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Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1–Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation., Graphical Abstract Graphical AbstractEnhancer architecture-dependent multilayered transcriptional regulation at the Halr1–Hoxa1 locus that orchestrates RA signaling-induced early lineage differentiation of ESCs.

Background: Oral cancer is one of the most frequent solid cancers worldwide, and oral squamous cell carcinoma (OSCC) constitutes approximately 90% of oral cancers. The discovery of reliable prognostic indicators would be a potential strategy for OSCC treatment. In the present study, we aim to explore the underlying mechanism by which microRNA-145 (miR-145) affected OSCC. Methods: Forty-eight patients diagnosed with OSCC were enrolled to obtain the OSCC tissues and adjacent normal tissues. The targeting relationship between miR-145 and Homeobox A1 (HOXA1) was verified. In order to assess the effects of miR-145 in OSCC and the detailed regulatory mechanism, the SCC-9 cell line was adopted, in which expression of miR-145 and HOXA1 were altered by transfection. Then, a series of in vitro and in vivo experiments were performed to evaluate the cell viability, migration, invasion, and tumor growth. Results: miR-145 was poorly expressed and HOXA1 was highly expressed in OSCC. HOXA1 was verified as a target of miR-145 to mediate the activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway. In the circumstance of miR-145 elevation or HOXA1 depletion, the SCC-9 cell line manifested with inhibited cell viability, invasion, and migration in vitro, coupled with reduced tumor growth in vivo, with a decreased expression of ERK/MAPK signaling pathway-related genes/proteins. Conclusion: These findings suggested that miR-145 can inhibit HOXA1 to inactivate the ERK/MAPK signaling pathway, thereby suppressing OSCC cell proliferation, migration, and invasion to further inhibit the development of OSCC, highlighting a novel therapeutic target for the OSCC treatment.

MicroRNAs (miRNAs) are dysregulated in human ovarian carcinoma (OC). But the mechanism underlying miR‐10a‐5p in regulating the progression of OC need deeply explored. In the current study, we observed that miR‐10a‐5p was down‐expressed in OC samples and OC cell lines. In addition, miR‐10a‐5p restrained the viability, colony formation, migration ability and invasiveness of OC cells. We further ascertained Homeobox A1 (HOXA1) was a downstream gene of miR‐10a‐5p. Furthermore, HOXA1 was distinctly upregulated in OC samples. Finally, upregulation of HOXA1 abolished the suppressive effects of miR‐10a‐5p on OC cells. These observations suggested that miR‐10a‐5p suppressed the aggressive phenotypes of OC cells via regulating HOXA1.

Hypoxic-ischemic brain damage (HIBD) is a major cause of fatality and morbidity in neonates. However, current treatment approaches to alleviate HIBD are not effective. Various studies have highlighted the role of microRNAs (miRNAs) in various biological functions in multiple diseases. This study investigated the role of miR-339-5p in HIBD progression. Neonatal HIBD mouse model was induced by ligation of the right common carotid artery. Neuronal cell model exposed to oxygen-glucose deprivation (OGD) was also established. The miR-339-5p expression in mouse brain tissues and neuronal cells was quantified, and the effects of miR-339-5p on neuronal cell activity and apoptosis induced by hypoxia-ischemia were explored. The overexpression or knockdown of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in hippocampal neurons was used to determine the effect of lncRNA NEAT1 on the expression of miR-339-5p and homeobox A1 (HOXA1) and apoptosis. Short hairpin RNA targeting lncRNA NEAT1 and miR-339-5p antagomir were used in neonatal HIBD mice to identify their roles in HIBD. Our results revealed that miR-339-5p was downregulated in neonatal HIBD mice and neuronal cells exposed to OGD. Downregulated miR-339-5p promoted neuronal cell viability and suppressed apoptosis during hypoxia-ischemia. Moreover, lncRNA NEAT1 competitively bound to miR-339-5p to increase HOXA1 expression and inhibited neuronal cell apoptosis under hypoxic-ischemic conditions. The key observations of the current study present evidence demonstrating that lncRNA NEAT1 upregulated HOXA1 to alleviate HIBD in mice by binding to miR-339-5p.

The long non-coding RNA regulator of reprogramming (lncRNA ROR) is involved in atherosclerosis (AS), but the specific mechanism remains unclear. The expressions of lncRNA ROR, let-7b-5p, Homeobox A1 (HOXA1), and apoptosis-associated proteins in the serum of AS patients and human umbilical vein endothelial cells (HUVECs) were determined by quantitative real-time PCR (qRT-PCR) and Western blot. The relationships of lncRNA ROR, let-7b-5p, and HOXA1 were analyzed by Pearson's correlation test. The viability and the migration of HUVECs were measured by Cell Counting Kit-8, wound healing, and Transwell assays. The predicted target gene and the potential binding sites were confirmed by dual-luciferase reporter assay. lncRNA ROR was highly expressed in AS, which promoted the cell viability and migration of HUVECs, while lncRNA ROR silencing produced the opposite results. The expression of let-7b-5p, which bound to lncRNA ROR, was downregulated in AS, indicating that the two genes were negatively correlated. Besides this, let-7b-5p reversed the effects of upregulated lncRNA ROR expression on let-7b-5p expression, cell viability, and migration as well as the expressions of apoptosis-related proteins of ox-LDL-treated HUVECs. HOXA1 was targeted by let-7b-5p and upregulated in AS, with its expression being negatively correlated with let-7b-5p but positively correlated with lncRNA ROR. In ox-LDL-treated HUVECs, overexpressed HOXA1 reversed the effects of let-7b-5p, and HOXA1 silencing reversed the effects of lncRNA ROR. In AS, lncRNA ROR promoted the biological characteristics of oxidation of low-density lipoprotein-induced HUVECs via the let-7b-5p/HOXA1 axis.

Circular RNAs (circRNAs) serve important roles in tumorigenesis and may be used as novel molecular biomarkers for clinical diagnosis. However, the role and molecular mechanisms of circRNAs in cervical cancer (CC) remain unknown. In the present study, circRNA isoform of eukaryotic translation initiation factor 4γ2 (circEIF4G2) was revealed to be significantly upregulated in CC tissues and cell lines. Furthermore, increased expression of circEIF4G2 was associated with poor prognosis in patients with CC. circEIF4G2 knockdown suppressed the malignant features of CC cells, including cell proliferation, colony formation, migration and invasion. Additionally, circEIF4G2 was identified to serve as a sponge for microRNA‑218 (miR‑218), which targeted homeobox A1 (HOXA1). Furthermore, circEIF4G2 may increase the expression levels of HOXA1 by sponging miR‑218. Rescue experiments suggested that transfection with a miR‑218 inhibitor attenuated the inhibitory effects of circEIF4G2 knockdown on cell proliferation, migration and invasion. Furthermore, silencing HOXA1 reversed the effects of the miR‑218 inhibitor on CC cells. Collectively, the present findings suggested that circEIF4G2 promoted cell proliferation and migration via the miR‑218/HOXA1 pathway.

Hox genes, a highly conserved homolog in most animals, play vital functions in cell development and organ formation. In recent years, researchers have discovered that it can act as a tumor regulator, and its members can participate in tumorigenesis by regulating receptor signaling, cell differentiation, apoptosis, migration, EMT, and angiogenesis. Hox genes and which major members play a vital role in the progress of head and neck squamous cell carcinoma (HNSCC) is still unclear. After analyzing the expression differences and prognostic value of all Hox genes through the TCGA-HNSC database, we use histochemistry stains in 52 pairs of HNSCC slices to verify the expression level of the key member-HOXA1. In correlation analysis, we found that high HOXA1 expression is related to poor pathological grade (p= 0.0077), advanced T stage (p= 0.021) and perineural invasion (PNI) (p= 0.0019). Furthermore, we used Cox univariate and multivariate regression analysis to confirm the independent predictive power of HOXA1 expression. To explore the underlying mechanisms behind HOXA1, we ran GSVA and GSEA and found fourteen mutual signaling pathways, including neuroprotein secretion and transport, tumor-associated signaling pathways, cell adhere junction and metabolic reprogramming. Finally, we found that the high expression of HOXA1 is significantly related to the decrease of CD8+ T cell infiltration and the decline of DNA methylation level. Our findings demonstrated that HOXA1, as a notable member of the HOX family, maybe an independent prognostic indicator in HNSCC.

Hepatocellular carcinoma (HCC) represents a malignant tumor predominantly arising in the setting of cirrhosis and is the third most common cause of cancer-associated death on a global scale. The heterogeneous nature of HCC and limited well-recognized biomarkers may contribute to poor patient prognosis and treatment failure. In this study, we identified expression pattern of microRNA-202-3p (miR-202-3p) in HCC and characterized its functional role as well as related mechanisms. First, we collected 50 HCC tissues and 38 normal liver tissues, and after bioinformatics prediction, the expression of miR-202-3p and KDM3A was determined in the tissues. We found lowly expressed miR-202-3p and overexpressed KDM3A in HCC tissues. Then, dual-luciferase reporter gene assay was employed to test the presence of miR-202-3p binding sites in the 3’UTR of KDM3A and chromatin immunoprecipitation (ChIP) assay to homeobox A1 (HOXA1) interaction with KDM3A and MEIS3. It has been confirmed that miR-202-3p negatively regulated KDM3A responsible for increasing the expression of HOXA1 by eliminating the histone H3 lysine 9 (H3K9)me2 in HCC cells. HOXA1 could evidently increase H3K4me1 and H3K27ac enrichment in the MEIS3 enhancer region and enhance the expression of MEIS3. Functional assays were also performed with the results showing that upregulated miR-202-3p or downregulated KDM3A retarded HCC cell viability, migration, and invasion. In addition, HepG2 cells were xenografted into nude mice, and we demonstrated that upregulated miR-202-3p reduced the growth of human HCC cells in vivo. Taken together, the present study elicits a novel miR-202-3p/KDM3A/HOXA1/MEIS3 pathway in HCC, potentiating an exquisite therapeutic target for HCC.

Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES) cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

Jian-Gang Wang,1,2 Wu-Ping Tang,2 Ming-Chu Liao,1 Yan-Ping Liu,1 Xiao-Hong Ai1 1Department of Radiation Oncology, The First Affiliated Hospital of University of South China, Hengyang, 2Department of Oncology, Shaoyang Hospital of TCM, Shaoyang, People’s Republic of China Background: Recent studies reported that miRNAs play important roles in the carcinogenesis and progression of nasopharyngeal carcinoma (NPC). Therefore, further studies are warranted to better elucidate the function and mechanism of miRNAs in NPC.Methods: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the miR-99a expression in NPC cell lines and tissue samples. Wound healing, transwell migration and invasion, and lung metastatic colonization assays were performed to determine NPC cell migratory, invasive and metastatic abilities of NPC cells. Luciferase reporter assays, quantitative RT-PCR and Western blotting were used to validate the target of miR-99a.Results: We found that miR-99a was significantly downregulated in NPC cell lines and tissue samples. Ectopic overexpression of miR-99a significantly inhibited NPC cell migration and invasion in vitro, and suppressed lung macroscopic and microscopic metastatic colonization in vivo. Conversely, silencing of miR-99a significantly promoted the migratory and invasive abilities of NPC cells. Furthermore, HOXA1 was validated as a direct target of miR-99a, and ectopic expression of HOXA1 could rescue the suppressive effect of miR-99a overexpression on NPC cell migration and invasion.Conclusion: Together, these results indicated that miR-99a could inhibit NPC invasion and metastasis by targeting HOXA1, thus providing a novel potential target for miRNA-based treatment for NPC patients in the future. Keywords: miR-99a, homeobox A1, nasopharyngeal carcinoma, invasion, metastasis

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.

Homeobox genes constitute a large family of genes widely studied because of their role in the establishment of the body pattern. However, they are also involved in many other events during development and adulthood. The main olfactory epithelium (MOE) is an excellent model to study neurogenesis in the adult nervous system. Analyses of homeobox genes during development show that some of these genes are involved in the formation and establishment of cell diversity in the MOE. Moreover, the mechanisms of expression of odorant receptors (ORs) constitute one of the biggest enigmas in the field. Analyses of OR promoters revealed the presence of homeodomain binding sites in their sequences. Here we characterize the expression patterns of a set of 49 homeobox genes in the MOE with in situ hybridization. We found that seven of them (Dlx3, Dlx5, Dlx6, Msx1, Meis1, Isl1, and Pitx1) are zonally expressed. The homeobox gene Emx1 is expressed in three guanylate cyclase(+) populations, two located in the MOE and the third one in an olfactory subsystem known as Gruneberg ganglion located at the entrance of the nasal cavity. The homeobox gene Tshz1 is expressed in a unique patchy pattern across the MOE. Our findings provide new insights to guide functional studies that aim to understand the complexity of transcription factor expression and gene regulation in the MOE. J. Comp. Neurol. 524:2713-2739, 2016. © 2016 Wiley Periodicals, Inc.

Highlight This study demonstrates that overexpression of POTH15 (a class-I KNOX in potato) can alter multiple morphological traits, and identifies numerous POTH15 targets involved in diverse developmental processes in potato., Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX. Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA., Highlighted Article: Analysis of the expression pattern and targets of the human transcription factor LEUTX suggests a two-stage model for embryonic genome activation, also involving DRPX.

An increasing number of studies have indicated that the dysregulation of microRNAs (miRNAs/miR) is closely associated with non‑small cell lung cancer (NSCLC) development and progression by acting as tumor suppressors or oncogenes. Therefore, an in‑depth understanding of the biological roles of miRNAs in NSCLC may provide novel therapeutic methods for the treatment of patients with this disease. In the present study, reverse transcription‑quantitative polymerase chain reaction was used to detect miR‑577 expression in NSCLC tissues and cell lines. Cell Counting Kit‑8 and Transwell invasion assays were performed to determine the effects of miR‑577 on NSCLC cell proliferation and invasion. Luciferase reporter assays were used to demonstrate the relationship between miR‑577 and homeobox A1 (HOXA1) in NSCLC cells. The results revealed that miR‑577 was markedly downregulated in NSCLC tissues and cell lines. Additionally, restoration of miR‑577 expression significantly decreased the proliferation and invasion of NSCLC cells. Furthermore, miR‑577 negatively regulated HOXA1 expression in NSCLC cells by directly binding to its 3'‑untranslated region. HOXA1 was significantly upregulated in NSCLC tissues, and its upregulation was inversely correlated with miR‑577. Notably, restored HOXA1 expression abrogated the reduced proliferation and invasion of NSCLC cells caused by miR‑577 overexpression. Taken together, these results indicated that miR‑577 may have served tumor suppressive roles in NSCLC by directly targeting HOXA1. Therefore, this miRNA may be developed as a potential therapeutic target for the therapy of patients with NSCLC.

Research has confirmed that abnormally expressed miRNAs are involved in the occurrence and development of hepatocellular carcinoma (HCC). In the present study, we confirmed that miR‑577 expression both in HCC tissues and cell lines was markedly downregulated. Clinically, downregulated expression of miR‑577 is notably related to malignant clinicopathological features, such as venous invasion and advanced TNM stage. Additionally, miR‑577 may act as a valuable tumor marker to predict the prognosis of HCC patients. Through knockdown and overexpression of miR‑577, miR‑577 was identified as an inhibitor of cell metastatic ability and EMT progress in HCC. Furthermore, miR‑577 was able to directly bind to the 3'‑UTR of homeobox A1 (HOXA1) to regulate the expression of HOXA1. In addition, there existed a negative correlation between the expression of miR‑577 and HOXA1 in HCC specimens. Rescue experiments revealed that the influence of miR‑577 on the migration, invasion and EMT of HCC cells was reversed by HOXA1. Taken together, our findings demonstrated that miR‑577 functions as an anti‑oncogene to suppress the migration, invasion and EMT of HCC cells through direct interaction with HOXA1. miR‑577 may act as a valuable target for the molecular‑targeted therapy of HCC.

Homeobox A1 (HOXA1) serves an oncogenic role in multiple cancer types. However, the role of HOXA1 in non-small cell lung cancer (NSCLC) remains unclear. In the present study, use of reverse transcription-quantitative polymerase chain reaction and the databases of The Cancer Genome Atlas (TCGA), Oncomine, Gene Expression Profiling Interactive Analysis and the Multi Experiment Matrix were combined to assess the expression of HOXA1 and its co-expressed genes in NSCLC. Bioinformatic analyses, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network and protein-protein interaction analyses, were used to investigate the underlying molecular mechanism effected by the co-expressed genes. Additionally, the potential miRNAs targeting HOXA1 were investigated. The results showed that HOXA1 was upregulated in NSCLC. The area under the curve of HOXA1 indicated a moderate diagnostic value of the HOXA1 level in NSCLC. According to GO and KEGG analyses, the co-expressed genes may be involved in 'dGTP metabolic processes', 'network-forming collagen trimers', 'centromeric DNA binding' and 'the p53 signaling pathway'. Three miRNAs (miR-181b-5p, miR-28-5p and miR-181d-5p) targeting HOXA1 were each predicted by 10 algorithms; miR-181b and miR-181d levels were downregulated in LUSC tissues compared with those in normal lung tissues based on data from the TCGA database, and inverse correlations were found between HOXA1 and miR-181b (r=−0.205, P

International audience; On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Homeobox genes are compared between genomes in an attempt to understand the evolution of animal development. The ability of the protist, Dictyostelium discoideum, to shift between uni- and multicellularity makes this group ideal for studying the genetic changes that may have occurred during this transition. We present here the first genome-wide classification and comparative genomic analysis of the 14 homeobox genes present in D. discoideum. Based on the structural alignment of the homeodomains, they can be broadly divided into TALE and non-TALE classes. When individual homeobox genes were compared with members of known class or family, we could further classify them into 3 groups, namely, TALE, OTHER and NOVEL classes, but no HOX family was found. The 5 members of TALE class could be further divided into PBX, PKNOX, IRX and CUP families; 4 homeobox genes classified as NOVEL did not show any similarity to any known homeobox genes; while the remaining 5 were classified as OTHERS as they did show certain degree of similarity to few known homeobox genes. No unique RNA expression pattern during development of D. discoideum emerged for members of an individual group. Putative promoter analysis revealed binding sites for few homeobox transcription factors among many probable factors.

The aberrant expression of miR-433 has been validated in some types of cancers. However, the expression profile and the biological function of miR-433 on colon cancer are still elusive. This study was designed to investigate the function of miR-433 on the proliferation and invasion of colon cancer cells. We detected the expression of miR-433 in colon cancer tissues, adjacent normal tissues, and cell lines. CCK8 and Transwell assays were performed to explore the impact of miR-433 on colon cancer cell proliferation and invasion. The luciferase reporter assay was applied to identify the direct target of miR-433. The results demonstrated that miR-433 was downregulated in colon cancer tissues and cell lines when compared with the control. Overexpression of miR-433 significantly suppressed the ability of colon cancer cell proliferation and invasion, whereas knockdown of miR-433 remarkably enhanced cell proliferation and invasion. Homeobox A1 (HOXA1) was identified as a target of miR-433, and it mediated the functions of miR-433 on colon cancer cells. To conclude, we revealed that miR-433 was downregulated in colon cancer, and it inhibited colon cancer cell proliferation and invasion by directly targeting HOXA1.

BACKGROUND: Generation of disease-specific models and development of new treatments for non-WNT/non-SHH medulloblastomas (MB) is an urgent need. Group 3 MB is characterized by amplifications of MYC and/or OTX2. Furthermore, transcriptional profiling of group 3 MB shows dysregulation of GABAergic and/or photoreceptor gene expression. The distalless/DLX homeobox genes are required for regulation of GABAergic interneuron differentiation and migration. Furthermore, in the retinas of the Dlx1/Dlx2 double knockout (DKO) mouse, which die at birth, there is marked upregulation of the cone rod homeobox, Crx, as well as the related orthodenticle homeobox gene, Otx2. Moreover, OTX2 activates Crx expression. Otx2 is expressed in three domains of the developing central nervous system (CNS): at the hindbrain/midbrain boundary, the forebrain and retina. METHODS/RESULTS: Using chromatin immunoprecipitation (ChIP) of wild-type mouse embryonic retinas and a specific antibody to DLX2, we have identified regions within regulatory elements of the Crx and Otx2 promoters, respectively. DLX2 protein:Crx/Otx2 DNA binding specificity was confirmed by mobility shift assays, including supershifts using the DLX2 antibody, as well as through site-directed mutagenesis of candidate homeodomain DNA binding motifs. Reporter gene assays demonstrated repression of Crx and/or Otx2 expression when co-transfected with Dlx2 expression constructs in vitro. De-repression of CRX and/or OTX2 expression was confirmed in the Dlx1/Dlx2 DKO in vivo. In addition, crossing of a Crx lacZ reporter mouse with the Dlx1/Dlx2 DKO showed increased lacZ expression in the retinas of the reporter mouse with concurrent loss of of Dlx1 and Dlx2 function. CONCLUSIONS: DLX2 directly binds to both Crx and Otx2 promoters in the embryonic retina and represses expression of Crx and Otx2. We are exploring other upstream transcriptional regulators of OTX2 in the developing CNS towards the development of a group 3 MB mouse model for testing novel therapeutic approaches for children with an intermediate to poor prognosis.

Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). The molecular events leading to HCC following chronic HCV infection remain poorly defined. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, and their deregulation is associated with different viral infections. In this study, we observed that HCV infection of hepatocytes transcriptionally downregulates miR-181c expression by modulating CCAAT/enhancer binding protein β (C/EBP-β). Reduced expression of the pri-miR-181c transcript was noted following HCV infection. In silico prediction suggests that homeobox A1 (HOXA1) is a direct target of miR-181c. HOXA1 is a member of the homeodomain-containing transcription factor family and possesses pivotal roles in normal growth, development, and differentiation of mammalian tissues. Our results demonstrated that HOXA1 expression is enhanced in HCV-infected hepatocytes. Exogenous expression of the miR-181c mimic inhibits HOXA1 and its downstream molecules STAT3 and STAT5, which are involved in cell growth regulation. Interestingly, overexpression of miR-181c inhibited HCV replication by direct binding with E1 and NS5A sequences. Furthermore, accumulation of HCV genotype 2a RNA with miR-181c was observed in an RNA-induced silencing complex in Huh7.5 cells. Our results provide new mechanistic insights into the role of miR-181c in HCV-hepatocyte interactions, and miR-181c may act as a target for therapeutic intervention. IMPORTANCE Chronic HCV infection is one of the major causes of end-stage liver disease, including hepatocellular carcinoma. An understanding of the molecular mechanisms of HCV-mediated hepatocyte growth promotion is necessary for therapeutic intervention against HCC. In this study, we have provided evidence of HCV-mediated transcriptional downregulation of miR-181c. HCV-infected liver biopsy specimens also displayed lower expression levels of miR-181c. We have further demonstrated that inhibition of miR-181c upregulates homeobox A1 (HOXA1), which is important for hepatocyte growth promotion. Exogenous expression of miR-181c inhibited HCV replication by directly binding with HCV E1 and NS5A sequences. Taken together, our results provided new mechanistic insights for an understanding of the role of miR-181c in HCV-hepatocyte interactions and revealed miR-181c as a potential target for therapeutic intervention.

We recently reported a role of polycomb repressive complex 2 (PRC2) and PRC2 trimethylation of histone 3 lysine 27 (H3K27me3) in the regulation of homeobox (HOX) (Marcinkiewicz and Gudas, 2013, Exp Cell Res) gene transcript levels in human oral keratinocytes (OKF6-TERT1R) and tongue squamous cell carcinoma (SCC) cells. Here, we assessed both the levels of various histone modifications at a subset of homeobox genes and genome wide DNA methylation patterns in OKF6-TERT1R and SCC-9 cells by using ERRBS (enhanced reduced representation bisulfite sequencing). We detected the H3K9me3 mark at HOXB7, HOXC10, HOXC13, and HOXD8 at levels higher in OKF6-TERT1R than in SCC-9 cells; at IRX1 and SIX2 the H3K9me3 levels were conversely higher in SCC-9 than in OKF6-TERT1R. The H3K79me3 mark was detectable only at IRX1 in OKF6-TERT1R and at IRX4 in SCC-9 cells. The levels of H3K4me3 and H3K36me3 marks correlate with the transcript levels of the assessed homeobox genes in both OKF6-TERT1R and SCC-9. We detected generally lower CpG methylation levels on DNA in SCC-9 cells at annotated genomic regions which were differentially methylated between OKF6-TERT1R and SCC-9 cells; however, some genomic regions, including the HOX gene clusters, showed DNA methylation at higher levels in SCC-9 than OKF6-TERT1R. Thus, both altered histone modification patterns and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human oral cavity SCC cells, and this dysregulation potentially plays a role in the neoplastic phenotype of oral keratinocytes.

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.

NUP98 is among the most promiscuously translocated genes in hematological diseases. Among the 28 known fusion partners, there are two categories: homeobox genes and non-homeobox genes. The homeobox fusion partners are well-studied in animal models, resulting in HoxA cluster overexpression and hematological disease. The non- homeobox fusion partners are less well studied. We created transgenic animal models for three NUP98 fusion genes (one homeobox, two non- homeobox), and show that in this system, the NUP98-homeobox fusion promotes self-renewal and aberrant gene expression to a significantly greater extent. We conclude that homeobox partners create more potent NUP98 fusion oncogenes than do non-homeobox partners.

Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of xenobiotic detoxification genes and is a critical mediator of gene–environment interactions. Many AHR target genes identified by genome-wide gene expression profiling have morphogenetic functions, suggesting that AHR may play a role in embryonic development. Objectives: To characterize the developmental functions of the AHR, we studied the consequences of AHR activation by the agonist 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD), and the result of its repression by the antagonists 6,2,4-trimethoxyflavone and CH 223191 or by short-hairpin RNA (shRNA)-mediated Ahr knockdown during spontaneous differentiation of embryonic stem (ES) cells into cardiomyocytes. Methods: We generated an AHR-positive cardiomyocyte lineage differentiated from mouse ES cells that expresses puromycin resistance and enhanced green fluorescent protein (eGFP) under the control of the Cyp1a1 (cytochrome P450 1a1) promoter. We used RNA sequencing (RNA.Seq) to analyze temporal trajectories of TCDD-dependent global gene expression in these cells during differentiation. Results: Activation, inhibition, and knockdown of Ahr significantly inhibited the formation of contractile cardiomyocyte nodes. Global expression analysis of AHR-positive cells showed that activation of the AHR/TCDD axis disrupted the concerted expression of genes that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation, including dozens of genes encoding homeobox transcription factors and Polycomb and trithorax group proteins. Conclusions: Disruption of AHR expression levels resulted in gene expression changes that perturbed cardiomyocyte differentiation. The main function of the AHR during development appears to be the coordination of a complex regulatory network responsible for attainment and maintenance of cardiovascular homeostasis. Citation: Wang Q, Chen J, Ko CI, Fan Y, Carreira V, Chen Y, Xia Y, Medvedovic M, Puga A. 2013. Disruption of aryl hydrocarbon receptor homeostatic levels during embryonic stem cell differentiation alters expression of homeobox transcription factors that control cardiomyogenesis. Environ Health Perspect 121:1334–1343; http://dx.doi.org/10.1289/ehp.1307297

OBJECTIVE: Different vascular beds show differing susceptibility to the development of atherosclerosis, but the molecular mechanisms underlying these differences are incompletely understood. This study aims to identify factors that contribute to the phenotypic heterogeneity of distinct regions of the adult vasculature. APPROACH AND RESULTS: High-throughput mRNA profiling in adult mice reveals higher expression of the homeobox paralogous genes 6 to 10 (Hox6-10) in the athero-resistant thoracic aorta (TA) than in the athero-susceptible aortic arch (AA). Higher homeobox gene expression also occurs in rat and porcine TA, and is maintained in primary smooth muscle cells isolated from TA (TA-SMCs) compared with cells from AA (AA-SMCs). This region-specific homeobox gene expression pattern is also observed in human embryonic stem cells differentiated into neuroectoderm-SMCs and paraxial mesoderm-SMCs, which give rise to AA-SMCs and TA-SMCs, respectively. We also find that, compared with AA and AA-SMCs, TA and TA-SMCs have lower activity of the proinflammatory and proatherogenic nuclear factor-κB (NF-κB) and lower expression of NF-κB target genes, at least in part attributable to HOXA9-dependent inhibition. Conversely, NF-κB inhibits HOXA9 promoter activity and mRNA expression in SMCs. CONCLUSION: Our findings support a model of Hox6-10-specified positional identity in the adult vasculature that is established by embryonic cues independently of environmental factors and is conserved in different mammalian species. Differential homeobox gene expression contributes to maintaining phenotypic differences between SMCs from athero-resistant and athero-susceptible regions, at least in part through feedback regulatory mechanisms involving inflammatory mediators, for example, reciprocal inhibition between HOXA9 and NF-κB. The authors’ laboratories Sources of Funding supported are by grants from the Ministerio de Economía y Competitividad (MINECO; SAF201016044), Instituto de Salud Carlos III (ISCIII; RD/06/0014/0021, RD12/0042/0028), the Belgian Society of Cardiology (Dr. Léon Dumont Prize 2010), the European Commission (Liphos-317916), the Wellcome Trust (WT078390MA), and the Cambridge Biomedical Research Center. C. Cheung was sponsored by the Agency for Science, Technology and Research (Singapore). J.M. GonzálezGranado and P. Fernández received salary support from the ISCIII (CP11/00145 and CD07/00021, respectively). The CNIC is supported by MINECO and Pro-CNIC Foundation. Sí

Polycomb-repressive complexes (PRCs) ensure the correct spatiotemporal expression of numerous key developmental regulators. Despite their pivotal role, how PRCs are recruited to specific targets remains largely unsolved, particularly in plants. Here we show that the Arabidopsis ASYMMETRIC LEAVES complex physically interacts with PRC2 and recruits this complex to the homeobox genes BREVIPEDICELLUS and KNAT2 to stably silence these stem cell regulators in differentiating leaves. The recruitment mechanism resembles the Polycomb response element-based recruitment of PRC2 originally defined in flies and provides the first such example in plants. Combined with recent studies in mammals, our findings reveal a conserved paradigm to epigenetically regulate homeobox gene expression during development.

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx–Lbx and NK3–NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila .

Knotted1 defined the first homeobox gene family to be isolated in plants and was identified from dominant maize mutants that produced 'knots' of mis-specified tissue on the leaf. The Knotted1-like homeobox (KNOX) gene family expanded last year to include members lacking the defining homeobox with exciting implications for KNOX gene regulation and function. Recent evidence for direct KNOX regulation by myb-related ARP proteins and epigenetic silencing by polycomb repressive complexes have also shed light on the mechanisms defining KNOX gene expression.

Approximately 300 homeobox loci were identified in the euchromatic regions of the human genome, of which 235 are probable functional genes and 65 are likely pseudogenes. Many of these genes play important roles in embryonic development and cell differentiation. Dysregulation of homeobox gene expression is a frequent occurrence in cancer. Accumulating evidence suggests that as genetics disorders, epigenetic modifications alter the expression of oncogenes and tumor suppressor genes driving tumorigenesis and perhaps play a more central role in the evolution and progression of this disease. Here, we described the current knowledge regarding homeobox gene DNA methylation in human cancer and describe its relevance in the diagnosis, therapeutic response and prognosis of different types of human cancers.

The homeobox encodes a DNA-binding domain found in transcription factors regulating key developmental processes. The most notable examples of homeobox containing genes are the Hox genes, arranged on chromosomes in the same order as their expression domains along the body axis. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Here we report the discovery of a conserved structural signature of the 180-base pair DNA fragment comprising the homeobox. We demonstrate that the homeobox DNA has a characteristic 3-base-pair periodicity in the hydroxyl radical cleavage pattern. This periodic pattern is significant in most of the 39 mammalian Hox genes and in other homeobox-containing transcription factors. The signature is present in segmented bilaterian animals as evolutionarily distant as humans and flies. It remains conserved despite the fact that it would be disrupted by synonymous mutations, which raises the possibility of evolutionary selective pressure acting on the structure of the coding DNA. The homeobox coding DNA may therefore have a secondary function, possibly as a regulatory element. The existence of such element may have important consequences for understanding how these genes are regulated.

Homeobox transcription factors are well known regulators of plant growth and development. In this study, we carried out functional analysis of two candidate stress-responsive HD-ZIP I class homeobox genes from rice, OsHOX22, and OsHOX24. These genes were highly up-regulated under various abiotic stress conditions at different stages of rice development, including seedling, mature and reproductive stages. The transcript levels of these genes were enhanced significantly in the presence of plant hormones, including abscisic acid (ABA), auxin, salicylic acid, and gibberellic acid. The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus. Electrophoretic mobility shift assay established the binding of these homeobox proteins with specific DNA sequences, AH1 (CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG). Transactivation assays in yeast revealed the transcriptional activation potential of full-length OsHOX22 and OsHOX24 proteins. Homo- and hetero-dimerization capabilities of these proteins have also been demonstrated. Further, we identified putative novel interacting proteins of OsHOX22 and OsHOX24 via yeast-two hybrid analysis. Over-expression of OsHOX24 imparted higher sensitivity to stress hormone, ABA, and abiotic stresses in the transgenic Arabidopsis plants as revealed by various physiological and phenotypic assays. Microarray analysis revealed differential expression of several stress-responsive genes in transgenic lines as compared to wild-type. Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways. Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses.

Homeobox genes represent a family of highly conserved transcription factors originally discovered to regulate organ patterning during development. More recently, several homeobox genes were shown to affect processes in adult tissue, including angiogenesis and wound healing. Whereas a subset of members of the Hox-family of homeobox genes activate growth and migration to promote angiogenesis or wound healing, other Hox genes function to restore or maintain quiescent, differentiated tissue function. Pathological tissue remodeling is linked to differential expression of activating or stabilizing Hox genes and dysregulation of Hox expression can contribute to disease progression. Studies aimed at understanding the role and regulation of Hox genes have provided insight into how these potent morphoregulatory genes can be applied to enhance tissue engineering or limit cancer progression.

To develop in vivo assays for homeobox gene function in neural development, we generated transgenic mice in which the expression of a homeobox gene is altered only within the nervous system, in neurons or neuronal precursor cells. Transgenic expression of Hoxc8 did not result in gross abnormalities, while a Hoxd4 transgene caused death shortly after birth. In neural progenitor cells, the motorneuron-specific homeodomain transcription factor Isl1 induced early developmental defects, including absence of anterior neural structures, profound defects in the neuroepithelium and defective neural tube closure. A fraction of Isl1 transgenic mice exhibited spina bifida. Isl1 transgene expression was also associated with decreased proliferation and increased Pbx1 expression in the ventral neural tube. Our results suggest a function for some homeobox genes in development of the nervous system, and that cell-type- and region-specific transgenic models will be useful to identify the cellular and molecular targets of homeobox transcription factors in nervous system development.

Human adenovirus type 5 (HAdV5) E4orf6 (early region 4 open reading frame 6 protein) is a multifunctional early viral protein promoting efficient replication and progeny production. E4orf6 complexes with E1B-55K to assemble cellular proteins into a functional E3 ubiquitin ligase complex that not only mediates proteasomal degradation of host cell substrates but also facilitates export of viral late mRNA to promote efficient viral protein expression and host cell shutoff. Recent findings defined the role of E4orf6 in RNA splicing independent of E1B-55K binding. To reveal further functions of the early viral protein in infected cells, we used a yeast two-hybrid system and identified the homeobox transcription factor HoxB7 as a novel E4orf6-associated protein. Using a HoxB7 knockdown cell line, we observed a positive role of HoxB7 in adenoviral replication. Our experiments demonstrate that the absence of HoxB7 leads to inefficient viral progeny production, as HAdV5 gene expression is highly regulated by HoxB7-mediated activation of various adenoviral promoters. We have thus identified a novel role of E4orf6 in HAdV5 gene transcription via regulation of homeobox protein-dependent modulation of viral promoter activity.

Homeobox genes encode transcription factors that regulate diverse developmental events. The largest known homeobox gene cluster – the X-linked mouse reproductive homeobox (Rhox) cluster – harbors genes whose expression patterns and functions are largely unknown. Here, we report that a member of this cluster,Rhox10, is expressed in male germ cells.Rhox10is highly transcribed in spermatogoniain vivoand is upregulated in response to the differentiation-inducing agent retinoic acidin vitro. Using a specific RHOX10 antiserum that we generated, we found that RHOX10 protein is selectively expressed in fetal gonocytes, germline stem cells, spermatogonia, and early spermatocytes. RHOX10 protein undergoes a dramatic shift in subcellular localization as germ cells progress from mitotically arrested gonocytes to mitotic spermatogonia and from mitotic spermatogonia to early meiotic spermatocytes, consistent with RHOX10 performing different functions in these stages.

LAY ABSTRACT—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. SCIENTIFIC ABSTRACT—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-byage interaction term, p=0.03), and driven by genotype-dependent differences in the rate of bilateral

Despite evidence that consuming alcohol during pregnancy is harmful for the developing fetus, more than 10% of women in the United States report at least some alcohol use during pregnancy (Tan et al., 2015). The prevalence of fetal alcohol syndrome in the United States ranges from 0.5 to 2.0 per 1,000 live births in the general population to 1.4 to 9.8 per 1,000 live births in high‐risk groups (May and Gossage, 2001). Based on active case ascertainment methods, the prevalence of the entire continuum of fetal alcohol spectrum disorder (FASD) has been estimated to be as high as 24 to 48 per 1,000 live births, or 2 to 5% in the United States (May et al., 2014). However, the identification of children adversely affected by prenatal alcohol exposure (PAE) can be difficult because many of them do not exhibit any of the physical features associated with PAE and mainly manifest with neurocognitive deficits later in life. In the absence of the physical features and without accurate information on PAE, it is difficult to make a diagnosis of FASD. While ethanol (EtOH) biomarkers, as an objective measure, can help to overcome the limitations of self‐report on alcohol use during pregnancy, none of the existing biomarkers of alcohol consumption are 100% sensitive or specific (Bakhireva and Savage, 2011). Given these limitations, the identification of more sensitive and specific biomarkers that either alone or in combination with other clinical and self‐report measures can provide more accurate information on PAE would result in better identification of children with FASD and increase the opportunities for earlier diagnosis and interventions. MicroRNAs (miRNAs) are approximately 22‐nucleotide single‐stranded non‐protein‐coding RNA molecules that serve primarily to silence the expression of mRNA transcripts to which they hybridize by base pairing. miRNA biogenesis involves the processing of primary miRNA transcripts (pri‐miRNA) to hairpin precursor structures (pre‐miRNA), which are then exported to the cytoplasm where either strand may be loaded into the RNA‐induced silencing complex (RISC). The strand previously called the miRNA star (miRNA*) was generally thought to be rapidly degraded. However, miRNA* sequences have been found in RISC and shown to impact disease states such as cancer (Jazdzewski et al., 2009). The nomenclature is now changing to describe from which arm of the precursor the miRNA originates, 5′ (5p) or 3′ (3p). Mature miRNAs can bind thousands of transcripts in a cell, making them “master regulators” of gene expression (Bartel, 2004). Moreover, miRNAs have been shown to control many complex biological processes, from embryonic development to the promotion (or inhibition) of various pathological conditions, including cancers and neurodevelopmental disorders (Sayed and Abdellatif, 2011). Recent animal model and cell culture studies suggest that miRNAs play an important role in the mechanisms underlying the deleterious effects of PAE (Balaraman et al., 2013). EtOH suppresses miRs‐9, ‐21, ‐153, and ‐335 in cultured fetal mouse neural stem cells, coordinately regulating genes that make cells resistant to apoptosis, with increased proliferation and aberrant differentiation (Sathyan et al., 2007). miRs‐9(‐5p), 9*(‐3p), and ‐153 are repressed by EtOH exposure in the developing zebrafish brain (Tal et al., 2012). EtOH exposure in the developing mouse brain increases the expression of miRs‐10a and ‐10b, with a concomitant decrease in the target gene homeobox A1 (HOXA1; Wang et al., 2009), which is essential for normal embryonic development. Alterations in miRNA expression were also found in primary neuronal cultures from the cortex of mice at embryonic day 15 following chronic intermittent EtOH exposure and withdrawal (Guo et al., 2012). Finally, differences in miRNA expression were seen in the amygdala and striatum of rats at postnatal day 42 that were exposed to EtOH prenatally and subjected to social enrichment training postnatally (Ignacio et al., 2014). In addition to working inside the cell, miRNAs can be secreted and transferred between cells, indicating that they participate in cell‐to‐cell communication (Valadi et al., 2007). miRNAs are present not only in blood plasma and serum but also in a variety of cell‐free body fluids including urine and saliva, making them attractive as potential biomarkers. In the circulation, they are found in vesicles such as exosomes, microvesicles, and apoptotic bodies (Valadi et al., 2007; Zernecke et al., 2009) as well as in association with proteins (Vickers et al., 2011). Because of their association with vesicles and proteins, circulating miRNAs are resistant to degradation and can be accurately measured even when present at very low levels (Mitchell et al., 2008). Thus, circulating miRNAs have great promise as biomarkers of a variety of disease conditions from cancer to myocardial infarction (Etheridge et al., 2011). Although extracellular miRNAs have been investigated in animal models of PAE (Balaraman et al., 2014), the utility of these biomarkers in humans has not been reported thus far. The goal of the present study was to identify alterations in miRNA expression in maternal serum that could predict maternal alcohol consumption in pregnant women and further elucidate the mechanisms of fetal alcohol effects.

MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.

The mammary gland, the unique organ that primarily form at puberty, is an ideal model to study the functions of homeobox (HB) genes in both development and tumorigenesis. HB genes comprise a large family of developmental regulators that have a critical role in cell growth and differentiation. In the normal mammary gland, homeobox genes are involved in ductal formation, epithelial branching, and lobulo-alveolar development by regulating epithelial proliferation and differentiation. The HB genes are controlled in a spatial and temporal manner in both stromal and epithelial cells. They are coordinately regulated by hormones and extracellular matrix, suggesting that many signaling pathways are involved in homeobox gene functions. When homeobox genes are misexpressed in animal models, different defects are displayed in mammary gland development. Aberrant expression of homeobox genes, overexpressed or downregulated, is found in primary carcinomas and in breast cancer. The Otx1 HB gene is a classic regulatory of nervous system development during embryogenesis. Postnatally Otx1 is transcribed in the anterior pituitary gland, where activates transcription of the pituitary hormones, and plays a role in hematopoiesis, enhancing pluripotent cells, and erythroid differentiation. Otx1 can still be detected in mature cells of the erythroid and megacaryocytic lineage. During cyclical development of mammary gland, the Otx1 gene is overexpressed in lactation, confirming a role of this transcription factor in cell differentiation. Recent studies report that Otx1 is overexpressed in breast cancer. Otx1 is expressed during embryogenesis, and it is expressed again during carcinogenesis, implying its possible function in differentiation of neoplastic cells.

Background/Aims: Midgut formation in Drosophilamelanogasteris dependent upon the integrity of a signaling loop in the endoderm which requires the TGFβ-related peptide, Decapentaplegic, and the Hoxtranscription factor, Labial. Interestingly, although Labial-like homeobox genes are present in mammals, their participation in endoderm morphogenesis is not clearly understood. Methods: We report the cloning, expression, localization, TGFβ inducibility, and biochemical properties of the mammalian Labial-like homeobox, HoxA1, in exocrine pancreatic cells that are embryologically derived from the gut endoderm. Results: HoxA1 is expressed in pancreatic cell populations as two alternatively spliced messages, encoding proteins that share their N-terminal domain, but either lack or include the homeobox at the C-terminus. Transcriptional regulatory assays demonstratethat the shared N-terminal domain behaves as a strong transcriptional activator in exocrine pancreatic cells. HoxA1 is an early response gene for TGFβ 1 in pancreatic epithelial cell populations and HoxA1 protein co-localizes with TGFβ 1 receptors in the embryonic pancreatic epithelium at a time when exocrine pancreatic morphogenesis occurs (days E16 and E17). Conclusions: These results report a role for HoxA1 in linking TGFβ-mediated signaling to gene expression in pancreatic epithelial cell populations, thus suggesting a high degree of conservation for a TGFβ/labial signaling loop in endoderm-derived cells between Drosophila and mammals.