells to halt tumor initiation, progression, and resistance to treatment.

NUP98-HBO1–fusion generates phenotypically and genetically relevant chronic myelomonocytic leukemia pathogenesis

AbstractChronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115+ Ly6Chigh monocytes (an equivalent subpopulation to human classical CD14+ CD16− monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1–mediated transcriptional signature in human CD34+ cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased HOXA9 gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.