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Homeobox proteins play critical roles in controlling processes such as morphogenesis and organogenesis in many organisms. Some of these proteins are known to aff ect the formation, development and regeneration of the liver. In this context, the present study was aimed at demonstrating the localization and expression intensity of some homeobox proteins in the bovine fetal liver during the diff erent stages of gestation, determining whether or not these proteins are found in the structural components of the liver, and identifying their potential physiological roles. Th e study material comprised of 27 clinically healthy bovine fetuses, which were obtained from slaughterhouses and belonged to diff erent stages of gestation. Th e fetuses were grouped according to their crown-rump length (CRL) measurements. Liver samples were taken from each study group and subjected to routine histological processing, followed by immunohistochemical staining. Th e staining results showed that, throughout gestation, the expression intensities of the homeobox proteins HOXA10, HOXA11, HOXB6, TLX1, Dlx-5 and HLX were stronger in the hepatocytes, compared to the hepatic artery, vena interlobularis and bile ducts. However, the expression intensity of HLX was determined to have significantly decreased during the second and third trimesters of gestation, compared to the first trimester. In conclusion, the expression of the investigated homeobox proteins at diff ering and similar levels in the hepatocytes, hepatic artery, vena interlobularis and bile ducts of the bovine fetal liver during gestation could be interpreted as an important indicator of these proteins being involved in the development and physiological activity of the fetal liver.

Homeobox proteins play critical roles in controlling processes such as morphogenesis and organogenesis in many organisms. Some of these proteins are known to affect the formation, development and regeneration of the liver. In this context, the present study was aimed at demonstrating the localization and expression intensity of some homeobox proteins in the bovine fetal liver during the different stages of gestation, determining whether or not these proteins are found in the structural components of the liver, and identifying their potential physiological roles. The study material comprised of 27 clinically healthy bovine fetuses, which were obtained from slaughterhouses and belonged to different stages of gestation. The fetuses were grouped according to their crown-rump length (CRL) measurements. Liver samples were taken from each study group and subjected to routine histological processing, followed by immunohistochemical staining. The staining results showed that, throughout gestation, the expression intensities of the homeobox proteins HOXA10, HOXA11, HOXB6, TLX1, Dlx-5 and HLX were stronger in the hepatocytes, compared to the hepatic artery, vena interlobularis and bile ducts. However, the expression intensity of HLX was determined to have significantly decreased during the second and third trimesters of gestation, compared to the first trimester. In conclusion, the expression of the investigated homeobox proteins at differing and similar levels in the hepatocytes, hepatic artery, vena interlobularis and bile ducts of the bovine fetal liver during gestation could be interpreted as an important indicator of these proteins being involved in the development and physiological activity of the fetal liver. [ABSTRACT FROM AUTHOR]

Abstract Background An increasing number of studies have described the aberrant expression of homeobox (HOX) proteins in gastric cancer (GC), which is critically associated with the prognosis and clinicopathological characteristics of GC. This study was conducted to investigate the clinical value and action mechanisms of HOX proteins in GC. Methods A comprehensive search of PubMed, Embase, Web of Science and Cochrane Library was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. The pooled hazard ratio (HR) with its 95% confidence interval (95% CI) and the pooled odds ratio (OR) with its 95% CI were used to assess the effect of HOX protein expression on the prognosis and clinicopathological features of GC, respectively. Results Nineteen studies containing 3775 patients were selected for this study. Heterogeneity among HRs of overall survival (OS) was markedly high (I2 = 90.5%, p = 0.000). According to the subgroup analysis, increased expression of HOX protein in the downregulated subgroup was associated with a good prognosis for patients with GC (pooled HR: 0.46, 95% CI: 0.36–0.59, I2 = 3.1%, p = 0.377), while overexpression of HOX protein in the upregulated subgroup was correlated with a reduced OS (pooled HR: 2.59, 95% CI: 1.79–3.74, I2 = 73.5%, p = 0.000). The aberrant expression of HOX protein was crucially related to the TNM stage, depth of tumour invasion, tumour size, lymph node metastasis, distant metastasis, vascular invasion, histological differentiation and Lauren classification in patients with GC. In addition, the molecular mechanisms by which HOX proteins regulate tumorigenesis and development of GC were also explored. Conclusions HOX proteins play vital roles in GC progression, which might serve as prognostic markers and therapeutic targets for GC.

Recent studies have shown that homeobox proteins play an important role in the formation and development of tissues and organs in the embryonic period. In our study, the distribution of Dlx-5 and TLX proteins, which are members of the homeobox family, in the testis, epididymis and ductus deferens ducts of some cat breeds were investigated. For this purpose, in the study, 18 testes younger than six months (immature) and older than one year (mature) were examined under a light microscope using an immunohistochemical method (indirect streptavidin-biotin complex). While it was determined that Dlx-5 and TLX1 proteins were expressed at varying levels in cells in immature and mature cat testicles, epithelial cells of ductus epididymis and ductus deferens, and smooth muscle cells of ductus deferens, no differences were observed between cat breeds. Dlx-5 immunoreactivity was more intense in the testes, epididymis and deferens ducts of immature and mature compared to TLX1. These results suggested that both proteins play important roles in the development of male feline genital organs and in the secretion and differentiation of cells, and also further observation of Dlx-5 expression suggested that this protein may be more effective than TLX1 in testicular development and physiological processes. [ABSTRACT FROM AUTHOR]

Talaromyces marneffei is a thermally dimorphic fungus that can cause life-threatening systemic mycoses, particularly in immunocompromised individuals. Fungal homeobox transcription factors control various developmental processes, including the regulation of sexual reproduction, morphology, metabolism, and virulence. However, the function of homeobox proteins in T. marneffei has not been fully explored. Here, we searched the T. marneffei genome for the total homeobox transcription factors and predicted their biological relevance by performing gene expression analysis in different cell types, including conidia, mycelia, yeasts, and during phase transition. RfeB is selected for further computational analysis since (i) its transcripts were differentially expressed in different phases of T. marneffei, and (ii) this protein contains the highly conserved protein-protein interaction region (IR), which could be important for pathobiology and have therapeutic application. To assess the structure-function of the IR region, in silico alanine substitutions were performed at three-conserved IR residues (Asp276, Glu279, and Gln282) of RfeB, generating a triple RfeB mutated protein. Using 3D modeling and molecular dynamics simulations, we compared the protein complex formation of wild-type and mutated RfeB proteins with the putative partner candidate TmSwi5. Our results demonstrated that the mutated RfeB protein exhibited increased free binding energy, elevated protein compactness, and a reduced number of atomic contacts, suggesting disrupted protein stability and interaction. Notably, our model revealed that the IR residues primarily stabilized the RfeB binding sites located in the central region (CR). This computational approach for protein mutagenesis could provide a foundation for future experimental studies on the functional characterization of RfeB and other homeodomain-containing proteins in T. marneffei. [ABSTRACT FROM AUTHOR]

Background: T-cell leukemia homeobox protein 1 (TLX1) has been revealed as a hub transcription factor in leukemia, while its function in hepatocellular carcinoma (HCC) has not been well described. Here, we investigated the regulation and function of TLX1 in HCC. Methods: TLX1 and its possible upstream and downstream molecules in HCC were identified using bioinformatics tools, which were then verified by RT-qPCR assay. CCK-8, wound healing, and Transwell invasion assays were performed to detect the effects of TLX1 knockdown on HCC cells. The interactions between TLX1 and trafficking protein particle complex subunit 9 (TRAPPC9) or Zinc finger protein 69 homolog B (ZFP69B) were further probed by ChIP and luciferase reporter assays. Rescue experiments were finally conducted in vitro and in vivo. Results: TLX1 was highly expressed in HCC cells, and the knockdown of TLX1 led to reduced malignant biological behavior of HCC cells. TLX1 bound to the promoter region of TRAPPC9, thereby promoting TRAPPC9 expression. Overexpression of TRAPPC9 attenuated the effect of TLX1 reduction on suppressing malignant behavior of HCC cells. ZFP69B was also highly expressed in HCC cells and bound to the promoter region of TLX1 to induce TLX1 expression. Knockdown of ZFP69B inhibited the viability and mobility of HCC cells in vitro and tumor growth in vivo, and overexpression of TLX1 rescued this inhibition. Conclusion: These findings suggest that ZFP69B promotes the proliferation of HCC cells by directly upregulating the expression of TLX1 and the ensuing TRAPPC9. [ABSTRACT FROM AUTHOR]

SUMMARY: Gibberellins (GAs) play crucial roles in regulating plant architecture and grain yield of crops. In rice, the inactivation of endogenous bioactive GAs and their precursors by GA 2‐oxidases (GA2oxs) regulates stem elongation and reproductive development. However, the regulatory mechanisms of GA2ox gene expression, especially in rice reproductive organs, are unknown. The BEL1‐like homeodomain protein OsBLH4, a negative regulatory factor for the rice OsGA2ox1 gene, was identified in this study. Loss of OsBLH4 function results in decreased bioactive GA levels and pleiotropic phenotypes, including reduced plant height, decreased grain number per panicle, and delayed heading date, as also observed in OsGA2ox1‐overexpressing plants. Consistent with the mutant phenotype, OsBLH4 was predominantly expressed in shoots and young spikelets; its encoded protein was exclusively localized in the nucleus. Molecular analysis demonstrated that OsBLH4 directly bound to the promoter region of OsGA2ox1 to repress its expression. Genetic assays revealed that OsBLH4 acts upstream of OsGA2ox1 to control rice plant height, grain number, and heading date. Taken together, these results indicate a crucial role for OsBLH4 in regulating rice plant architecture and yield potential via regulation of bioactive GA levels, and provide a potential strategy for genetic improvements of rice. Significance Statement: GA 2‐oxidases (GA2oxs) play crucial roles in regulating plant architecture and yield by inactivating endogenous bioactive GAs and their precursors, but the regulatory mechanisms of GA2ox gene expression remain unclear. This study identifies that OsBLH4, a member of the BEL1‐like homeodomain proteins, acts upstream of OsGA2ox1, modulates endogenous bioactive GA levels, and regulates stem elongation and reproductive development in rice. [ABSTRACT FROM AUTHOR]

In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor (TF) is required for specifying the sepals and petals identities and confers a major A-function to antagonize the C-function in the outer floral whorls. In the asterid species Petunia, the AP2-type ROB TFs are required for perianth and pistil development, as well as repressing the B-function together with TOE-type TF BEN. In Long-homostyle (LH) Fagopyrum esculentum, VIGS-silencing showed that FaesAP2 is mainly involved in controlling filament and style length, but FaesTOE is mainly involved in regulating filament length and pollen grain development. Both FaesAP2 (AP2-type) and FaesTOE (TOE-type) are redundantly involved in style and/or filament length determination instead of perianth development. However, neither FaesAP2 nor FaesTOE could directly repress the B and/or C class genes in common buckwheat. Moreover, the FaesAP1_2 silenced flower showed tepal numbers, and filament length decreased obviously. Interestingly, yeast one-hybrid (Y1H) and dual-luciferase reporter (DR) further suggested that FaesTOE directly up-regulates FaesAP1_2 to be involved in filament length determination in LH common buckwheat. Moreover, the knockdown of FaesTOE expression could result in expression down-regulation of the directly target FaesAP1_2 in the FaesTOE-silenced LH plants. Our findings uncover a stamen development pathway in common buckwheat and offer deeper insight into the functional evolution of AP2 orthologs in the early-diverging core eudicots. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: This study shed light on regulatory mechanisms of plastid development in fruits by demonstrating that the class II knotted‐like homeodomain protein SlKN5 negatively regulates chloroplast number and chlorophyll accumulation in tomato fruit development. SlKN5 interacts with BEL1‐like homeodomain protein SlBLH7 and its paralogs to suppress SlAPRR2‐like1 and chlorophyll biosynthetic genes, promoting the developmental shift from growth phase to onset of ripening. This highlights the crucial role of the SlKN5‐BLH module in the regulation of tomato fruit coloration. [ABSTRACT FROM AUTHOR]

Background: Coiled-coil domain-containing protein 178 (CCDC178) has been revealed to exert metastasis-promoting properties in hepatocellular carcinoma, whereas its function in gastric cancer (GC) has not been fully understood. Aims: We evaluated its role in GC and the molecular mechanism. Methods: The differentially expressed genes in datasets related to GC metastasis were intersected with survival-related genes in GC, followed by prognostic significance prediction. Loss- and gain-of-function assays were conducted to examine the involvement of CCDC178, Homeobox protein BarH-like 1 (BARX1), and the extracellular signal-regulated kinase (ERK) pathway in GC cell malignant phenotype and the polarization of tumor-associated macrophages (TAM). The corresponding functions were verified in the in vivo animal experiment. Results: High CCDC178 expression predicted a poor prognosis for GC patients, and CCDC178 correlated significantly with macrophage infiltration in GC tissues. CCDC178 activated the ERK pathway in GC. Silencing of CCDC178 reduced the colony formation, migratory and invasive potential of GC cells, and the M2-like polarization of TAM, which was reversed by TBHQ (an ERK activator). BARX1 bound to the promoter region of CCDC178, thus inducing its transcriptional level. Silencing of BARX1 suppressed the M2-type polarization of TAM in vitro and in vivo, and CCDC178 mitigated the repressing role of BARX1 knockdown. Conclusions: BARX1 activates the transcription of CCDC178 to induce the ERK pathway, thereby supporting macrophage recruitment and M2-like polarization in GC. [ABSTRACT FROM AUTHOR]

Background and Aim: Circular ubiquitin‐like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1–G9a–ubiquitin‐like, containing PHD and ring finger domains 1 (UHRF1) mRNA–eukaryotic translation initiation factor 4A3 (EIF4A3)–PDZ and LIM domain 1 (PDLIM1) network in HCC. Methods: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial–mesenchymal transition (EMT)‐related proteins, and Hippo–Yap pathway components was determined by quantitative polymerase chain reaction (Q‐PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit‐8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull‐down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co‐immunoprecipitation was performed to examine the binding between UHRF1 and G9a. Results: Circular ubiquitin‐like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription. Conclusion: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo–Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1–G9a–UHRF1 mRNA–EIF4A3–PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment. [ABSTRACT FROM AUTHOR]

KNOXs, a type of homeobox genes that encode atypical homeobox proteins, play an essential role in the regulation of growth and development, hormonal response, and abiotic stress in plants. However, the KNOX gene family has not been explored in sweet potato. In this study, through sequence alignment, genomic structure analysis, and phylogenetic characterization, 17, 12 and 11 KNOXs in sweet potato (I. batatas, 2n = 6x = 90) and its two diploid relatives I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30) were identified. The protein physicochemical properties, chromosome localization, phylogenetic relationships, gene structure, protein interaction network, cis-elements of promoters, tissue-specific expression and expression patterns under hormone treatment and abiotic stresses of these 40 KNOX genes were systematically studied. IbKNOX4, -5, and − 6 were highly expressed in the leaves of the high-yield varieties Longshu9 and Xushu18. IbKNOX3 and IbKNOX8 in Class I were upregulated in initial storage roots compared to fibrous roots. IbKNOXs in Class M were specifically expressed in the stem tip and hardly expressed in other tissues. Moreover, IbKNOX2 and − 6, and their homologous genes were induced by PEG/mannitol and NaCl treatments. The results showed that KNOXs were involved in regulating growth and development, hormone crosstalk and abiotic stress responses between sweet potato and its two diploid relatives. This study provides a comparison of these KNOX genes in sweet potato and its two diploid relatives and a theoretical basis for functional studies. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection (CHB) is a risk factor for the development of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Covalently closed circular DNA serves as the sole transcription template for all viral RNAs and viral transcription is driven and enhanced by viral promoter and enhancer elements, respectively. Interactions between transcription factors and these cis-elements regulate their activities and change the production levels of viral RNAs. Here, we report the identification of homeobox protein MSX-1 (MSX1) as a novel host restriction factor of HBV in liver. In both HBV-transfected and HBV-infected cells, MSX1 suppresses viral gene expression and genome replication. Mechanistically, MSX1 downregulates enhancer II/core promoter (EnII/Cp) activity via direct binding to an MSX1 responsive element within EnII/Cp, and such binding competes with hepatocyte nuclear factor 4α binding to EnII/Cp due to partial overlap between their respective binding sites. Furthermore, CHB patients in immune active phase express higher levels of intrahepatic MSX1 but relatively lower levels of serum and intrahepatic HBV markers compared to those in immune tolerant phase. Finally, MSX1 was demonstrated to induce viral clearance in two mouse models of HBV persistence, suggesting possible therapeutic potential for CHB. IMPORTANCE Covalently closed circular DNA plays a key role for the persistence of hepatitis B virus (HBV) since it serves as the template for viral transcription. Identification of transcription factors that regulate HBV transcription not only provides insights into molecular mechanisms of viral life cycle regulation but may also provide potential antiviral targets. In this work, we identified host MSX1 as a novel restriction factor of HBV transcription. Meanwhile, we observed higher intrahepatic MSX1 expression in chronic hepatitis B virus (CHB) patients in immune active phase compared to those in immune tolerant phase, suggesting possible involvement of MSX1 in the regulation of HBV activity by the host. Lastly, intrahepatic overexpression of MSX1 delivered by recombinant adenoviruses into two mouse models of HBV persistence demonstrated MSX1-mediated repression of HBV in vivo, and MSX1-induced clearance of intrahepatic HBV DNA in treated mice suggested its potential as a therapeutic target for the treatment of CHB. [ABSTRACT FROM AUTHOR]

How our brain generates diverse neuron types that assemble into precise neural circuits remains unclear. Using Drosophila lamina neuron types (L1-L5), we show that the primary homeodomain transcription factor (HDTF) brain-specific homeobox (Bsh) is initiated in progenitors and maintained in L4/L5 neurons to adulthood. Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates while repressing the HDTF Zfh1 to prevent ectopic L1/L3 fates (control: L1-L5; Bsh-knockdown: L1-L3), thereby generating lamina neuronal diversity for normal visual sensitivity. Subsequently, in L4 neurons, Bsh and Ap function in a feed-forward loop to activate the synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. Expression of a Bsh:Dam, specifically in L4, reveals Bsh binding to the DIP-β locus and additional candidate L4 functional identity genes. We propose that HDTFs function hierarchically to coordinate neuronal molecular identity, circuit formation, and function. Hierarchical HDTFs may represent a conserved mechanism for linking neuronal diversity to circuit assembly and function. [ABSTRACT FROM AUTHOR]

Hox genes are a family of homeodomain transcription factors that regulate specialized morphological structures along the anterior-posterior axis of metazoans. Over the past few decades, researchers have focused on defining how Hox factors with similar in vitro DNA binding activities achieve sufficient target specificity to regulate distinct cell fates in vivo. In this review, we highlight how protein interactions with other transcription factors, many of which are also homeodomain proteins, result in the formation of transcription factor complexes with enhanced DNA binding specificity. These findings suggest that Hox-regulated enhancers utilize distinct combinations of homeodomain binding sites, many of which are low-affinity, to recruit specific Hox complexes. However, low-affinity sites can only yield reproducible responses with high transcription factor concentrations. To overcome this limitation, recent studies revealed how transcription factors, including Hox factors, use intrinsically disordered domains (IDRs) to form biomolecular condensates that increase protein concentrations. Moreover, Hox factors with altered IDRs have been associated with altered transcriptional activity and human disease states, demonstrating the importance of IDRs in mediating essential Hox output. Collectively, these studies highlight how Hox factors use their DNA binding domains, protein-protein interaction domains, and IDRs to form specific transcription factor complexes that yield accurate gene expression. [Display omitted] • Hox complex formation with other homeodomain factors increase DNA specificity. • Different Hox complexes have distinct DNA binding site requirements. • Low-affinity DNA sites increase Hox binding specificity. • Hox factors are concentrated within distinct nuclear sub-compartments. • Hox factor intrinsically disordered regions (IDRs) form biomolecular condensates. • Hox factors with altered IDRs have been associated with human diseases. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is a degenerative muscle disease caused by loss of epigenetic silencing and ectopic reactivation of the embryonic double homeobox protein 4 gene (DUX4) in skeletal muscle. The p38 MAP kinase inhibitor losmapimod is currently being tested in FSHD clinical trials due to the finding that p38 inhibition suppresses DUX4 expression in preclinical models. However, the role of p38 in regulating DUX4 at different myogenic stages has not been investigated. We used genetic and pharmacologic tools in FSHD patient-derived myoblasts/myocytes to explore the temporal role of p38 in differentiation-induced DUX4 expression. Deletion of MAPK14/11 or inhibition of p38α/β caused a significant reduction in early differentiation-dependent increases in DUX4 and DUX4 target gene expression. However, in MAPK14/11 knockout cells, there remains a differentiation-associated increase in DUX4 and DUX4 target gene expression later in differentiation. Furthermore, pharmacologic inhibition of p38α/β only partially decreased DUX4 and DUX4 target gene expression in late differentiating myotubes. In xenograft studies, p38α/β inhibition by losmapimod failed to suppress DUX4 target gene expression in late FSHD xenografts. Our results show that while p38 is critical for DUX4 expression during early myogenesis, later in myogenesis a significant level of DUX4 expression is independent of p38α/β activity. [ABSTRACT FROM AUTHOR]

The maritime pine (Pinus pinaster Ait.) is a highly valuable Mediterranean conifer. However, recurrent drought events threaten its propagation and conservation. P. pinaster populations exhibit remarkable differences in drought tolerance. To explore these differences, we analyzed stem transcriptional profiles of grafts combining genotypes with contrasting drought responses under well-watered and water-stress regimes. Our analysis underscored that P. pinaster drought tolerance is mainly associated with constitutively expressed genes, which vary based on genotype provenance. However, we identified key genes encoding proteins involved in water stress response, abscisic acid signaling, and growth control including a PHD chromatin regulator, a histone deubiquitinase, the ABI5-binding protein 3, and transcription factors from Myb-related, DOF NAC and LHY families. Additionally, we identified that drought-tolerant rootstock could enhance the drought tolerance of sensitive scions by regulating the accumulation of transcripts involved in carbon mobilization, osmolyte biosynthesis, flavonoid and terpenoid metabolism, and reactive oxygen species scavenging. These included genes encoding galactinol synthase, CBL-interacting serine/threonine protein kinase 5, BEL1-like homeodomain protein, dihydroflavonol 4-reductase, and 1-deoxy-D-xylulose-5-phosphate. Our results revealed several hub genes that could help us to understand the molecular and physiological response to drought of conifers. Based on all the above, grafting with selected drought-tolerant rootstocks is a promising method for propagating elite recalcitrant conifer species, such as P. pinaster. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant genetic disease, which is caused by the mistaken expression of double homeobox protein 4 protein 4 (DUX4) in skeletal muscle. Patients with FSHD are usually accompanied by degenerative changes in the face, shoulders, and upper muscles, gradually accumulating in the lower limb muscles. The severity of patients is quite different, and most patients end up using wheelchairs and losing their self-care ability. At present, the exploration of treatment strategies for FSHD has shifted from relieving symptoms to gene therapy, which brings hope to the future of patients, but the current gene therapy is only in the clinical trial stage. Here, we conducted a comprehensive search of the relevant literature using the keywords FSHD, DUX4, and gene therapy methods including ASOs, CRISPR, and RNAi in the PubMed and Web of Science databases. We discussed the current advancements in treatment strategies for FSHD, as well as ongoing preclinical and clinical trials related to FSHD. Additionally, we evaluated the advantages and limitations of various gene therapy approaches targeting DUX4 aimed at correcting the underlying genetic defect. [ABSTRACT FROM AUTHOR]

Homeodomain‐only protein homeobox (HOPX) mainly exerts its transcriptional repression by physically sequestering the serum co‐repressor and recruiting histone deacetylase (HDAC), possessing important potential as a prognostic gene in acute myeloid leukemia (AML). HDACs play crucial roles in cell growth, gene regulation, and metabolism, and they are also important factors in promoting AML progression. Therefore, this project attempts to investigate whether HOPX affects AML progression by interacting with HDAC2 protein. Bioinformatics analysis was employed to identify potential prognostic genes in AML. Flow cytometry and MTT assays were performed to analyze the cellular biological functions of the AML prognostic marker HOPX. The interaction network of HOPX was analyzed using the Search Tool for the Retrieval of Interacting Genes database, and the interaction between HOPX and HDAC2 was observed using endogenous and exogenous immunoprecipitation. HOPX is highly expressed in AML cells. Further research uncovered that low expression of HOPX can repress the proliferation activity, anti‐apoptotic ability, and differentiation blockage of AML cells. Moreover, mechanistically, HOPX induced AML differentiation blockage and malignant progression through interaction with HDAC. HOPX can serve as a prognostic marker for AML and can interact with HDAC2 to induce AML differentiation blockage and malignant progression. [ABSTRACT FROM AUTHOR]

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein‐encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST‐EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down‐regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation‐induced genes. [ABSTRACT FROM AUTHOR]

PBX1 is a transcription factor that can promote the occurrence of various tumors and play a reg-ulatory role in tumor growth, metastasis, invasion, and drug resistance. Furthermore, a variant generated by fusion of E2A and PBX1, E2A-PBX1, has been found in 25% of patients with childhood acute lymphoblastic leukemia. Thus, PBX1 is a potential therapeutic target for many cancers. Here, we describe the structure of PBX1 and E2A-PBX1 as well as the molecular mecha-nisms whereby these proteins promote tumorigenesis to provide future research directions for developing new treatments. We show that PBX1 and E2A-PBX1 induce the development of highly malignant and difficult-to-treat solid and blood tumors. The development of specific drugs against their targets may be a good therapeutic strategy for PBX1-related cancers. Furthermore, we strongly recommend E2A-PBX1 as one of the genes for prenatal screening to reduce the incidence of childhood hematological malignancies. [ABSTRACT FROM AUTHOR]

Skeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120 kb upstream of the Mrf4-Myf5 locus. An enhancer, located at −57/−58 kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development.

A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, we examined whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap -positive neurons, but it is not necessarily involved in the development of all ap -positive neurons. • Drosophila apterous (ap) is essential for embryonic and larval neurodevelopment. • ap is expressed in the adult mushroom body (MB) and Pdf neurons. • ap knockout flies showed severe structural defects of MB neurons. • No morphological defects of Pdf neurons were detected in ap knockout flies. • ap does not necessarily regulate the development of all ap -positive neurons in adults. [ABSTRACT FROM AUTHOR]

Drought stress impairs plant growth and other physiological functions. MeHDZ14, a homeodomainleucine zipper I transcription factor, is strongly induced by drought stress in various cassava cultivars. However, the role of MeHDZ14 in cassava growth regulation has remained unclear. Here we report that MeHDZ14 affected plant height, such that a dwarf phenotype and altered internode elongation were observed in transgenic cassava lines. MeHDZ14 was found to negatively regulate the biosynthesis of lignin. Its overexpression resulted in abaxially rolled leaves. The morphogenesis of leaf epidermal cells was inhibited by overexpression of MeHDZ14, with decreased auxin and gibberellin and increased cytokinin contents. MeHDZ14 was found to regulate many drought-responsive genes, including genes involved in cell wall synthesis and expansion. MeHDZ14 bound to the promoter of caffeic acid 3-Omethyltransferase 1 (MeCOMT1), acting as a transcriptional repressor of genes involved in cell wall development. MeHDZ14 appears to act as a negative regulator of internode elongation and epidermal cell morphogenesis during cassava leaf development. [ABSTRACT FROM AUTHOR]

Pre-B cell leukemia factor 1 (PBX1) is a Three Aminoacid Loop Extension (TALE) homeodomain-containing transcription factor playing crucial roles in organ pattering during embryogenesis, through the formation of nuclear complexes with other TALE class and/or homeobox proteins to regulate target genes. Its contribution to the development of several organs has been elucidated mainly through the study of murine knockout models. A crucial role for human development has been recently highlighted through the discovery of different de novo pathogenic PBX1 variants in children affected by developmental defects. In the adult, PBX1 is expressed in selected tissues such as in the brain, in the gastro-intestinal and urinary systems, or in hematopoietic stem and progenitor cells, while in other organs is barely detectable. When involved in the t(1;19) chromosomal translocation it acts as an oncogene, since the resulting fusion protein drives pre-B cell leukemia, due to the induction of target genes not normally targeted by the native protein. Its aberrant expression has been associated to tumor development, progression, or therapy-resistance as in breast cancer, ovarian cancer or myeloproliferative neoplasm (MPN). On the other hand, in colorectal cancer PBX1 functions as a tumor suppressor, highlighting its context-dependent role. We here discuss differences and analogies of PBX1 roles during embryonic development and in cancer, focusing mainly on the most recent discoveries. [ABSTRACT FROM AUTHOR]

Objectives: To comprehensively investigate the potential association between prostate cancer (PCa) and the G84E mutation within the Homeobox Protein B13 (HOXB13) gene among individuals of Turkish descent, our study aims to undertake a prospective examination. Methods: We evaluated 300 patients (150 diagnosed with prostate cancer, 150 controls) who presented in our clinic. Data collected were prospectively examined. DNA isolation was performed using an isolation kit. The HOXB13-G84E mutation (rs138213197) was analyzed in the obtained samples. Data encoding and statistical analysis were performed. Results: The pathological allele for the G84E mutation was T. According to the findings, no mutations were detected in the control group, while the G84E mutation was detected in 17 patients in the patient group, all of whom had the TC genotype. The analysis showed that having the CC genotype reduced the risk of prostate cancer by 0.47 times (OR=0.47, CI=0.415-0.532). Our results did not support a trend toward family history or earlier-onset disease in comparisons between carriers and non-carriers of HOXB13 G84E mutation. Individuals with a positive family history exhibited a higher frequency of the G84E mutation. Conclusions: We concluded that HOXB13 gene mutation is indeed linked to PCa in Turkish men. However, we did not find a relationship between the HOXB13 gene G84E mutation carrier status and either early-onset PCa or familial PCa in Turkish men. [ABSTRACT FROM AUTHOR]

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (non-IRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression. [ABSTRACT FROM AUTHOR]

The first TALE homeodomain transcription factor gene to be described in plants was maize knotted1 (kn1). Dominant mutations in kn1 disrupt leaf development, with abnormal knots of tissue forming in the leaf blade. kn1 was found to be expressed in the shoot meristem but not in a peripheral region that gives rise to leaves. Furthermore, KN1 and closely related proteins were excluded from initiating and developing leaves. These findings were a prelude to a large body of work wherein TALE homeodomain proteins have been identified as vital regulators of meristem homeostasis and organ development in plants. KN1 homologues are widely represented across land plant taxa. Thus, studying the regulation and mechanistic action of this gene class has allowed investigations into the evolution of diverse plant morphologies. This review will focus on the function of TALE homeodomain transcription factors in leaf development in eudicots. Here, we discuss how TALE homeodomain proteins contribute to a spectrum of leaf forms, from the simple leaves of Arabidopsis thaliana to the compound leaves of Cardamine hirsuta and species beyond the Brassicaceae. [ABSTRACT FROM AUTHOR]

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The evolution of major taxa is often associated with the emergence of new gene families. In all multicellular animals except sponges and comb jellies, the genomes contain Hox genes, which are crucial regulators of development. The canonical function of Hox genes involves colinear patterning of body parts in bilateral animals. This general function is implemented through complex, precisely coordinated mechanisms, not all of which are evolutionarily conserved and fully understood. We suggest that the emergence of this regulatory complexity was preceded by a stage of cooperation between more ancient morphogenetic programs or their individual elements. Footprints of these programs may be present in modern animals to execute non-canonical Hox functions. Non-canonical functions of Hox genes are involved in maintaining terminal nerve cell specificity, autophagy, oogenesis, pre-gastrulation embryogenesis, vertical signaling, and a number of general biological processes. These functions are realized by the basic properties of homeodomain protein and could have triggered the evolution of ParaHoxozoa and Nephrozoa subsequently. Some of these non-canonical Hox functions are discussed in our review. [ABSTRACT FROM AUTHOR]

Cells regulate gene expression by the specific binding of transcription regulators to cis- regulatory sequences. Pair-wise cooperativity between regulators—whereby two different regulators physically interact and bind DNA in a cooperative manner—is common and permits complex modes of gene regulation. Over evolutionary timescales, the formation of new combinations of regulators represents a major source of pheno- typic novelty, facilitating new network structures. How functional, pair-wise cooperative interactions arise between regulators is poorly understood, despite the abundance of examples in extant species. Here, we explore a protein–protein interaction between two ancient transcriptional regulators—the homeodomain protein Matα2 and the MADS box protein Mcm1—that was gained approximately 200 million y ago in a clade of ascomycete yeasts that includes Saccharomyces cerevisiae. By combining deep muta- tional scanning with a functional selection for cooperative gene expression, we tested millions of possible alternative evolutionary solutions to this interaction interface. The artificially evolved, functional solutions are highly degenerate, with diverse amino acid chemistries permitted at all positions but with widespread epistasis limiting success. Nonetheless, approximately ~45% of the random sequences sampled function as well or better in controlling gene expression than the naturally evolved sequence. From these variants (which are unconstrained by historical contingency), we discern structural rules and epistatic constraints governing the emergence of cooperativity between these two transcriptional regulators. This work provides a mechanistic basis for long-standing observations of transcription network plasticity and highlights the importance of epista- sis in the evolution of new protein–protein interactions. [ABSTRACT FROM AUTHOR]

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis. [ABSTRACT FROM AUTHOR]

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells. Author summary: We demonstrate with double, triple and quadruple Six KO mouse embryos that specific Six combinations are required for proper myogenesis depending on the level of the mouse embryonic body axis. We show that the Six1 and Six2 genes are required for craniofacial myogenesis by controlling the engagement of unsegmented cranial paraxial mesodermal cells in the myogenic pathway. We also show that hypaxial somitic dermomyotomal cells from embryos mutant for the Six1 and Six4 genes are unable to engage in the skeletal muscle lineage. Last, we show that embryos mutant for the four Six genes expressed in the myogenic lineage exhibit a defect in self-renewal of PAX7+ stem cells present in their residual muscle masses, and that SIX proteins interact directly with several enhancer elements at the Pax7 locus to control its expression. We have thus characterized new functions of SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades that govern the genesis of myogenic stem cells. [ABSTRACT FROM AUTHOR]

Background. The objectives of this study were to determine the spatiotemporal distribution of human caudal-type homeobox proteins CDX1, CDX2 and CDX4 during development of the hindgut and anorectum in the embryo and to explore the possible roles of CDX genes during morphogenesis of the hindgut and anorectum. Methods. Embryos (89) were cut into sections serially and sagittally. From gestation weeks 4–9, CDX1, CDX2 and CDX4 proteins were detected on the caudal midline by immunohistochemical staining. Results. During week 4, extensive immunoreactivity of CDX1, CDX2 and CDX4 was detected in the dorsal urorectal septum, urogenital sinus and hindgut. From weeks 5–7, CDX1-, CDX2- and CDX4- positive cells were detected mainly in the mesenchyme of the urorectal septum and hindgut. The levels of CDX2 and CDX4 immunoreactivity were lower compared to CDX1. During weeks 8 and 9, the anorectal epithelium stained positive for CDX1 and CDX4, and the anal epithelium was positive for CDX2. Conclusions. The CDX proteins are constantly distributed during development of the hindgut and anorectum and exhibit overlapping distribution patterns in the cloaca/hindgut, suggesting they are important in the morphogenesis of the human hindgut and anorectum. CDX genes might be involved in development of the anorectal epithelium after the rectum has separated from the urorectal septum.

Background: We have previously demonstrated that double homeobox 4 centromeric (DUX4C) encoded for a functional DUX4c protein upregulated in dystrophic skeletal muscles. Based on gain- and loss-of-function studies we have proposed DUX4c involvement in muscle regeneration. Here, we provide further evidence for such a role in skeletal muscles from patients affected with facioscapulohumeral muscular dystrophy (FSHD). Methods: DUX4c was studied at RNA and protein levels in FSHD muscle cell cultures and biopsies. Its protein partners were co-purified and identified by mass spectrometry. Endogenous DUX4c was detected in FSHD muscle sections with either its partners or regeneration markers using co-immunofluorescence or in situ proximity ligation assay. Results: We identified new alternatively spliced DUX4C transcripts and confirmed DUX4c immunodetection in rare FSHD muscle cells in primary culture. DUX4c was detected in nuclei, cytoplasm or at cell–cell contacts between myocytes and interacted sporadically with specific RNA-binding proteins involved, a.o., in muscle differentiation, repair, and mass maintenance. In FSHD muscle sections, DUX4c was found in fibers with unusual shape or central/delocalized nuclei (a regeneration feature) staining for developmental myosin heavy chain, MYOD or presenting intense desmin labeling. Some couples of myocytes/fibers locally exhibited peripheral DUX4c-positive areas that were very close to each other, but in distinct cells. MYOD or intense desmin staining at these locations suggested an imminent muscle cell fusion. We further demonstrated DUX4c interaction with its major protein partner, C1qBP, inside myocytes/myofibers that presented features of regeneration. On adjacent muscle sections, we could unexpectedly detect DUX4 (the FSHD causal protein) and its interaction with C1qBP in fusing myocytes/fibers. Conclusions: DUX4c upregulation in FSHD muscles suggests it contributes not only to the pathology but also, based on its protein partners and specific markers, to attempts at muscle regeneration. The presence of both DUX4 and DUX4c in regenerating FSHD muscle cells suggests DUX4 could compete with normal DUX4c functions, thus explaining why skeletal muscle is particularly sensitive to DUX4 toxicity. Caution should be exerted with therapeutic agents aiming for DUX4 suppression because they might also repress the highly similar DUX4c and interfere with its physiological role. [ABSTRACT FROM AUTHOR]