Homeobox Research Articles

Clinical Case of Rare Genetic Disorder (Proud Syndrome) in a Child

Background. Proud syndrome is rare inherited disease with X-linked inheritance associated with mutations in the homeobox gene ARX. Typical clinical signs of this syndrome are severe mental retardation, intractable epilepsy, agenesis (dysgenesis) of corpus callosum. Less common features are genital abnormalities, microcephaly, facial dysmorphia, and skeletal malformations. Clinical case description. The article describes the clinical findings of Proud syndrome in girl A., admitted to Children’s City Clinical Hospital No. 1 in Nizhny Novgorod. The girl was born without asphyxia at term through natural vaginal delivery after the first uneventful pregnancy. Body weight at birth was 2600 g. The genealogical history is burdened by the presence of epilepsy in girl’s father relatives. There were no deviations in psychomotor skills development before disease onset. Hemi-convulsive seizures (switching sides) have appeared at the age of 6 months. These attacks had status course and were resistant to anticonvulsant therapy. Neuroimaging has revealed agenesis of corpus callosum. Regression of psychomotor development, new behavioral disorders (stereotypes and auto-aggression), hyperexcitability, and sleep disorders were observed in dynamics. The diagnosis of Proud syndrome was confirmed by identification of probably pathogenic mutation in the ARX gene (c.1111C>T, p. Arg 371*64). The features of anticonvulsant management were demonstrated in the patient. Conclusion. This clinical case presents typical clinical picture of Proud syndrome. The disease is non-curable. Such patients should be administered with syndromic therapy: constant anticonvulsant therapy, correction of behavioral disorders (classes with specialist on mental defects), neurologist, epileptologist, psychiatrist observations. Parental examination is crucial ARX mutation search in order to determine the prognosis for further child-bearing (parents refused to perform genetic study at the time of article writing).

Hox Gene Collinearity with Pulling Physical Forces Creates a Hox Gene Clustering in Embryos of Vertebrates and Invertebrates: Complete or Split Clusters

Hox gene clusters are crucial in embryogenesis. It was observed that some Hox genes are located in order along the telomeric to centromeric direction of the DNA sequence: Hox1, Hox2, Hox3…. These genes are expressed in the same order in the ontogenetic units of the Drosophila embryo along the anterior–posterior axis. The two entities (genome and embryo) differ significantly in linear size and in-between distance. This strange phenomenon was named spatial collinearity (SP). Later, it was observed that, particularly in the vertebrates, a temporal collinearity (TC) coexists: first Hox1 is expressed, later Hox2, and later on Hox3…. According to a biophysical model (BM), pulling forces act at the anterior end of the cluster while a cluster fastening applies at the posterior end. Hox clusters are irreversibly elongated along the force direction. During evolution, the elongated Hox clusters are broken at variable lengths, thus split clusters may be created. An empirical rule was formulated, distinguishing development due to a complete Hox cluster from development due to split Hox clusters. BM can explain this empirical rule. In a spontaneous mutation, where the cluster fastening is dismantled, a weak pulling force automatically shifts the cluster inside the Hox activation domain. This cluster translocation can probably explain the absence of temporal collinearity in Drosophila.

Genome-wide analysis of the poplar homeobox gene family and the identification of PtHB180 as an important regulator of plant developmental processes and drought stress response

Homeobox (HB) transcription factors have been reported to regulate various developmental processes and hormone signaling and mediate plant adaption to abiotic stresses, including drought. As a major subfamily of HB family, KNOX genes have been widely studied in herbaceous plants. However, the behaviors of KNOX genes in regulating plant developmental processes and stress resistance in woody plants remain poorly understood. Here, we found a KNOX family gene in the model tree species Populus trichocarpa (hereafter “poplar”), PtHB180, which enhances drought tolerance by regulating developmental processes through auxin and gibberellin pathways and mediating the ABA signaling pathway leading to rapid stomatal closure. Overexpression lines of PtHB180 (PtHB180-OE) displayed pleiotropic phenotypes, including increased plant height and decreased leaf area by affecting auxin and/or gibberellin contents and signaling. PtHB180-OE plants also displayed improved drought tolerance under short- and long-term drought stress. Results of transcriptome sequencing suggested the involvement of genes related to ABA signaling in regulating drought tolerance in PtHB180-OE. Specifically, PtHB180-OE plants showed significantly enhanced stomatal sensitivity to exogenous ABA. Taken together, our results indicate that PtHB180 enhances drought tolerance in poplar via regulating plant architecture and mediating ABA signaling to close stomata. Therefore, PtHB180 can be used as a potential candidate gene to improve drought tolerance by genetic transformation in poplar and related plant species.

Genomic analysis of a spontaneous unifoliate mutant reveals gene candidates associated with compound leaf development in Vigna unguiculata [L] Walp

Molecular mechanisms which underpin compound leaf development in some legumes have been reported, but there is no previous study on the molecular genetic control of compound leaf formation in Vigna unguiculata (cowpea), an important dryland legume of African origin. In most studied species with compound leaves, class 1 KNOTTED-LIKE HOMEOBOX genes expressed in developing leaf primordia sustain morphogenetic activity, allowing leaf dissection and the development of leaflets. Other genes, such as, SINGLE LEAFLET1 in Medicago truncatula and Trifoliate in Solanum lycopersicum, are also implicated in regulating compound leaf patterning. To set the pace for an in-depth understanding of the genetics of compound leaf development in cowpea, we applied RNA-seq and whole genome shotgun sequence datasets of a spontaneous cowpea unifoliate mutant and its trifoliate wild-type cultivar to conduct comparative reference-based gene expression, de novo genome-wide isoform switch, and genome variant analyses between the two genotypes. Our results suggest that genomic variants upstream of LATE ELONGATED HYPOCOTYL and down-stream of REVEILLE4, BRASSINOSTERIOD INSENSITIVE1 and LATERAL ORGAN BOUNDARIES result in down-regulation of key components of cowpea circadian rhythm central oscillator and brassinosteroid signaling, resulting in unifoliate leaves and brassinosteroid-deficient-like phenotypes. We have stated hypotheses that will guide follow-up studies expected to provide more insights.

Msx genes delineate a novel molecular map of the developing cerebellar neuroepithelium.

In the early cerebellar primordium, there are two progenitor zones, the ventricular zone (VZ) residing atop the IVth ventricle and the rhombic lip (RL) at the lateral edges of the developing cerebellum. These zones give rise to the several cell types that form the GABAergic and glutamatergic populations of the adult cerebellum, respectively. Recently, an understanding of the molecular compartmentation of these zones has emerged. To add to this knowledge base, we report on the Msx genes, a family of three transcription factors, that are expressed downstream of Bone Morphogenetic Protein (BMP) signaling in these zones. Using fluorescent RNA in situ hybridization, we have characterized the Msx (Msh Homeobox) genes and demonstrated that their spatiotemporal pattern segregates specific regions within the progenitor zones. Msx1 and Msx2 are compartmentalized within the rhombic lip (RL), while Msx3 is localized within the ventricular zone (VZ). The relationship of the Msx genes with an early marker of the glutamatergic lineage, Atoh1, was examined in Atoh1-null mice and it was found that the expression of Msx genes persisted. Importantly, the spatial expression of Msx1 and Msx3 altered in response to the elimination of Atoh1. These results point to the Msx genes as novel early markers of cerebellar progenitor zones and more importantly to an updated view of the molecular parcellation of the RL with respect to the canonical marker of the RL, Atoh1.

HOXB5 promotes the progression and metastasis of osteosarcoma cells by activating the JAK2/STAT3 signalling pathway

ObjectiveTo investigate the involvement of the homeobox gene B5 (HOXB5) in the progression and metastasis of osteosarcoma. MethodsThe expression of HOXB5 in human osteosarcoma tissues and its correlation with clinical indicators were investigated using bioinformatics analysis and immunohistochemical labelling. Human osteosarcoma cells (HOS, MG63, U2OS, and Saos-2) and normal human osteoblasts (hFOB1.19) were cultivated. The expression of HOXB5 in these cells was detected using western blotting (WB) and RT‒PCR. Two cell lines exhibiting elevated HOXB5 expression were chosen and divided into three groups: the blank group (mock), control group (control) and transfection group (shHOXB5). The transfection group was infected with lentivirus expressing shRNAs targeting HOXB5. The transfection efficiency was detected by WB. Cell proliferation suppression was measured by CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays; the percentage of apoptotic cells was determined by flow cytometry; and cell migration and invasion were detected via the Transwell chamber test. WB was utilized to determine the protein expression of genes linked to metastasis (MMP2, MMP9), apoptosis (Bax, Bcl-2), and the JAK2/STAT3 pathway (JAK2, p-JAK2, STAT3, p-STAT3). ResultsIn osteosarcoma tissues, HOXB5 expression was elevated and strongly correlated with distant metastasis. Silencing HOXB5 reduced the proliferation, migration and invasion of osteosarcoma cells; prevented the progression and metastasis of tumours in tumour-bearing nude mice; and reduced the activation of key proteins in the JAK2/STAT3 signalling pathway. ConclusionThrough the JAK2/STAT3 signalling pathway, HOXB5 plays a crucial role in the malignant progression of osteosarcoma and is a promising target for osteosarcoma treatment.

Desflurane alleviates LPS-induced acute lung injury by modulating let-7b-5p/HOXA9 axis.

Acute lung injury (ALI) is characterized by acute respiratory failure with tachypnea and widespread alveolar infiltrates, badly affecting patients' health. Desflurane (Des) is effective against lung injury. However, its mechanism in ALI remains unknown. BEAS-2B cells were incubated with lipopolysaccharide (LPS) to construct an ALI cell model. Cell apoptosis was evaluated using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was employed to examine the levels of inflammatory cytokines. Interactions among let-7b-5p, homeobox A9 (HOXA9), and suppressor of cytokine signaling 2 (SOCS2) were verified using Dual luciferase activity, chromatin immunoprecipitation (ChIP), and RNA pull-down analysis. All experimental data of this study were derived from three repeated experiments. Des treatment improved LPS-induced cell viability, reduced inflammatory cytokine (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6)) levels, decreased cell apoptosis, down-regulated the pro-apoptotic proteins (Bcl-2-associated X protein (Bax) and cleaved caspase 3) expression, and up-regulated the anti-apoptotic protein B-cell-lymphoma-2 (Bcl-2) expression in LPS-induced BEAS-2B cells. Des treatment down-regulated let-7b-5p expression in LPS-induced BEAS-2B cells. Moreover, let-7b-5p inhibition improved LPS-induced cell injury. let-7b-5p overexpression weakened the protective effects of Des. Mechanically, let-7b-5p could negatively modulate HOXA9 expression. Furthermore, HOXA9 inhibited the NF-κB signaling by enhancing SOCS2 transcription. HOXA9 overexpression weakened the promotion of let-7b-5p mimics in LPS-induced cell injury. Des alleviated LPS-induced ALI via regulating let-7b-5p/ HOXA9/NF-κB axis.

PagWOX11/12a from hybrid poplar enhances drought tolerance by modulating reactive oxygen species

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar.

MicroRNA target homeobox messenger RNA in HIV induced hematopoietic inhibition.

Cytopenias are a common occurrence due to abnormal hematopoiesis persistent in patients suffering from and advancing with HIV/AIDS. In order to develop efficacious therapies against cytopenias, it is necessary to understand the mechanisms by which HIV infection affects the differentiation of hematopoietic stem-progenitor cells (HSPCs), causing hematopoietic inhibition, that leads to hematological disorders. Currently, only the antiretrovirals that are being used to treat HIV infection and indirectly lower the levels of virus replication also co-attenuate cytopenias. The evidence available suggests that this indirect efficacy may not prevail for the lifetime of the infected patients, and the acquired immunodeficiency can overtake the beneficial consequences of decreased virus replication. As cited in this article, we and our colleagues are the first to make a foray into the involvement of microRNAs and their use as potential interventional treatments for the cytopenias that occur with HIV/AIDS. Herein, we progressed further in the direction of the mechanisms of the involvement of homeobox gene regulation to cause cytopenias. We had previously shown that HIV-1 inhibits multi-lineage hematopoiesis of the CD34+ cells using SCID-hu Thy/Liv animals in vivo. Furthermore, we demonstrated that the virus-induced hematopoietic inhibition occurred despite the CD34+ cells being resistant to HIV-1 infection. We set out to search for the specific host factors secreted by CD4+ T-cells that likely participate in the inhibition of hematopoiesis of the HIV infection-resistant CD34+ cells. More recently, we reported the identification of virus-infected CD4+ thymocyte-secreted miRNA-15a and miRNA-24 and that their differential expression following HIV infection causes the indirect inhibition of hematopoiesis. We then hypothesized that the observed miRNA differential expression in the virus-infected T-cells causes the abnormal regulation of homeobox (HOX) gene-encoded transcriptomes in the CD34+ cells, affecting specific MAPK signaling and CD34+ cell fate, thereby disrupting normal hematopoiesis. We present that in HIV infection, miRNA-mediated post-transcriptional dysregulation of HOXB3 mRNA inhibits multi-lineage hematopoiesis, which translates into hematological disorders in virus-infected patients with HIV/AIDS. These observations portend specific microRNA candidates for potential efficacy against the virus-induced cytopenias that are otherwise not treatable by the existing HAART/ART regimens, which are primarily designed and applicable for the attenuation of virus replication.

HOXA-AS2 Epigenetically Inhibits HBV Transcription by Recruiting the MTA1-HDAC1/2 Deacetylase Complex to cccDNA Minichromosome.

Persistent transcription of HBV covalently closed circular DNA (cccDNA) is critical for chronic HBV infection. Silencing cccDNA transcription through epigenetic mechanisms offers an effective strategy to control HBV. Long non-coding RNAs (lncRNAs), as important epigenetic regulators, have an unclear role in cccDNA transcription regulation. In this study, lncRNA sequencing (lncRNA seq) is conducted on five pairs of HBV-positive and HBV-negative liver tissue. Through analysis, HOXA-AS2 (HOXA cluster antisense RNA 2) is identified as a significantly upregulated lncRNA in HBV-infected livers. Further experiments demonstrate that HBV DNA polymerase (DNA pol) induces HOXA-AS2 after establishing persistent high-level HBV replication. Functional studies reveal that HOXA-AS2 physically binds to cccDNA and significantly inhibits its transcription. Mechanistically, HOXA-AS2 recruits the MTA1-HDAC1/2 deacetylase complex to cccDNA minichromosome by physically interacting with metastasis associated 1 (MTA1) subunit, resulting in reduced acetylation of histone H3 at lysine 9 (H3K9ac) and lysine 27 (H3K27ac) associated with cccDNA and subsequently suppressing cccDNA transcription. Altogether, the study reveals a mechanism to self-limit HBV replication, wherein the upregulation of lncRNA HOXA-AS2, induced by HBV DNA pol, can epigenetically suppress cccDNA transcription.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes

BackgroundHox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function.ResultIt found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter.ConclusionTaken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues.

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

Identification and Expression Analysis of the WOX Transcription Factor Family in Foxtail Millet (Setaria italica L.).

WUSCHEL-related homeobox (WOX) transcription factors are unique to plants and play pivotal roles in plant development and stress responses. In this investigation, we acquired protein sequences of foxtail millet WOX gene family members through homologous sequence alignment and a hidden Markov model (HMM) search. Utilizing conserved domain prediction, we identified 13 foxtail millet WOX genes, which were classified into ancient, intermediate, and modern clades. Multiple sequence alignment results revealed that all WOX proteins possess a homeodomain (HD). The SiWOX genes, clustered together in the phylogenetic tree, exhibited analogous protein spatial structures, gene structures, and conserved motifs. The foxtail millet WOX genes are distributed across 7 chromosomes, featuring 3 pairs of tandem repeats: SiWOX1 and SiWOX13, SiWOX4 and SiWOX5, and SiWOX11 and SiWOX12. Collinearity analysis demonstrated that WOX genes in foxtail millet exhibit the highest collinearity with green foxtail, followed by maize. The SiWOX genes primarily harbor two categories of cis-acting regulatory elements: Stress response and plant hormone response. Notably, prominent hormones triggering responses include methyl jasmonate, abscisic acid, gibberellin, auxin, and salicylic acid. Analysis of SiWOX expression patterns and hormone responses unveiled potential functional diversity among different SiWOX genes in foxtail millet. These findings lay a solid foundation for further elucidating the functions and evolution of SiWOX genes.

A Genome-Wide Analysis of the WUSCHEL-Related Homeobox Transcription Factor Family Reveals Its Differential Expression Patterns, Response to Drought Stress, and Localization in Sweet Cherry (Prunus avium L.)

The WUSCHEL-related homeobox (WOX) gene family has a critical effect on plant development and abiotic stress. However, there have been no genome-wide studies on WOX genes within sweet cherry (Prunus avium L.). In the present work, eight PavWOX genes were discovered within sweet cherry at the genome-wide level, and they were mapped to six chromosomes. Based on phylogenetic relationships, these genes were classified into three groups, with genes in one group having similar gene structures and conserved motifs. Meanwhile, the PavWOX genes possessed cis-acting elements and functions associated with hormone responses, stress responses, and development. As revealed by expression patterns, certain PavWOX genes are specifically expressed within tissues, suggesting that they may have unique functions. Additionally, the gene family expression patterns under drought stress were analyzed. PavWOX4, PavWOX5, PavWOX13A, and PavWOX13B had increased expressions upon drought stress. In addition, the transcription factor of PavWOX4 and PavWOX13A was localized in the nucleus, confirming the estimated results. Our findings lay the foundation for determining the expression patterns and functions of the PavWOX gene family within sweet cherry and shed more light on the underlying regulatory mechanisms.

Rewiring of a KNOXI regulatory network mediated by UFO underlies the compound leaf development in Medicago truncatula

Class I KNOTTED-like homeobox (KNOXI) genes are parts of the regulatory network that control the evolutionary diversification of leaf morphology. Their specific spatiotemporal expression patterns in developing leaves correlate with the degrees of leaf complexity between simple-leafed and compound-leafed species. However, KNOXI genes are not involved in compound leaf formation in several legume species. Here, we identify a pathway for dual repression of MtKNOXI function in Medicago truncatula. PINNATE-LIKE PENTAFOLIATA1 (PINNA1) represses the expression of MtKNOXI, while PINNA1 interacts with MtKNOXI and sequesters it to the cytoplasm. Further investigations reveal that UNUSUAL FLORAL ORGANS (MtUFO) is the direct target of MtKNOXI, and mediates the transition from trifoliate to pinnate-like pentafoliate leaves. These data suggest a new layer of regulation for morphological diversity in compound-leafed species, in which the conserved regulators of floral development, MtUFO, and leaf development, MtKNOXI, are involved in variation of pinnate-like compound leaves in M. truncatula.

Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening intomato fruit.

Knotted1-like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA-seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein-protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1-clade of BEL1-like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9-mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5-SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast- and chlorophyll-related genes, including SlAPRR2-like1, SlCAB-1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5-SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine-tuning the color transition from immature green fruit to mature red fruit.

Helicobacter pylori infection induces gastric cancer cell malignancy by targeting HOXA-AS2/miR-509-3p/MMD2 axis.

Helicobacter pylori (Hp) infection is considered to be the strongest risk factor for gastric cancer (GC). Long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) has been indicated to be significantly related to Hp infection in GC patients. To investigate the detailed role and molecular mechanism of lncRNA HOXA-AS2 in Hp-induced GC. GC cells were treated with Hp filtrate for cell infection. Bioinformatics tools were utilized for survival analysis and prediction of HOXA-AS2 downstream molecules. Western blotting and RT-qPCR were utilized for assessing protein and RNA levels, respectively. Flow cytometry, colony formation and CCK-8 assays were implemented for testing HOXA-AS2 functions in Hp-infected GC cells. HOXA-AS2 localization in cells was determined by subcellular fractionation assay. The relationship between RNAs were measured by luciferase reporter assay. Hp infection induced HOXA-AS2 upregulation in GC cells. Knocking down HOXA-AS2 restrained cell proliferation but promoted cell apoptosis with Hp infection. HOXA-AS2 bound to miR-509-3p, and miR-509-3p targeted monocyte to macrophage differentiation associated 2 (MMD2). Overexpressing MMD2 reversed HOXA-AS2 depletion-mediated suppression on cell aggressiveness with Hp infection. Hp infection induces the aggressiveness of GC cells by regulating HOXA-AS2/miR-509-3p/MMD2 axis.

453 Changed Innervation Upon Loss of Mandibular Division of Trigeminal Nerve to Muscles of Mastication

INTRODUCTION: The gastrulation brain homeobox (Gbx) genes are essential for patterning and maintenance of neurons along the anteroposterior axis of the developing neural tube. Knockout (ko) of Gbx2 results in neonatal lethality associated with neurological and other defects. To understand pathologies, ko studies are not realistic as gene loss usually results in death before birth. Gbx2 neo/neo mutant mice express 6-10% of the wildtype Gbx2 expression levels. They have milder malformations than ko mice but lack the cerebellar vermis and mandibular division of the trigeminal nerve (3rd division of 5th cranial nerve = V3), among other defects. These Gbx2 neo/neo mutant mice die perinatally as they are unable to suckle due to lack of motor innervation via V3 to the muscles of mastication. Muscle cells develop largely without interaction with their motor nerve. However, the final differentiation of myocytes requires interactions with nerve fibers. Unexpectedly, the muscles of mastication appear normal in Gbx2 neo/neo mutant mice, despite the lack of V3. METHODS: We performed microdissections of neonate wildtype and Gbx2neo/neo mutant mice. Additionally, we embedded mutant and wildtype mouse embryos in paraffin, serial sectioned them (7 μm), stained the sections with Azan staining, and analyzed specimen microscopically. RESULTS: Current analysis of the data to identify where nerve fibers in the muscles of mastication originate is in process. We favor the origin of these fibers from the facial nerve (7th cranial nerve), which has several overlapping territories with the trigeminal nerve. CONCLUSIONS: Muscles require innervation for their final differentiation steps. The loss of a nerve can result in the invasion of another nerve into the territory of the lost one, which rescues muscle differentiation but not muscle function. IACUC Ziermann Med-20-02, Funding NSF #2000005 to Ziermann.

Molecular expression of Forkhead Box C2 gene (FOXC2) and Prospero homeobox gene (PROX-1) in oral squamous carcinoma and their correlation with clinicopathological parameters: A prospective cohort study.

Forkhead box C2 gene (FOXC2) acts as an epithelial-mesenchymal transition (EMT) inducer while Prospero homeobox 1 gene (PROX-1) function as a regulator of lymphangiogenesis and angiogenesis in oral squamous cell carcinoma (OSCC). It is presumed that PROX-1 has both tumour-suppressive and oncogenic effects. The main aim of this study is to evaluate the role of PROX-1 and FOXC2 in the invasion and progression of OSCC cases and to correlate their expression with various histopathological parameters. A prospective cohort study was conducted in a total sample size of 52 OSCC tissues and histologically tumour-free margins of 20. mRNA expression and protein levels of FOXC2 and PROX-1 were evaluated using real-time PCR and sandwich enzyme-linked immunosorbent assay techniques. Chi-square analysis and correlation analysis were done. Kaplan-Meier analysis evaluated the survival rate. Mean Ct values of FOXC2 were 1.915 ± 0.519 and PROX-1 was 0.061 ± 0.173. There was a significant 2-fold increase in the FOXC2 expression and a 0.5-fold decrease in the PROX-1 expression in OSCC tissue. Increased levels of FOXC2 protein and decreased levels of PROX-1 with a mean difference of 1.64 ± 0.73 ng/ml and 1.27 ± 0.33 ng/ml were observed in OSCC compared to histologically tumour-free margins. A significant positive correlation was found between the FOXC2 expression and clinicopathological parameters such as staging, perineural invasion (PNI) and lymphovascular invasion (LVI) whereas PROX-1 showed a significant negative correlation with histopathological parameters such as staging, PNI, LVI and tumour staging. There was a significant positive correlation between the PROX-1 and histologically tumour-free margins in disease-free survival patients (P-value = 0.03). FOXC2 and PROX-1 expressions were correlated with lymphovascular invasion, OSCC tumour staging and PNI. Thus, FOXC2 and PROX-1 could be possible therapeutic targets in the treatment of OSCC that can inhibit the EMT in OSCC and thereby favouring a better prognosis.