Characterization of 3D Organotypic Culture of Mouse Adipose-Derived Stem Cells.

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal-epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms.

Inducing positive inotropy in human iPSC-derived cardiac muscle by gene editing-based activation of the cardiac α-myosin heavy chain.

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Breast cancer malignancy is governed by regulation of the macroH2A2/TM4SF1 axis, the AKT/NF-κB pathway, and elevated MMP13 expression.

The histone variant, macroH2A (mH2A) influences gene expression through epigenetic regulation. Tumor suppressive function of mH2A isoforms has been reported in various cancer types, but few studies have investigated the functional role of mH2A2 in breast cancer pathophysiology. This study aimed to determine the significance of mH2A2 in breast cancer development and progression by exploring its downstream regulatory mechanisms. Knockdown of mH2A2 facilitated the migration and invasion of breast cancer cells, whereas its overexpression exhibited the opposite effect. In vivo experiments revealed that augmenting mH2A2 expression reduced tumor growth and lung metastasis. Microarray analysis showed that TM4SF1 emerged as a likely target linked to mH2A2 owing to its significant suppression in breast cancer cell lines where mH2A2 was overexpressed among the genes that exhibited over twofold upregulation upon mH2A2 knockdown. Suppressing TM4SF1 reduced the migration, invasion, tumor growth, and metastasis of breast cancer cells in vitro and in vivo. TM4SF1 depletion reversed the increased aggressiveness triggered by mH2A2 knockdown, suggesting a close interplay between mH2A2 and TM4SF1. Our findings also highlight the role of the mH2A2/TM4SF1 axis in activating the AKT/NF-κB pathway. Consequently, activated NF-κB signaling leads to increased expression and secretion of MMP13, a potent promoter of metastasis. In summary, we propose that the orchestrated regulation of the mH2A2/TM4SF1 axis in conjunction with the AKT/NF-κB pathway and the subsequent elevation in MMP13 expression constitute pivotal factors governing the malignancy of breast cancer.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Downregulation of complement factor H attenuates the stemness of MDA­MB­231 breast cancer cells via modulation of the ERK and p38 signaling pathways.

The complement system is a powerful innate immune system deployed in the immediate response to pathogens and cancer cells. Complement factor H (CFH), one of the regulators involved in the complement cascade, can interrupt the death of target cells. Certain types of cancer, such as breast cancer, can adopt an aggressive phenotype, such as breast cancer stem cells (BCSCs), through enhancement of the defense system against complement attack by amplifying various complement regulators. However, little is known about the association between CFH and BCSCs. In the present study, the roles of CFH in the CSC characteristics and radioresistance of MDA-MB-231 human breast cancer cells were investigated. CFH knockdown in MDA-MB-231 cells decreased the viability of the cells upon complement cascade activation. Notably, CFH knockdown also decreased cell survival and suppressed mammosphere formation, cell migration and cell invasion by attenuating radioresistance. Additionally, CFH knockdown further enhanced irradiation-induced apoptosis through G2/M cell cycle arrest. It was also discovered that CFH knockdown attenuated the aggressive phenotypes of cancer cells by regulating CSC-associated gene expression. Finally, by microarray analysis, it was found that the expression of erythrocyte membrane protein band 4.1-like 3 (EPB41L3) was markedly increased following CFH knockdown. EPB41L3 inhibited ERK and activated the p38 MAPK signaling pathway. Taken together, these results indicated that CFH knockdown attenuated CSC properties and radioresistance in human breast cancer cells via controlling MAPK signaling and through upregulation of the tumor suppressor, EPB41L3.

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

Genomic targets and selective inhibition of DNA methyltransferase isoforms.

BACKGROUND: DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. RESULTS: We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δß > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (ß: 0.2-0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δß > 0.1) was observed at 121 of the 353 loci associated with the Horvath 'epigenetic clock' model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5-15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. CONCLUSIONS: Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome.

G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer.

Epigenetic abnormalities affect tumor progression, as well as gene expression and function. Among the diverse epigenetic modulators, the histone methyltransferase G9a has been focused on due to its role in accelerating tumorigenesis and metastasis. Although epigenetic dysregulation is closely related to tumor progression, reports regarding the relationship between G9a and its possible downstream factors regulating breast tumor growth are scarce. Therefore, we aimed to verify the role of G9a and its presumable downstream regulators during malignant progression of breast cancer. G9a-depleted MCF7 and T47D breast cancer cells exhibited suppressed motility, including migration and invasion, and an improved response to ionizing radiation. To identify the possible key factors underlying these effects, microarray analysis was performed, and a TGF-ß superfamily member, BMP5, was selected as a prominent target gene. It was found that BMP5 expression was markedly increased by G9a knockdown. Moreover, reduction in the migration/invasion ability of MCF7 and T47D breast cancer cells was induced by BMP5. Interestingly, a G9a-depletion-mediated increase in BMP5 expression induced the phosphorylation of Smad proteins, which are the intracellular signaling mediators of BMP5. Accordingly, we concluded that the observed antitumor effects may be based on the G9a-depletion-mediated increase in BMP5 expression and the consequent facilitation of Smad protein phosphorylation.

A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency.

CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

Longitudinal Imaging of Liver Cancer Using MicroCT and Nanoparticle Contrast Agents in CRISPR/Cas9-Induced Liver Cancer Mouse Model.

INTRODUCTION: Micro-computed tomography with nanoparticle contrast agents may be a suitable tool for monitoring the time course of the development and progression of tumors. Here, we suggest a practical and convenient experimental method for generating and longitudinally imaging murine liver cancer models. METHODS: Liver cancer was induced in 6 experimental mice by injecting clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 plasmids causing mutations in genes expressed by hepatocytes. Nanoparticle agents are captured by Kupffer cells and detected by micro-computed tomography, thereby enabling longitudinal imaging. A total of 9 mice were used for the experiment. Six mice were injected with both plasmids and contrast, 2 injected with contrast alone, and one not injected with either agent. Micro-computed tomography images were acquired every 2- up to 14-weeks after cancer induction. RESULTS: Liver cancer was first detected by micro-computed tomography at 8 weeks. The mean value of hepatic parenchymal attenuation remained almost unchanged over time, although the standard deviation of attenuation, reflecting heterogeneous contrast enhancement of the hepatic parenchyma, increased slowly over time in all mice. Histopathologically, heterogeneous distribution and aggregation of Kupffer cells was more prominent in the experimental group than in the control group. Heterogeneous enhancement of hepatic parenchyma, which could cause image quality deterioration and image misinterpretation, was observed and could be due to variation in Kupffer cells distribution. CONCLUSION: Micro-computed tomography with nanoparticle contrast is useful in evaluating the induction and characteristics of liver cancer, determining appropriate size of liver cancer for testing, and confirming therapeutic response.

Low level DUX4 expression disrupts myogenesis through deregulation of myogenic gene expression.

Loss of silencing of the DUX4 gene on chromosome 4 causes facioscapulohumeral muscular dystrophy. While high level DUX4 expression induces apoptosis, the effects of low level DUX4 expression on human myogenic cells are not well understood. Low levels and sporadic expression of DUX4 have been reported in FSHD biopsy samples and myoblast cultures. Here, we show that a large set of human myogenic genes is rapidly deregulated by DUX4, including MYOD1 and MYF5, which are efficiently repressed even by low, non-toxic levels of DUX4. Human myoblasts modified to express low nontoxic levels of DUX4 were significantly impaired from differentiating into myotubes in vitro. Surprisingly, inhibition of differentiation does not require the transcriptional activation domain, thus is likely a feature of all mammalian DUX genes. DUX4 does not bind near the MYF5 gene, but has a prominent ChIP-seq peak within the MYF5 -118 kb enhancer. We find that when DUX4 binds at this site, it directs enhancer activity towards a nearby transcriptional start site for a noncoding nonfunctional RNA we name DIME (DUX4-induced MYF5 enhancer) transcript. These data highlight the anti-myogenic properties of DUX4 in human myogenic progenitor cells, and provide an example of enhancer disruption in the downregulation of MYF5.

Smek1/2 is a nuclear chaperone and cofactor for cleaved Wnt receptor Ryk, regulating cortical neurogenesis.

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

Downregulation of UHRF1 increases tumor malignancy by activating the CXCR4/AKT-JNK/IL-6/Snail signaling axis in hepatocellular carcinoma cells.

UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) plays a crucial role in DNA methylation, chromatin remodeling and gene expression and is aberrantly upregulated in various types of human cancers. However, the precise role of UHRF1 in cancer remains controversial. In this study, we observed that hypoxia-induced downregulation of UHRF1 contributes to the induction of the epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells. By negatively modulating UHRF1 expression, we further showed that UHRF1 deficiency in itself is sufficient to increase the migratory and invasive properties of cells via inducing EMT, increasing the tumorigenic capacity of cells and leading to the expansion of cancer stem-like cells. Epigenetic changes caused by UHRF1 deficiency triggered the upregulation of CXCR4, thereby activating AKT and JNK to increase the expression and secretion of IL-6. In addition, IL-6 readily activated the JAK/STAT3/Snail signaling axis, which subsequently contributed to UHRF1 deficiency-induced EMT. Our results collectively demonstrate that UHRF1 deficiency may play a pivotal role in the malignant alteration of cancer cells.

Smek promotes corticogenesis through regulating Mbd3's stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis.

The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG-binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs' self-renewal and neuronal differentiation during mammalian corticogenesis.

Transcriptional Inhibitors Identified in a 160,000-Compound Small-Molecule DUX4 Viability Screen.

Facioscapulohumeral muscular dystrophy is a genetically dominant, currently untreatable muscular dystrophy. It is caused by mutations that enable expression of the normally silent DUX4 gene, which encodes a pathogenic transcription factor. A screen based on Tet-on DUX4-induced mouse myoblast death previously uncovered compounds from a 44,000-compound library that protect against DUX4 toxicity. Many of those compounds acted downstream of DUX4 in an oxidative stress pathway. Here, we extend this screen to an additional 160,000 compounds and, using greater stringency, identify a new set of DUX4-protective compounds. From 640 hits, we performed secondary screens, repurchased 46 of the most desirable, confirmed activity, and tested each for activity against other cell death-inducing insults. The majority of these compounds also protected against oxidative stress. Of the 100 repurchased compounds identified through both screens, only SHC40, 75, and 98 inhibited DUX4 target genes, but they also inhibited dox-mediated DUX4 expression. Using a target gene readout on the 640-compound hit set, we discovered three overlooked compounds, SHC351, 540, and 572, that inhibit DUX4 target gene upregulation without nonspecific effects on the Tet-on system. These novel inhibitors of DUX4 transcriptional activity may thus act on pathways or cofactors needed by DUX4 for transcriptional activation in these cells.

Low-dose irradiation promotes Rad51 expression by down-regulating miR-193b-3p in hepatocytes.

Current evidence indicates that there is a relationship between microRNA (miRNA)-mediated gene silencing and low-dose irradiation (LDIR) responses. Here, alterations of miRNA expression in response to LDIR exposure in male BALB/c mice and three different types of hepatocytes were investigated. The miRNome of the LDIR-exposed mouse spleens (0.01 Gy, 6.5 mGy/h) was analyzed, and the expression of miRNA and mRNA was validated by qRT-PCR. Western blotting, chromatin immunoprecipitation (ChIP), and luciferase assays were also performed to evaluate the interaction between miRNAs and their target genes and to gain insight into the regulation of miRNA expression. The expression of miRNA-193b-3p was down-regulated in the mouse spleen and liver and in various hepatocytes (NCTC, Hepa, and HepG2 cell lines) in response to LDIR. The down-regulation of miR-193b-3p expression was caused by histone deacetylation on the miR-193b-3p promoter in the HepG2 cells irradiated with 0.01 Gy. However, the alteration of histone deacetylation and miR-193b-3p and Rad51 expression in response to LDIR was restored by pretreatment with N-acetyl-cyctein. In conclusion, we provide evidence that miRNA responses to LDIR include the modulation of cellular stress responses and repair mechanisms.

DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.

Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation.

DNA-binding sequence specificity of DUX4.

BACKGROUND: Misexpression of the double homeodomain transcription factor DUX4 results in facioscapulohumeral muscular dystrophy (FSHD). A DNA-binding consensus with two tandem TAAT motifs based on chromatin IP peaks has been discovered; however, the consensus has multiple variations (flavors) of unknown relative activity. In addition, not all peaks have this consensus, and the Pitx1 promoter, the first DUX4 target sequence mooted, has a different TAAT-rich sequence. Furthermore, it is not known whether and to what extent deviations from the consensus affect DNA-binding affinity and transcriptional activation potential. RESULTS: Here, we take both unbiased and consensus sequence-driven approaches to determine the DNA-binding specificity of DUX4 and its tolerance to mismatches at each site within its consensus sequence. We discover that the best binding and the greatest transcriptional activation are observed when the two TAAT motifs are separated by a C residue. The second TAAT motif in the consensus sequence is actually (T/C)AAT. We find that a T is preferred here. DUX4 has no transcriptional activity on "half-sites", i.e., those bearing only a single TAAT motif. We further find that DUX4 does not bind to the TAATTA motif in the Pitx1 promoter, that Pitx1 sequences have no competitive band shift activity, and that the Pitx1 sequence is transcriptionally inactive, calling into question PITX1 as a DUX4 target gene. Finally, by multimerizing binding sites, we find that DUX4 transcriptional activation demonstrates tremendous synergy and that at low DNA concentrations, at least two motifs are necessary to detect a transcriptional response. CONCLUSIONS: These studies illuminate the DNA-binding sequence preferences of DUX4.

Downregulation of UHRF1 promotes EMT via inducing CXCR4 in human cancer cells.

Activation of epithelial-mesenchymal transition (EMT) is important for malignant tumor progression exhibiting migratory and invasive properties. UHRF1 (ubiquitin-like, with PHD and RING finger domains 1), as an epigenetic regulator, plays a crucial role in DNA CpG methylation, chromatin remodeling and gene expression. Many studies demonstrated that UHRF1 is aberrantly expressed in various types of human cancer. However, the precise role of UHRF1 in human cancers remains highly controversial. In the present study, we found that downregulation of UHRF1 enhances the migratory and invasive properties of human cancer cells by inducing EMT, and that the CXCR4 signaling pathway is strictly necessary for UHRF1 deficiency-mediated induction of EMT. Downregulation of UHRF1 induced the expression of the EMT-regulating transcription factors, Zeb1, Slug and Snail and then led to decreased protein level of E-cadherin, and increased protein level of N-cadherin and vimentin, including increased migratory and invasive properties of human cancer cells. In addition, siRNA targeting of Zeb1 or Snail effectively attenuated UHRF1 deficiency-induced EMT, but siRNA targeting of Slug did not, indicating that Zeb1 and Snail play key roles in this event. Moreover, downregulation of UHRF1 induced the expression of CXCR4 in HepG2 cells. siRNA targeting of CXCR4 greatly suppressed the UHRF1 deficiency-induced EMT, as evidenced by a reversal of expression patterns of Snail and Zeb1, and by reduced migratory and invasive properties of HepG2 cells. In conclusion, our results demonstrate that downregulation of UHRF1 contributes to the induction of EMT in human cancer cells via the activation of CXCR4 signaling pathway. Our observation also suggests that UHRF1 may play a pivotal role in suppressing the malignant alteration of cancer cells.