Hox Proteins Research Articles

An update on epigenetic mechanisms in endometriosis.

The etiopathogenesis of endometriosis, a chronic debilitating disease affecting nearly 10% of women, has evaded elucidation until the recent epigenetic discoveries. Although still deemed multifactorial, endometriosis is likely predisposed in women with genetic and epigenetic alterations, which are activated by environmental factors. There are many epigenetic changes that have recently been associated with endometriosis: DNA methylation and phosphorylation, modifications to histones and non-coding RNA, and chromatin remodeling and organization. Gene markers, such as HOXA10, SF-1, and GATA transcription factors, are also debatably correlated to endometriosis. An improved understanding of the etiopathogenesis of endometriosis may propel our field toward our objectives: sooner and more efficient detection as well as targeted therapy. In this comprehensive review, we will identify and discuss the current literature on epigenetic changes seen in endometriosis. A primary computerized search was performed on PubMed and Google scholar of publications from 1990 to 2022. We searched for keyword terms such as "endometriosis" and "endometriosis epigenetics." We also looked through the references of prior articles to find other relevant articles to this topic. Articles were categorized by type of epigenetic change found such as DNA hypo- or hyper- methylation, histone hyper- or hypo-acetylation, chromatin remodeling, and non-coding RNA-mediated down-regulation and the research was elaborated in sections based on the type of epigenetic change. There are many articles on TET, DNMTs, EZH2, HDACs, HATs, let-7 family, miRNAs, Hox proteins, GATA family, sirtuins (e.g. SIRT1, SIRT3), ARID1A, SF-1, USF1, USF2, STRA6, ESR1, ESR2, PGR, ALDHIA2, and CTCF; however, the studies analyzed in this review were heterogeneous in comparison populations, analysis methods, tissues types (e.g. endometriomas, ectopic endometriotic tissue, eutopic endometrial tissue). Due to this, it is difficult to synthesize over-arching conclusions based on the current literature; however, there are many epigenetic changes and genes linked to endometriosis as noted in the literature.

The Hox protein Antennapedia orchestrates Drosophila adult flight muscle development.

Muscle development and diversity require a large number of spatially and temporally regulated events controlled by transcription factors (TFs). Drosophila has long stood as a model to study myogenesis due to the highly conserved key TFs involved at all stages of muscle development. While many studies focused on the diversification of Drosophila larval musculature, how distinct adult muscle types are generated is much less characterized. Here, we identify an essential regulator of Drosophila thoracic flight muscle development, the Hox TF Antennapedia (Antp). Correcting a long-standing belief that flight muscle development occurs without the input of Hox TFs, we show that Antp intervenes at several stages of flight muscle development, from the establishment of the progenitor pool in the embryo to myoblast differentiation in the early pupa. Furthermore, the precisely regulated clearance of Hox in the developing flight muscle fibers is required to allow for fibrillar muscle fate diversification, setting these muscles apart from all other adult tubular muscle types.

Multimodal Hox5 activity generates motor neuron diversity

Motor neurons (MNs) are the final output of circuits driving fundamental behaviors, such as respiration and locomotion. Hox proteins are essential in generating the MN diversity required for accomplishing these functions, but the transcriptional mechanisms that enable Hox paralogs to assign distinct MN subtype identities despite their promiscuous DNA binding motif are not well understood. Here we show that Hoxa5 modifies chromatin accessibility in all mouse spinal cervical MN subtypes and engages TALE co-factors to directly bind and regulate subtype-specific genes. We identify a paralog-specific interaction of Hoxa5 with the phrenic MN-specific transcription factor Scip and show that heterologous expression of Hoxa5 and Scip is sufficient to suppress limb-innervating MN identity. We also demonstrate that phrenic MN identity is stable after Hoxa5 downregulation and identify Klf proteins as potential regulators of phrenic MN maintenance. Our data identify multiple modes of Hoxa5 action that converge to induce and maintain MN identity.

Genome-wide identification of DNA methylation marks associated with resistance to Streptococcus agalactiae in the GIFT strain of Nile Tilapia (Oreochromis niloticus)

Epigenetic mechanism plays a pivotal role in the immune responses of fish. Streptococcus agalactiae (SA) infection is a significant challenge to tilapia aquaculture. DNA methylation involved in immune responses in tilapia against SA is unclear. Here, by using a QTLseq strategy integrated with whole-genome bisulfite sequencing-based DNA methylation analysis (methyl-QTLseq) and RNA sequencing (RNA-seq), we identified 3608 differentially methylated regions (DMRs) and 4355 differentially expressed genes (DEGs) in the spleen of Nile tilapia Oreochromis niloticus. Compared to the susceptible group, the up-regulated DMRs (2449) were approximately twice as many as the down-regulated DMRs (1159). Compared to the susceptible group, lower methylation levels for CpG, CHG and CHH in TEs and for CHG and CHH in genes and significant decreased expression for 8 of the 11 DNA methyltransferase genes in the resistant group were detected. 46 DEGs with differentially methylated promoter regions (DMPs) were significantly enriched in immune processes such as T cell differentiation and adaptive immunity. We detected 4 methyl-QTLs located on LG8:5332501–5,336,400, LG14:2307301–2,353,500, LG16:16889101–16,903,500, and LG22:25238401–2,536,880 across the tilapia genome. The methyl-QTL region located within the chromosome LG22:25238401–25,368,800 contains the most DMRs (91) and DMGs (7). Interestingly, most of the annotated genes that located within or under the peak of the methyl-QTL interval belong to the HOX protein family including hoxa1, hoxa2, hoxa4a and hoxa5a. Bisulfite sequencing PCR and expression analysis indicated that DNA methylation in the promoter regions of hoxa4a, lcp2a, and ccr9a regulated gene expression in spleen in response to SA infection. Overall, our study provides epigenetics evidences involving in immune responses against SA infection in tilapia.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Affinity-optimizing enhancer variants disrupt development

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1–3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4–6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

The Impact of Early Genes and Gene Editing Technology Associated with Clustered Repeats on the Development of Human Body Organs

Early growth response 1 (EGR-1) and homeobox (Hox) proteins play crucial roles in regulating genes within the body, promoting cell renewal, and expediting the wound healing process. Conversely, Clustered Regularly Interspaced Palindromic Re-peats/Associated (CRISPR-Cas) technology is primarily involved in gene editing. However, significant gaps persist in understanding the mechanisms of key genes like EGR-1, CRISPR/Cas, and HOX genes in organ development. Unveiling their mechanisms is crucial for advancing organ development and discovering new therapeutic strategies. This study aims to investigate the roles of EGR-1, CRISPR/Cas, and HOX genes in organ development and growth. The study used CRISPR/Cas to investigate the impact of catalase mutations on organ regeneration. Early embryonic mutants were generated by injecting gRNAs and Cas9 protein into zygotes, followed by tail amputation in larvae. Additionally, the study explored the role of specific Hox genes in axon elongation and Wnt signaling regulation. EGR-1, induced by TGF-β1, enhanced collagen production, underscoring its importance in wound healing. Integration of EGR-1, HOX proteins, and CRISPR-Cas revealed a regulatory complex influencing organ development. The integration of EGR-1, HOX proteins, and CRISPR-Cas revealed a regulatory complex. EGR-1 aids wound healing, HOX proteins influence fetal development and organ formation, and CRISPR-Cas enables precise genome modifications.

Hox genes and patterning the vertebrate body.

The diversity of vertebrate body plans is dizzying, yet stunning for the many things they have in common. Vertebrates have inhabited virtually every part of the earth from its coldest to warmest climates. They locomote by swimming, flying, walking, slithering, or climbing, or combinations of these behaviors. And they exist in many different sizes, from the smallest of frogs, fish and lizards to giraffes, elephants, and blue whales. Despite these differences, vertebrates follow a remarkably similar blueprint for the establishment of their body plan. Within the relatively small amount of time required to complete gastrulation, the process through which the three germ layers, ectoderm, mesoderm, and endoderm are created, the embryo also generates its body axis and is simultaneously patterned. For the length of this axis, the genes that distinguish the neck from the rib cage or the trunk from the sacrum are the Hox genes. In vertebrates, there was evolutionary pressure to maintain this set of genes in the organism. Over the past decades, much has been learned regarding the regulatory mechanisms that ensure the appropriate expression of these genes along the main body axes. Genetic functions continue to be explored though much has been learned. Much less has been discerned on the identity of co-factors used by Hox proteins for the specificity of transcriptional regulation or what downstream targets and pathways are critical for patterning events, though there are notable exceptions. Current work in the field is demonstrating that Hox genes continue to function in many organs long after directing early patterning events. It is hopeful continued research will shed light on remaining questions regarding mechanisms used by this important and conserved set of transcriptional regulators.

Stability and dynamics of extradenticle modulates its function

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔGG→A of −2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely compromised by the quenching of its flexibility and enhancement of its stability and DNA-binding affinity.

Myeloid Ecotropic Viral Integration Site (MEIS) Proteins: Illuminating Cancer Diagnosis and Biomarker Significance

Context: The MEIS proteins, originally described as the "helper groups" of HOX proteins, are now becoming recognized as significant and incredibly diverse controllers of cellular behavior. The MEIS agents can incorporate a variety of epigenetic modulators into chromatin to aid in embryonic growth or tissue homeostasis by adult stem cells. The MEIS gene's expression, binding, stability, post-translational protein modification, and controlled intracellular availability are all fully regulated, and they all react to external stimuli. Objectives: We explored the diverse roles of MEIS proteins in cellular behavior and epigenetic regulation and investigated the potential of MEIS proteins as diagnostic and therapeutic biomarkers in cancers. Results: The MEIS proteins can be diagnostic and therapeutic biomarkers for cancer. The MEIS-PBX heterotrimeric is involved in neurogenesis processes, and the formation of the MEIS2 heterodimer with PAX6 and DLX2 can proliferate periplomerular dopaminergic neurons in the olfactory bulb. The estrogen receptor in breast cancer upregulates MEIS1 and FOXP3. The estrogen receptor pathway's cancer-related genes can express themselves more strongly when MEIS1-FOXP3 interacts with a positive feedback mechanism. Because MEIS1 expression is decreased in clear renal cell carcinoma (ccRCC) cell lines, MEIS1 may function as a tumor suppressor in the development of ccRCC. Conclusions: MEIS1 is recognized as one of the main contributors to the development of leukemias. The MEIS proteins influence the c-MYC signaling pathway, cell proliferation, and invasion of PC by contributing to its development.

A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus

BackgroundCells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation.ResultsTo expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other factors that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF).ConclusionsOur foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

Hox gene clusters in the mussel Mytilus coruscus: Implications for bivalves' evolution

Hox genes encode transcription factors that are crucial regulators of various developmental processes, including patterning of the body axis and differentiation of cells, tissues and organs. They are organized in clusters in a wide range of Metazoans and expressed during embryonic development in a collinear manner from the 3′ to the 5′ end of these genomic units. Hox genes are part of the larger homeobox gene super-class, and other gene clusters beyond Hox are being increasingly discovered. This transcriptional dynamic generates spatiotemporal expression patterns throughout the developing body that are required for the establishment of fields of cell identity. Here we suggest that subcluster temporal collinearity may occur during the development of a bivalve species, as demonstrated for other Bilateran species. We performed a genome-wide analysis of the Mytilus coruscus Hox gene family, including gene structure, synteny, and expression patterns. It was shown that 11 Hox genes were evenly distributed within one chromosome in M. coruscus. Phylogenetic analysis showed that 11 Hox proteins were divided into four groups and each of them contained different conserved motifs. Besides, sequence alignment and analysis showed the conservation of 11 Hox genes of M. coruscus. Selection pressure analysis revealed the Ka/Ks ratios of Hox1, Hox4, Lox4, Post2, and Post1 were less than 1 in Bivalvia, indicating that these five Hox genes suffered significant purifying selection. Notably, Hox1, Hox4, Post2 were overlapped with the leading gene in the subcluster collinearity. This discovery suggests the potential existence of subcluster temporal collinearity in M. coruscus, which adds new insights into Hox gene organization in Bivalvia or Mollusca.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology.

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, we further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. We found that repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, we identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. We then used transcription factor (TF) binding motif analysis in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer. We also tested the role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs. We found several TFs that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together our results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology.

Homeobox protein A1-like and DNA methylation regulate embryo-specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori.

DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryonic stages through adulthood, in the nervous system. However, the functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Finally, we identify a two-component design principle for homeostatic control of Hox gene expression in adult MNs: Hox transcriptional autoregulation is counterbalanced by negative UNC-3 feedback. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

Specificity of the Hox member Deformed is determined by transcription factor levels and binding site affinities

Hox proteins have similar binding specificities in vitro, yet they control different morphologies in vivo. This paradox has been partially solved with the identification of Hox low-affinity binding sites. However, anterior Hox proteins are more promiscuous than posterior Hox proteins, raising the question how anterior Hox proteins achieve specificity. We use the AP2x enhancer, which is activated in the maxillary head segment by the Hox TF Deformed (Dfd). This enhancer lacks canonical Dfd-Exd sites but contains several predicted low-affinity sites. Unexpectedly, these sites are strongly bound by Dfd-Exd complexes and their conversion into optimal Dfd-Exd sites results only in a modest increase in binding strength. These small variations in affinity change the sensitivity of the enhancer to different Dfd levels, resulting in perturbed AP-2 expression and maxillary morphogenesis. Thus, Hox-regulated morphogenesis seems to result from the co-evolution of Hox binding affinity and Hox dosage for precise target gene regulation.

Characterizing Hox genes in mayflies (Ephemeroptera), with Hexagenia limbata as a new mayfly model

BackgroundHox genes are key regulators of appendage development in the insect body plan. The body plan of mayfly (Ephemeroptera) nymphs differs due to the presence of abdominal appendages called gills. Despite mayflies’ phylogenetic position in Paleoptera and novel morphology amongst insects, little is known of their developmental genetics, such as the appendage-regulating Hox genes. To address this issue we present an annotated, early instar transcriptome and embryonic expression profiles for Antennapedia, Ultrabithorax, and Abdominal A proteins in the mayfly Hexagenia limbata, identify putative Hox protein sequences in the mayflies H. limbata, Cloeon dipterum, and Ephemera danica, and describe the genomic organization of the Hox gene cluster in E. danica.ResultsTranscriptomic sequencing of early instar H. limbata nymphs yielded a high-quality assembly of 83,795 contigs, of which 22,975 were annotated against Folsomia candida, Nilaparvata lugens, Zootermopsis nevadensis and UniRef90 protein databases. Homeodomain protein phylogeny and peptide annotations identified coding sequences for eight of the ten canonical Hox genes (excluding zerknüllt/Hox3 and fushi tarazu) in H. limbata and C. dipterum, and all ten in E. danica. Mayfly Hox protein sequences and embryonic expression patterns of Antp, Ubx, and Abd-A appear highly conserved with those seen in other non-holometabolan insects. Similarly, the genomic organization of the Hox cluster in E. danica resembles that seen in most insects.ConclusionsWe present evidence that mayfly Hox peptide sequences and the embryonic expression patterns for Antp, Ubx, and Abd-A are extensively conserved with other insects, as is organization of the mayfly Hox gene cluster. The protein data suggest mayfly Antp, Ubx, and Abd-A play appendage promoting and repressing roles during embryogenesis in the thorax and abdomen, respectively, as in other insects. The identified expression of eight Hox genes, including Ubx and abd-A, in early instar nymphs further indicates a post-embryonic role, possibly in gill development. These data provide a basis for H. limbata as a complementary Ephemeridae model to the growing repertoire of mayfly model species and molecular techniques.

Hox genes collaborate with helix-loop-helix factor Grainyhead to promote neuroblast apoptosis along the anterior-posterior axis of the Drosophila larval central nervous system.

Hox genes code for a family of a homeodomain containing transcription factors that use TALE-HD containing factors Pbx/Exd and Meis/Homothorax to specify the development of the anterior-posterior axis of an organism. However, the absence of TALE-HD containing factors from specific tissues emphasizes the need to identify and validate new Hox cofactors. In Drosophila central nervous system, Hox executes segment-specific apoptosis of neural stem cells (neuroblasts) and neurons. In abdominal segments of larval central nervous system, Hox gene Abdominal-A mediates neuroblast apoptosis with the help of Extradenticle and bHLH factor Grainyhead using a 717-bp apoptotic enhancer. In this study, we show that this enhancer is critical for abdominal neuroblast apoptosis and relies on 2 separable set of DNA-binding motifs responsible for its initiation and maintenance. Our results also show that Abdominal-A and Grainyhead interact through their highly conserved DNA-binding domains, and the DNA-binding specificity of Abdominal-A-homeodomain is important for it to interact with Grainyhead and essential for it to execute neuroblast apoptosis in central nervous system. We also establish that Grainyhead is required for Hox-dependent neuroblast apoptosis in Labial and Sex Combs Reduced expressing regions of the central nervous system, and it can physically interact with all the Hox proteins in vitro. Our biochemical and functional data collectively support the idea that Grainyhead can function as a Hox cofactor and help them carry out their in vivo roles during development.