Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

DLX genes and proteins in mammalian forebrain development.

The vertebrate Dlx gene family encode homeobox transcription factors that are related to the Drosophila Distal-less (Dll) gene and are crucial for development. Over the last ∼35 years detailed information has accrued about the redundant and unique expression and function of the six mammalian Dlx family genes. DLX proteins interact with general transcriptional regulators, and co-bind with other transcription factors to enhancer elements with highly specific activity in the developing forebrain. Integration of the genetic and biochemical data has yielded a foundation for a gene regulatory network governing the differentiation of forebrain GABAergic neurons. In this Primer, we describe the discovery of vertebrate Dlx genes and their crucial roles in embryonic development. We largely focus on the role of Dlx family genes in mammalian forebrain development revealed through studies in mice. Finally, we highlight questions that remain unanswered regarding vertebrate Dlx genes despite over 30 years of research.

Measuring transcription factor function with cell type-specific somatic transgenesis in chicken embryos.

Retroviral-mediated misexpression in chicken embryos has been a powerful research tool for developmental biologists in the last two decades. In the RCASBP retroviral vectors that are widely used for in vivo somatic transgenesis, a coding sequence of interest is under the transcriptional control of a strong viral promoter in the long terminal repeat. While this has proven to be effective for studying secreted signalling proteins, interpretation of the mechanisms of action of nuclear factors is more difficult using this system since it is not clear whether phenotypic effects are cell-autonomous or not, and therefore whether they represent a function of the endogenous protein. Here, we report the consequences of retroviral expression using the RCANBP backbone, in which the transcription factor Dlx5 is expressed under the control of chondrocyte-specific regulatory sequences from the Col2a1 gene. To our knowledge, this is the first demonstration of a tissue-specific phenotype in the chicken embryo.

DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression.

In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.

Dlx1/2-dependent expression of Meis2 promotes neuronal fate determination in the mammalian striatum.

The striatum is a central regulator of behavior and motor function through the actions of D1 and D2 medium-sized spiny neurons (MSNs), which arise from a common lateral ganglionic eminence (LGE) progenitor. The molecular mechanisms of cell fate specification of these two neuronal subtypes are incompletely understood. Here, we found that deletion of murine Meis2, which is highly expressed in the LGE and derivatives, led to a large reduction in striatal MSNs due to a block in their differentiation. Meis2 directly binds to the Zfp503 and Six3 promoters and is required for their expression and specification of D1 and D2 MSNs, respectively. Finally, Meis2 expression is regulated by Dlx1/2 at least partially through the enhancer hs599 in the LGE subventricular zone. Overall, our findings define a pathway in the LGE whereby Dlx1/2 drives expression of Meis2, which subsequently promotes the fate determination of striatal D1 and D2 MSNs via Zfp503 and Six3.

A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum.

SignificanceOutside the neurogenic niches, the adult brain lacks multipotent progenitor cells. In this study, we performed a series of in vivo screens and reveal that a single factor can induce resident brain astrocytes to become induced neural progenitor cells (iNPCs), which then generate neurons, astrocytes, and oligodendrocytes. Such a conclusion is supported by single-cell RNA sequencing and multiple lineage-tracing experiments. Our discovery of iNPCs is fundamentally important for regenerative medicine since neural injuries or degeneration often lead to loss/dysfunction of all three neural lineages. Our findings also provide insights into cell plasticity in the adult mammalian brain, which has largely lost the regenerative capacity.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

BACKGROUND: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. RESULTS: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. CONCLUSIONS: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

Developmental origin of the mammalian premaxilla.

The evolution of jaws has played a major role in the success of vertebrate expansion into a wide variety of ecological niches. A fundamental, yet unresolved, question in craniofacial biology is about the origin of the premaxilla, the most distal bone present in the upper jaw of all amniotes. Recent reports have suggested that the mammalian premaxilla is derived from embryonic maxillary prominences rather than the frontonasal ectomesenchyme as previously shown in studies of chicken embryos. However, whether mammalian embryonic frontonasal ectomesenchyme contributes to the premaxillary bone has not been investigated and a tool to trace the contributions of the frontonasal ectomesenchyme to facial structures in mammals is lacking. The expression of the Alx3 gene is activated highly specifically in the frontonasal ectomesenchyme, but not in the maxillary mesenchyme, from the beginning of facial morphogenesis in mice. Here, we report the generation and characterization of a novel Alx3CreERT2 knock-in mouse line that express tamoxifen-inducible Cre DNA recombinase from the Alx3 locus. Tamoxifen treatment of Alx3CreERT2/+;Rosa26mTmG/+ embryos at E7.5, E8.5, E9.5, and E10.5, each induced specific labeling of the embryonic medial nasal and lateral nasal mesenchyme but not the maxillary mesenchyme. Lineage tracing of Alx3CreERT2-labeled frontonasal mesenchyme from E9.5 to E16.5 clearly showed that the frontonasal mesenchyme cells give rise to the osteoblasts generating the premaxillary bone. Furthermore, we characterize a Dlx1-Cre BAC transgenic mouse line that expresses Cre activity in the embryonic maxillary but not the frontonasal mesenchyme and show that the Dlx1-Cre labeled embryonic maxillary mesenchyme cells contribute to the maxillary bone as well as the soft tissues lateral to both the premaxillary and maxillary bones but not to the premaxillary bone. These results clearly demonstrate the developmental origin of the premaxillary bone from embryonic frontonasal ectomesenchyme cells in mice and confirm the evolutionary homology of the premaxilla across amniotes.

NFIB-MLL1 complex is required for the stemness and Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Despite significant progress in our understanding of the molecular mechanism of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here, we show that the NFIB-MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB-MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis showed that the NFIB-MLL1 complex mediates the deposition of trimethylated histone H3K4 at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased trimethylated histone H3K4 and results in elevated trimethylated histone H3K9 at those developmental genes. Furthermore, gene expression profiling and ChIP-sequencing analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB-MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 MSCs and support the epigenetic regulatory mechanism underlying the stemness and plasticity of MSCs.

Long non-coding RNA RP11-197K6.1 as ceRNA promotes colorectal cancer progression via miR-135a-5p/DLX5 axis.

BACKGROUND: Colorectal cancer (CRC) remains a major global health challenge, with high incidence and mortality rates. The role of long noncoding RNAs (lncRNAs) in cancer progression has received considerable attention. The present study aimed to investigate the function and mechanisms underlying the role of lncRNA RP11-197K6.1, microRNA-135a-5p (hsa-miR-135a-5p), and DLX5 in CRC development. METHODS: We analyzed RNA sequencing data from The Cancer Genome Atlas Colorectal Cancer dataset to identify the association between lncRNA RP11-197K6.1 and CRC progression. The expression levels of lncRNA RP11-197K6.1 and DLX5 in CRC samples and cell lines were determined by real-time quantitative PCR and western blotting assays. Fluorescence in situ hybridization was used to confirm the cellular localization of lncRNA RP11-197K6.1. Cell migration capabilities were assessed by Transwell and wound healing assays, and flow cytometry was performed to analyze apoptosis. The interaction between lncRNA RP11-197K6.1 and miR-135a-5p and its effect on DLX5 expression were investigated by the dual-luciferase reporter assay. Additionally, a xenograft mouse model was used to study the in vivo effects of lncRNA RP11-197K6.1 on tumor growth, and an immunohistochemical assay was performed to assess DLX5 expression in tumor tissues. RESULTS: lncRNA RP11-197K6.1 was significantly upregulated in CRC tissues and cell lines as compared to that in normal tissues, and its expression was inversely correlated with patient survival. It promoted the migration and metastasis of CRC cells by interacting with miR-135a-5p, alleviated suppression of DLX5 expression, and facilitated tumor growth. CONCLUSION: This study demonstrated the regulatory network and mechanism of action of the lncRNA RP11-197K6.1/miR-135a-5p/DLX5 axis in CRC development. These findings provided insights into the molecular pathology of CRC and suggested potential therapeutic targets for more effective treatment of patients with CRC.

DLX6-AS1 regulates odonto/osteogenic differentiation in dental pulp cells under the control of BMP9 via the miR-128-3p/MAPK14 axis: A laboratory investigation.

AIM: The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal-like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6-AS1) in odonto/osteogenic differentiation induced by BMP9. METHODOLOGY: Custom RT2 profiler PCR array, quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of DLX6-AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t-test or one-way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p-value of less than .05 considered statistically significant. RESULTS: DLX6-AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR-128-3p participated in BMP9-induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR-128-3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6-AS1. Lastly, miR-128-3p directly interacted with both MAPK14 and DLX6-AS1. CONCLUSIONS: DLX6-AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR-128-3p/MAPK14 axis.

LncRNA HOTTIP facilitates osteogenic differentiation in bone marrow mesenchymal stem cells and induces angiogenesis via interacting with TAF15 to stabilize DLX2.

AIM: The molecular mechanism of differentiation in bone marrow mesenchymal stem cells (BMSCs) preserves to be further elucidated. LncRNA HOTTIP has been proven to accelerate osteogenic differentiation, but the regulation mechanism is still unclear. METHODS: The human BMSCs (hBMSCs) were isolated and identified by the antigen CD29, CD34, CD44, CD45, and CD90 through flow cytometry. The osteogenic state was determined by the ALP Detection Kit and Alizarin red staining. The tube formation was observed under a microscope. HOTTIP expression level, DLX2 and TAF15, Wnt/ß-catenin pathway, and transcriptional markers in osteogenesis and angiogenesis were examined with Western blot and RT-qPCR, respectively. The combination of TAF15 with lncRNA HOTTIP and DLX2 was detected by RNA immunoprecipitation (RIP) and RNA pulldown assays. RESULTS: The outcomes revealed that HOTTIP was noticeably up-regulated accompanied by the osteogenic transcriptional factor in the process of osteoblast differentiation and angiogenesis. Besides, HOTTIP enhanced alkaline phosphatase (ALP) activity, accelerated osteogenic differentiation and angiogenesis along with up-regulation of osteogenic and angiogenic-related gene expression, by interaction with TAF15 to stabilize DLX2. CONCLUSION: Taken together, our outcomes reveal that lncRNA HOTTIP accelerated osteogenic differentiation and angiogenesis by interaction with TAF15 to stabilize DLX2.

JMJD3 is Involved in Intracranial Aneurysm Development by Regulating DLX2 Expression through H3K27me3 Modification.

Intracranial aneurysms are dilatations in the arteries that supply blood to the brain. Rupture of an intracranial aneurysm leads to a subarachnoid hemorrhage, which is fatal in about 50% of the cases. Microarray-based mRNA expression studies provide unbiased information about molecular mechanisms of intracranial aneurysm and the foundation for functional studies. In this study, by using a Gene Expression Omnibus (GEO) dataset, we identified distal-less homeobox 2 (DLX2) as a significantly upregulated gene in intracranial aneurysms and set to dissect its functional role and upstream mechanism. Here, we found that DLX2 expression was elevated in intracranial aneurysm patients. Silencing of DLX2 suppressed the proliferative capacity of human aortic vascular smooth muscle cells (HA-VSMC) and promoted their apoptosis. Moreover, loss of DLX2 promoted collagen I and collagen III and inhibited the levels of MMP2/9 and pro-inflammatory factors. Additionally, jumonji domain-containing protein 3 demethylase (JMJD3) promoted DLX2 expression by inhibiting H3K27me3 modification. Depletion of JMJD3 exerted the same function as DLX2 in vitro and in vivo, whereas overexpression of DLX2 in the presence of JMJD3 knockdown led to accentuated intracranial aneurysm progression and enhanced HA-VSMC survival. We conclude that JMJD3 promotes DLX2 expression through inhibition of H3K27me3 modification, thereby promoting intracranial aneurysm formation.

Co-option of the limb patterning program in cephalopod eye development.

BACKGROUND: Across the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod. RESULTS: Here we identify the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. We show radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exd, Meis/hth, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. We assess the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and find the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss. CONCLUSION: This regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, our study suggests that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures.

DLX5/6 GABAergic Expression Affects Social Vocalization: Implications for Human Evolution.

DLX5 and DLX6 are two closely related transcription factors involved in brain development and in GABAergic differentiation. The DLX5/6 locus is regulated by FoxP2, a gene involved in language evolution and has been associated with neurodevelopmental disorders and mental retardation. Targeted inactivation of Dlx5/6 in mouse GABAergic neurons (Dlx5/6VgatCre mice) results in behavioral and metabolic phenotypes notably increasing lifespan by 33%. Here, we show that Dlx5/6VgatCre mice present a hyper-vocalization and hyper-socialization phenotype. While only 7% of control mice emitted more than 700 vocalizations/10 min, 30% and 56% of heterozygous or homozygous Dlx5/6VgatCre mice emitted more than 700 and up to 1,400 calls/10 min with a higher proportion of complex and modulated calls. Hyper-vocalizing animals were more sociable: the time spent in dynamic interactions with an unknown visitor was more than doubled compared to low-vocalizing individuals. The characters affected by Dlx5/6 in the mouse (sociability, vocalization, skull, and brain shape…) overlap those affected in the "domestication syndrome". We therefore explored the possibility that DLX5/6 played a role in human evolution and "self-domestication" comparing DLX5/6 genomic regions from Neanderthal and modern humans. We identified an introgressed Neanderthal haplotype (DLX5/6-N-Haplotype) present in 12.6% of European individuals that covers DLX5/6 coding and regulatory sequences. The DLX5/6-N-Haplotype includes the binding site for GTF2I, a gene associated with Williams-Beuren syndrome, a hyper-sociability and hyper-vocalization neurodevelopmental disorder. The DLX5/6-N-Haplotype is significantly underrepresented in semi-supercentenarians (>105 years of age), a well-established human model of healthy aging and longevity, suggesting their involvement in the coevolution of longevity, sociability, and speech.

The insufficiency of Dlx5 for ventral patterning in post-migratory neural crest cells reveals a loss of plasticity in early jaw-forming tissue.

The articulated jaws of vertebrates arise from the first pharyngeal arch, the most rostral of several transient ventral structures in pharyngeal stage embryos. Migratory cranial neural crest cells from the caudal midbrain and rostral hindbrain populate the first arch as ectomesenchyme and supply the progenitors of skeletal and soft tissues that form the upper (maxillary) and lower (mandibular) jaws. Dlx genes encode key transcriptional regulators that profoundly influence jaw development through their actions in first pharyngeal arch patterning. The broadly conserved nested expression of Dlx paralogues in vertebrate embryos points to a retained ancestral role in patterning first arch tissue. Loss-of-function experiments consistently highlight the necessity of Dlx gene function for jaw morphogenesis. Specifically, the combined effects of Dlx5 and Dlx6 are required to specify ventral/mandibular fate and forced expression of Dlx5 in migrating neural crest cells results in the ectopic upregulation of ventral markers in the maxillary arch. Here, we ask whether Dlx5 is also sufficient to respecify post-migratory ectomesenchyme in the maxillary branch as mandibular. Unexpectedly, we show that Dlx5 is not sufficient to activate mandibular marker genes in the maxillary branch of PA1, highlighting a loss of plasticity in post-migratory first arch ectomesenchyme.

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.

LncRNA HOTAIR impairs the prognosis of papillary thyroid cancer via regulating cellular malignancy and epigenetically suppressing DLX1.

PURPOSE: Papillary thyroid cancer (PTC) is the most common endocrine malignancy with a fast-growing incidence in recent decades. HOTAIR as a long non-coding RNA has been shown to be highly expressed in papillary thyroid cancer tissues with only a limited understanding of its functional roles and downstream regulatory mechanisms in papillary thyroid cancer cells. METHODS: We applied three thyroid cancer cell lines (MDA-T32, MDA-T41 and K1) to investigate the phenotypic influence after gain or loss of HOTAIR. The Cancer Genome Atlas (TCGA) database were utilised to select candidate genes possibly regulated by HOTAIR with validation in the cellular system and immunohistochemical (IHC) staining of PTC tissues. RESULTS: We observed HOTAIR was highly expressed in MDA-T32 cells but presents significantly decreased levels in MDA-T41 and K1 cells. HOTAIR knockdown in MDA-T32 cells significantly suppressed proliferation, colony formation, migration with cell cycle retardation at G1 phase. On the contrary, HOTAIR overexpression in MDA-T41 cells dramatically enhanced proliferation, colony formation, migration with cell cycle driven toward S and G2/M phases. Similar phenotypic effects were also observed as overexpressing HOTAIR in K1 cells. To explore novel HOTAIR downstream mechanisms, we analyzed TCGA transcriptome in PTC tissues and found DLX1 negatively correlated to HOTAIR, and its lower expression associated with reduced progression free survival. We further validated DLX1 gene was epigenetically suppressed by HOTAIR via performing chromatin immunoprecipitation. Moreover, IHC staining shows a significantly stepwise decrease of DLX1 protein from normal thyroid tissues to stage III PTC tissues. CONCLUSIONS: Our study pointed out that HOTAIR is a key regulator of cellular malignancy and its epigenetic suppression on DLX1 serves as a novel biomarker to evaluate the PTC disease progression.

LncRNA Dlx6os1 Accelerates Diabetic Nephropathy Progression by Epigenetically Repressing SOX6 via Recruiting EZH2.

INTRODUCTION: Diabetic nephropathy (DN) is the leading cause of kidney failure worldwide. To explore the pathogenesis and effective biological target of DN is beneficial to seeking novel treatment strategies. OBJECTIVE: This study aimed to investigate the role of the lncRNA Dlx6os1/SOX6/EZH2 axis in DN progression. METHODS: PAS staining was performed to evaluate extracellular matrix accumulation; ELISA was carried out to assess the levels of urine microalbumin and blood glucose concentration; RT-qPCR was carried out to detect the levels of lncRNA Dlx6os1, TNF-α, IL-1ß, IL-6, SOX6, and EZH2. Western blot was performed to assess the levels of Col-IV, FN, TGF-ß1, and SOX6 proteins. RIP assay was carried out to verify the interaction between lncRNA Dlx6os1 and EZH2. ChIP-qPCR was conducted to verify the interaction between EZH2 and SOX6 promoter. RESULTS: Our results illustrated that lncRNA Dlx6os1 was highly expressed in DN mice and HG-induced SV40 MES13 cells. LncRNA Dlx6os1 knockdown inhibited HG-induced SV40 MES13 cell proliferation, fibrosis, and inflammatory cytokine release. LncRNA Dlx6os1 inhibited SOX6 expression by recruiting EZH2 in HG-SV40 MES13 cells, and SOX6 mediated the effects of lncRNA Dlx6os1 on proliferation, fibrosis, and inflammatory factor release of HG-induced SV40 MES13 cells. CONCLUSION: LncRNA Dlx6os1 accelerates the progression of DN by epigenetically repressing SOX6 via recruiting EZH2.

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.