Homeobox Protein Research Articles

Circ ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 increases the stability of G9a and ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 messenger RNA through recruiting eukaryotic translation initiation factor 4A3, transcriptionally inhibiting PDZ and homeobox protein domain protein 1, and promotes the metastasis of hepatocellular carcinoma.

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. 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. 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. 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.

Hsa-miR-1301-3p Promotes the Proliferation and Migration of Nonsmall Cell Lung Cancer Cells and Reduces Radiosensitivity via Targeting Homeodomain-Only Protein Homeobox.

Background: There is increasing evidence that abnormal expression of microRNAs is involved in the occurrence and progression of tumors. In previous experiments, we found that the content of hsa-miR-1301-3p in tumor tissues of patients with nonsmall cell lung cancer (NSCLC) showed an obvious upward trend compared with that in normal tissues. We performed a detailed study on the impact and underlying mechanism of hsa-miR-1301-3p in NSCLC cells. Methods: The impact of hsa-miR-1301-3p on NSCLC cell proliferation, apoptosis, migration, and invasion was examined using colony formation, flow cytometry, modified Boyden chamber, and wound healing assays. Different doses of radiation were applied to NSCLC cells to investigate their sensitivity to radiotherapy. The potential target gene of hsa-miR-1301-3p was determined by dual-luciferase reporter assay and immunoblotting. Result: hsa-miR-1301-3p was upregulated in NSCLC tissues and cells. hsa-miR-1301-3p effectively promoted the rapid proliferation, migration, and invasion of NSCLC cells, while inhibiting apoptosis. It also induced radioresistance in NSCLC cells. hsa-miR-1301-3p targeted the homeodomain-only protein homeobox (HOPX) mRNA 3' untranslated region and inhibited its transcription in NSCLC cells. Exogenous HOPX overexpression antagonized the mechanism by which hsa-miR-1301-3p regulates NSCLC cell proliferation, metastasis, and apoptosis. Conclusions: hsa-miR-1301-3p plays an oncogenic role in the occurrence and development of NSCLC. By targeting HOPX, hsa-miR-1301-3p can not only promote the proliferation and metastasis of NSCLC cells, but also alleviate apoptosis and reduce radiosensitivity.

Methyltransferase-like 3-mediated m6A modification of miR-1908-5p contributes to nasopharyngeal carcinoma progression by targeting homeodomain-only protein homeobox.

N6-methyladenosine (m6A) modification interacting microRNAs (miRNAs) have been confirmed to participate in nasopharyngeal carcinoma (NPC) progression. This research investigated miR-1908-5p's function and regulatory mechanism in the tumorigenesis of NPC via m6A modification and targeting a key gene. The levels of miR-1908-5p, homeodomain-only protein homeobox (HOPX), and methyltransferase-like 3 (METTL3) expressions were detected via RT-qPCR. The correlation between miR-1908-5p and the HOPX/METTL3 axis, as well as their regulatory mechanism, was investigated by dual luciferase reporter, western blotting, and MeRIP assays. Moreover, the bio-functions of miR-1908-5p, HOPX, and METTL3 in NPC were explored through CCK8, transwell, caspase-3 activity, and xenograft tumor assays. RT-qPCR results indicated a miR-1908-5p upregulation in NPC. Knocking down miR-1908-5p diminished the NPC cell viability and migration in vitro. In vivo, downregulating miR-1908-5p repressed NPC cell tumor growth. Moreover, HOPX was specifically targeted by miR-1908-5p, and HOPX downregulation led to reversal of the anti-tumor impact of the miR-1908-5p inhibitor against NPC cell malignancy. Also, METTL3 could mediate the m6A modification of miR-1908-5p to regulate its influence on NPC cells. This study demonstrated that the METTL3-mediated m6A modification of miR-1908-5p enhanced the tumorigenesis of NPC by targeting HOPX. These findings propose new insights for NPC diagnosis and therapy.

Pancreatic beta cell regenerative potential of Zanthoxylum chalybeum Engl. Aqueous stem bark extract

Ethnopharmacological relevanceZanthoxylum chalybeum Engl. is endemic to Africa and has been used traditionally to treat diabetes mellitus. Moreover, its pharmacological efficacy has been confirmed experimentally using in vitro and in vivo models of diabetes. However, the effects of Z. chalybeum extracts and its major constituent compounds on beta cell and islet regeneration are not clear. Further, the mechanisms associated with observed antidiabetic effects at the beta cell level are not fully elucidated. Aim of the studyWe determined the beta cell regenerative efficacy of Z. chalybeum aqueous stem bark extract, identified the chemical compounds in Z. chalybeum aqueous stem bark extracts and explored their putative mechanisms of action. Materials and methodsPhytochemical profiling of the Z. chalybeum extract was achieved using ultra high-performance liquid chromatography hyphenated to high-resolution mass spectrometry. Thereafter, molecular interactions of the compounds with beta cell regeneration targets were evaluated via molecular docking. In vitro, effects of the extract on cell viability, proliferation, apoptosis and oxidative stress were investigated in RIN-5F beta cells exposed to palmitate or streptozotocin. In vivo, pancreas tissue sections from streptozotocin-induced diabetic male Wistar rats treated with Z. chalybeum extract were stained for insulin, glucagon, pancreatic duodenal homeobox protein 1 (Pdx-1) and Ki-67. ResultsBased on ligand target and molecular docking interactions diosmin was identified as a dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) inhibitor. In vitro, Z. chalybeum augmented cell viability and cell proliferation while in palmitate-pre-treated cells, the extract significantly increased cell activity after 72 h. In vivo, although morphometric analysis showed decreased islet and beta cell size and density, observation of increased Pdx-1 and Ki-67 immunoreactivity in extract-treated islets suggests that Z. chalybeum extract has mild beta cell regenerative potential mediated by increased cell proliferation. ConclusionsOverall, the mitogenic effects observed in vitro, were not robust enough to elicit sufficient recovery of functional beta cell mass in our in vivo model, in the context of a sustained diabetic milieu. However, the identification of diosmin as a potential Dyrk1A inhibitor merits further inquiry into the attendant molecular interactions.

Integrative Single-Cell Tracking of Clonal Evolution, Leukemia Phenotypes and Donor Engraftment Following Allogeneic Stem Cell Transplant Using Mitochondrial DNA Mutations

Mitochondrial DNA (mtDNA) mutations can distinguish cells from unrelated individuals and their spectrum per patient sample can evolve following therapeutic bottlenecks. As such, these natural barcodes can potentially enable integrated single cell tracking of chimeric cellular populations and clonal evolution following unrelated allogeneic hematopoietic stem cell transplantation (HSCT). However, the feasibility of mtDNA-based donor-recipient deconvolution or identification of leukemia-specific phenotypes and the extent of co-evolution with somatic mutations remains unknown. If confirmed, post-transplant monitoring using mtDNA mutations could enable detection of early post-HSCT relapse and provide qualitative information on leukemia phenotypes, clonal evolution, or residual donor engraftment via a combined single cell assay. To investigate the extent of co-evolution of somatic nuclear mutations with mtDNA mutations, we generated 33,796 single cell DNA sequencing profiles (Tapestri) from 3 sample pairs of circulating chronic lymphocytic leukemia (CLL) collected before and after HSCT. The samples were characterized using a primer panel (n=67 nuclear and 21 mtDNA) targeting single nucleotide variants previously identified by whole-exome sequencing. We detected 8 mtDNA mutations that defined CLL subclones together with a median of 14 (8-19) somatic mutations from these cases. Clonal shifts defined by somatic mutations discernable pre- or post-HSCT tracked with analogous changes in mtDNA mutations and included clonal replacement, selection of a resistant subclone or skewing of CLL subclones. We thus provide clear evidence of co-segregation between somatic nuclear and mtDNA mutations. To assess the sensitivity of mtDNA-based donor-recipient deconvolution, we performed an in-silico titration and spiked 1-1,000 single cell mtDNA profiles (mtscATAC-seq) from one CLL case ('CLL4') into 7,579 profiles from another ('CLL5'). Overall, we recovered 98.3-100% of CLL4, identifying up to 1 in 7,579 cells (0.13%). Only 123 cells (1%) failed deconvolution, due to insufficient mtDNA coverage. In an in-vitro titration of expanded T cells (donor1) with peripheral blood mononuclear cells (donor2) at ratios of 1:3, 1:30 and 1:300, we recovered 1,449 of 5,345 (27%), 150 of 4,262 (3.5%) and 14 of 4,381 (0.3%) T cells of donor1. In sum, with current sequencing throughput, mtDNA-based donor-recipient deconvolution was assessed to have a sensitivity of ~0.1%. To determine if mtDNA-based deconvolution can track early AML relapse and identify leukemia phenotypes, we analyzed peripheral blood and bone marrow of 4 patients with disease recurrence 87-185 days post-HSCT and subsequent reinstatement of remission 105-184 days after tapering of immunosuppression (IST). We obtained 19,143 ATAC with select antigen profiling by sequencing (ASAP-seq) profiles at incipient relapse (pre-IST) and following response to IST (post-IST). Donor-recipient deconvolution detected recipient-derived cells in progenitor, erythroid and monocytic populations pre-IST but no recipient-derived cells post-IST, consistent with elimination of AML. Overall, chromatin accessibility of donor- and recipient-derived cells was very similar, reflecting that AML recapitulates hematopoietic differentiation states. Nevertheless, we observed differences in accessibility of genes such as IL1B, CD36, chemokine or homeobox protein genes in recipient- compared to donor-derived cells. Together, mtDNA-based donor-recipient deconvolution reflected clinical responses and identified leukemia-specific chromatin accessibility profiles. Finally, we tracked clonal evolution and donor-derived hematopoiesis in two non-responder cases of overt AML relapse following 3-10 cycles of decitabine and ipilimumab (NCT02890329). We defined 12 and 7 mtDNA-based AML subclones and observed contraction and expansion of subclones during treatment. Donor-derived non-T cells were exceedingly rare (12 of 10,708 [0.1%] and 34 of 5,863 [0.6%]), mainly of erythroid origin (12 and 23 cells) and disappeared during treatment. Thus, mtDNA mutations can monitor donor- and recipient-derived hematopoiesis and resolve short-term changes in AML subpopulations during therapy. As sequencing throughput improves, we envision single cell-based post-transplant monitoring as a powerful approach for guiding clinical decision making.

Identification and expression analysis of the KNOX genes during organogenesis and stress responseness in Camellia sinensis (L.) O. Kuntze.

Tea plant (Camellia sinensis L.), whose leaves are the major reproductive organs, has been cultivated and consumed widely for its economic and health benefits. The Knotted1-like Homeobox (KNOX) proteins play significant roles in leaf morphology formation and development. However, the functions of KNOX proteins in tea plants are still unknown. Here, 11 CsKNOX genes from the tea plants were cloned and divided into Class I, II, and KNATM clades based on their protein sequences. These 11 CsKNOX genes were mapped on 8 out of 15 tea plant chromosomes, all localized in the nucleus. Specific spatiotemporal expression patterns of CsKNOX genes were found in various tissues and different development periods of buds, flowers, and roots of tea plants. Meanwhile, transcript levels of CsKNOX in tea leaves were strongly correlated with the accumulation of flavan-3-ols and proanthocyanidins. It was found that most of the CsKNOX genes could respond to drought, salt, cold, and exogenous MeJA and GA3 by analysis of transcriptomics data and promoter elements. The protein interaction analysis showed that CsKNOX could cooperate with CsAS1 and other critical functional proteins. In conclusion, this research provided the basic information for the functions of the CsKNOX family during organogenesis and stress response in tea plants, which was necessary for further functional characterization verification.

HD-Zip I protein LlHOX6 antagonizes homeobox protein LlHB16 to attenuate basal thermotolerance in lily.

Homeodomain-leucine zipper (HD-Zip) I transcription factors are crucial for plant responses to drought, salt, and cold stresses. However, how they are associated with thermotolerance remains mostly unknown. We previously demonstrated that lily (Lilium longiflorum) LlHB16 (HOMEOBOX PROTEIN 16) promotes thermotolerance, whereas the roles of other HD-Zip I members are still unclear. Here, we conducted a transcriptomic analysis and identified a heat-responsive HD-Zip I gene, LlHOX6 (HOMEOBOX 6). We showed that LlHOX6 represses the establishment of basal thermotolerance in lily. LlHOX6 expression was rapidly activated by high temperature, and its protein localized to the nucleus. Heterologous expression of LlHOX6 in Arabidopsis (Arabidopsis thaliana) and overexpression in lily reduced their basal thermotolerance. In contrast, silencing LlHOX6 in lily elevated basal thermotolerance. Cooverexpressing or cosilencing LlHOX6 and LlHB16 in vivo compromised their functions in modulating basal thermotolerance. LlHOX6 interacted with itself and with LlHB16, although heterologous interactions were stronger than homologous ones. Notably, LlHOX6 directly bounds DNA elements to repress the expression of the LlHB16 target genes LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2) and LlMBF1c (MULTIPROTEIN BRIDGING FACTOR 1C). Moreover, LlHB16 activated itself to form a positive feedback loop, while LlHOX6 repressed LlHB16 expression. The LlHOX6-LlHB16 heterooligomers exhibited stronger DNA binding to compete for LlHB16 homooligomers, thus weakening the transactivation ability of LlHB16 for LlHSFA2 and LlMBF1c and reducing its autoactivation. Altogether, our findings demonstrate that LlHOX6 interacts with LlHB16 to limit its transactivation, thereby impairing heat stress responses in lily.

Targeting the SOX2/CDP protein complex with a peptide suppresses the malignant progression of esophageal squamous cell carcinoma

Emerging evidence indicates that SOX2 is an oncogene for esophageal squamous cell carcinoma (ESCC). However, direct targeting of SOX2 is not feasible given that this transcription factor plays important roles in the maintenance of tissues such as the brain. Here, we identified CDP (Homeobox protein cut-like 1 or CASP) as a unique SOX2 binding partner enriched in ESCC with Duolink proximity ligation assay, bimolecular fluorescence complementation (BiFc) and immunoprecipitation. We then screened a peptide aptamer library using BiFc and immunoprecipitation and identified several peptide aptamers, including P58, that blocked the CDP/SOX2 interaction, leading to the inhibition of ESCC progress in vitro and in vivo. Upon administration, synthetic peptide P58, containing the YGRKKRRQRRR cell-penetrating peptide and the fluorophore TAMRA, also blocked the growth and metastasis of ESCC in both mice and zebrafish. Therefore, targeting the SOX2 binding partner CDP with peptide P58 offers an alternative avenue to treat ESCC with increased SOX2 levels.

O-GlcNAcylation regulates OTX2’s proteostasis

O-GlcNAcylation is a key post-translational modification, playing a vital role in cell signaling during development, especially in the brain. In this study, we investigated the role of O-GlcNAcylation in regulating the homeobox protein OTX2, which contributes to various brain disorders, such as combined pituitary hormone deficiency, retinopathy, and medulloblastoma. Our research demonstrated that, under normal physiological conditions, the proteasome plays a pivotal role in breaking down endogenous OTX2. However, when the levels of OTX2 rise, it forms oligomers and/or aggregates that require macroautophagy for clearance. Intriguingly, we demonstrated that O-GlcNAcylation enhances the solubility of OTX2, thereby limiting the formation of these aggregates. Additionally, we unveiled an interaction between OTX2 and the chaperone protein CCT5 at the O-GlcNAc sites, suggesting a potential collaborative role in preventing OTX2 aggregation. Finally, our study demonstrated that while OTX2 physiologically promotes cell proliferation, an O-GlcNAc-depleted OTX2 is detrimental to cancer cells.

The Efficacy of Vitamin D Supplement in the Expression and Protein Levels of Endometrial Decidualization Factors in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a challenging situation for infertility specialists, and its treatment is introduced as a difficult case in the field of assisted reproductive technology (ART). Vitamin D (VD) is one of the supplements that have been suggested to improve the implantation process. In the present study, the effect of VD on the expression and protein levels of VD receptor (VDR), progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1), and homeobox protein A10 (HOXA10) in the endometrial cells of unknown RIF women with and without VD deficiency were assessed by qRT-PCR and immunohistochemistry. Twelve women with unknown RIF and VD deficiency (≤ 20 ng/ml) and twelve women with unknown RIF without VD deficiency (≥ 30 ng/ml) from 2021 to 2022 were identified. Endometrial specimens were collected in the mid-luteal stage before treatment or pregnancy. In the group with VD deficiency, oral medication of VD 50,000 units was prescribed for 2 to 3 months and their serum levels of VD were re-measured, then an endometrial biopsy at the same stage of the menstrual cycle was performed. The expression and protein levels of VDR, PR, PRL, IGFBP1, and HOXA10 in RIF patients with VD deficiency were lower than the RIF patients without VD deficiency (P value < 0.05). Our findings suggest that VD can play a key role in the pregnancy process, especially during embryo implantation and decidualization of the endometrial cells.IRCT registration number: IRCT20220528055006N1, Registration date: 2022-10-15, Registration timing: retrospective.

The ameliorating effects of mesenchymal stem cells compared to α-tocopherol on apoptosis and autophagy in streptozotocin-induced diabetic rats: Implication of PI3K/Akt signaling pathway and entero-insular axis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are considered a novel regenerative therapy that holds much potential. This study aimed to examine and compare the ameliorative effects of BM-MSCs compared to α-tocopherol (α-Toc) on apoptosis, autophagy, and β-cell function in a rat model of streptozotocin (STZ)-induced diabetes and further analyzed the implications and interrelations of the entero-insular axis, and type I phosphoinositide 3-kinase(PI3K)/Akt signaling.Forty adult male albino rats were categorized into four groups (n = 10, in each): control group, STZ-induced diabetic group (single i.p. injection of STZ 45 mg/kg), diabetic and treated with BM-MSCs injection, diabetic and treatment with α-Toc p.o. The serum glucose, insulin, nitric oxide (NO), and catalase (CAT) were measured. Histopathological examination of the pancreas, the expression levels of insulin, CD44, caspase-3, autophagy markers, P13K/Akt, and pancreas/duodenum homeobox protein 1, in pancreatic tissue, and glucose-dependent insulinotropic polypeptide (GIP) in the duodenum were detected by hematoxylin and eosin staining, immunofluorescence labeling, and by quantitative real-time polymerase chain reaction.The diabetic rats showed reduced insulin, hyperglycemia, nitrosative stress (NO, CAT), augmented apoptosis (caspase 3), impaired autophagy (p62/SQSTM1, LC3), downregulated PI3K/Akt pathway and increased GIP expression, and degeneration of pancreatic islets. Treatment with either BM-MSCs or α-Toc suppressed the nitrosative stress, reduced apoptosis, recovered autophagy, upregulated PI3K/Akt pathway, and subsequently increased insulin levels, decreased blood glucose, and downregulated GIP expression with partial restoration of pancreatic islets.Based on our findings, the cytoprotective effects of BM-MSCs and α-Toc in type 1-induced diabetes appeared to be related to repaired autophagy and recovered PI3K/Akt signaling. Moreover, we reported their novel effects on reversing intestinal GIP expression level. The effect of BM-MSCs was notably superior to that of α-Toc.

Long non-coding RNA Homeobox D gene cluster antisense growth-associated long noncoding RNA/microRNA-182-5p/Homeobox protein A10 alleviates postmenopausal osteoporosis via accelerating osteoblast differentiation of bone marrow mesenchymal stem cells

BackgroundStudies have illuminated that long non-coding RNA (lncRNA) influences bone cell differentiation and formation. Nevertheless, whether lncRNA Homeobox D gene cluster antisense growth-associated long noncoding RNA (HAGLR) was implicated in postmenopausal osteoporosis (PMOP) was yet uncertain.PurposeThe research was to explore HAGLR’s role in the osteogenic differentiation (OD) process of bone marrow mesenchymal stem cells (BMSCs).MethodsBMSCs were isolated from mouse bone marrow tissues and identified by electron microscope and flow cytometry. HAGLR, microRNA (miR)-182-5p, and homeobox protein A10 (Hoxa10) levels in BMSCs were detected. Mouse BMSC OD process was induced, and calcium deposition and alkaline phosphatase content were analyzed, as well as expressions of runt-related transcription factor 2, osteopontin, and osteocalcin, and cell apoptosis. Bilateral ovaries were resected from mice to construct the ovariectomized model and bone mineral density, maximum bending stress, maximum load, and elastic modulus of the femur were tested, and the femur was histopathologically evaluated. Chondrocyte apoptosis in the articular cartilage of mice was analyzed. Analysis of the interaction of HAGLR, miR-182-5p with Hoxa10 was conducted.ResultsHAGLR and Hoxa10 were down-regulated and miR-182-5p was elevated in PMOP patients. During the BMSC OD process, HAGLR and Hoxa10 levels were suppressed, while miR-182-5p was elevated. Promotion of HAGLR or suppression of miR-182-5p accelerated OD of BMSCs. Inhibition of miR-182-5p reversed the inhibitory effect of HAGLR on BMSC OD. In in vivo experiments, up-regulating HAGLR alleviated PMOP, while silencing Hoxa10 reversed the effects of upregulating HAGLR. HAGLR performed as a sponge for miR-182-5p, while miR-182-5p targeted Hoxa10.ConclusionIn general, HAGLR boosted the OD process of BMSCs and relieved PMOP via the miR-182-5p/Hoxa10 axis. These data preliminarily reveal the key role of HAGLR in PMOP, and the research results have a certain reference for the treatment of PMOP.

The transcription factor DUX4 orchestrates translational reprogramming by broadly suppressing translation efficiency and promoting expression of DUX4-induced mRNAs.

Translational control is critical for cell fate transitions during development, lineage specification, and tumorigenesis. Here, we show that the transcription factor double homeobox protein 4 (DUX4), and its previously characterized transcriptional program, broadly regulates translation to change the cellular proteome. DUX4 is a key regulator of zygotic genome activation in human embryos, whereas misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and is associated with MHC-I suppression and immune evasion in cancer. We report that translation initiation and elongation factors are disrupted downstream of DUX4 expression in human myoblasts. Genome-wide translation profiling identified mRNAs susceptible to DUX4-induced translation inhibition, including those encoding antigen presentation factors and muscle lineage proteins, while DUX4-induced mRNAs were robustly translated. Endogenous expression of DUX4 in human FSHD myotubes and cancer cell lines also correlated with reduced protein synthesis and MHC-I presentation. Our findings reveal that DUX4 orchestrates cell state conversion by suppressing the cellular proteome while maintaining translation of DUX4-induced mRNAs to promote an early developmental program.

Methyltransferase-like 3 facilitates the stem cell properties of esophageal cancer by upregulating patched homolog 1 via N6-methyladenosine methylation.

Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.

Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.).

WUSCHEL-related homeobox (WOX) proteins are very important in controlling plant development and stress responses. However, the WOX family members and their role in response to abiotic stresses are largely unknown in melon (Cucumis melo L.). In this study, 11 WOX (CmWOX) transcript factors with conserved WUS and homeobox motif were identified and characterized, and subdivided into modern clade, ancient clade and intermediate clade based on bioinformatic and phylogenetic analysis. Evolutionary analysis revealed that the CmWOX family showed protein variations in Arabidopsis, tomato, cucumber, melon and rice. Alignment of protein sequences uncovered that all CmWOXs had the typical homeodomain, which consisted of conserved amino acids. Cis-element analysis showed that CmWOX genes may response to abiotic stress. RNA-seq and qRT-PCR results further revealed that the expression of partially CmWOX genes are associated with cold and drought. CmWOX13a and CmWOX13b were constitutively expressed under abiotic stresses, CmWOX4 may play a role in abiotic processes during plant development. Taken together, this study offers new perspectives on the CmWOX family's interaction and provides the framework for research on the molecular functions of CmWOX genes.

Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections

The balance of contralateral and ipsilateral retinogeniculate projections is critical for binocular vision, but the transcriptional programs regulating this process remain ill defined. Here we show that the Pou class homeobox protein POU3F1 is expressed in nascent mouse contralateral retinal ganglion cells (cRGCs) but not ipsilateral RGCs (iRGCs). Upon Pou3f1 inactivation, the proportion of cRGCs is reduced in favor of iRGCs, leading to abnormal projection ratios at the optic chiasm. Conversely, misexpression of Pou3f1 in progenitors increases the production of cRGCs. Using CUT&RUN and RNA sequencing in gain- and loss-of-function assays, we demonstrate that POU3F1 regulates expression of several key members of the cRGC gene regulatory network. Finally, we report that POU3F1 is sufficient to induce RGC-like cell production, even in late-stage retinal progenitors of Atoh7 knockout mice. This work uncovers POU3F1 as a regulator of the cRGC transcriptional program, opening possibilities for optic nerve regenerative therapies.

Three-amino acid loop extension homeodomain proteins regulate stress responses and encystation in Entamoeba.

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.

Identification and Evolutionary Analysis of Cotton (Gossypium hirsutum) WOX Family Genes and Their Potential Function in Somatic Embryogenesis.

WUSCHEL-related homeobox (WOX) proteins participate profoundly in plant development and stress responses. As the difficulty of somatic embryogenesis severely constrains cotton genetic modification, in this study, we identified and comprehensively analyzed WOX genes in cotton. As a result, 40 WOX genes were identified in the upland cotton genome. All these cotton WOX genes were classified into three clades, ancient, intermediate, and modern clades, based on the phylogenetic analysis of previous studies. The majority (24) of the cotton WOX genes belonged to the modern clade, in which all gene members contain the vital functional domain WUS-box, which is necessary for plant stem cell regulation and maintenance. Collinearity analysis indicated that the WOX gene family in cotton expanded to some degree compared to Arabidopsis, especially in the modern clade. Genome duplication and segmental duplication may greatly contribute to expansion. Hormone-response- and abiotic-stress-response-related cis-acting regulatory elements were widely distributed in the promoter regions of cotton WOX genes, suggesting that the corresponding functions of stress responses and the participation of development processes were involved in hormone responses. By RNA sequencing, we profiled the expression patterns of cotton WOX genes in somatic embryogenesis. Only about half of cotton WOX genes were actively expressed during somatic embryogenesis; different cotton WOX genes may function in different development stages. The most representative, GhWOX4 and GhWOX13, may function in almost all stages of somatic embryogenesis; GhWOX2 and GhWOX9 function in the late stages of embryo patterning and embryo development during cotton somatic embryogenesis. Co-expression analysis showed that the cotton WOXs co-expressed with genes involved in extensive genetic information processing, including DNA replication, DNA repair, homologous recombination, RNA transport, protein processing, and several signaling and metabolism pathways, in which plant hormones signal transduction, MAPK signaling pathways, phosphatidylinositol signaling systems, and ABC transporters, as well as the metabolism of fatty acid; valine, leucine, and isoleucine biosynthesis; and cutin, suberine, and wax biosynthesis, were most significantly enriched. Taken together, the present study provides useful information and new insights into the functions of cotton WOX genes during somatic embryogenesis. The specific regulatory roles of some WOX genes in somatic embryogenesis are worthy of further functional research.

Expression and clinicopathological characteristics of PDX1, PTF1A, and SALL4 in large and small ducts of ectopic pancreas located in gastro-duodenum and jejunum

An ectopic pancreas is defined as pancreatic tissue outside its normal location, anatomically separated from the pancreas.The transcription factor pancreas/duodenum homeobox protein 1 (PDX1) is involved in maintaining the pancreas and functions in early pancreatic development, beta cell differentiation, and endocrine non beta cells. Pancreatic transcription factor 1 subunit alpha (PTF1A) affects exocrine cell formation and regulation of acinar cell identity, and is expressed in exocrine cells as a transcription factor. The depletion of SALL4 disrupts self-renewal and induces differentiation.To clarify which of PDX1, PTF1A, or SALL4 determines the difference in Heinrich's classification, we examined the localization and number of positive cells. We analyzed the differential expression of PDX1, PTF1A, and SALL4 in large and small ducts in ectopic pancreas by immunohistochemistry. Results showed that the number of PTF1A-positive cells in large ducts was more widespread in type I than in type II in the gastro-duodenum, and more SALL4-positive cells were noticed in large ducts than in small ducts in the gastro-duodenum of type II. Our results revealed that PTF1A might promote exocrine differentiation in developing the pancreatic tissues, and that those with widespread expression differentiate into exocrine cells.

Hypoxia causes pancreatic β-cell dysfunction and impairs insulin secretion by activating the transcriptional repressor BHLHE40.

Hypoxia can occur in pancreatic β-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on β-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human β-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or β-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic β-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in β-cells that inhibit insulin secretion by suppressing MAFA expression.