Oncogenic Transformation Drives DNA Methylation Loss and Transcriptional Activation at Transposable Element Loci.

Transposable elements (TE) are typically silenced by DNA methylation and repressive histone modifications in differentiated healthy human tissues. However, TE expression increases in a wide range of cancers and is correlated with global hypomethylation of cancer genomes. We assessed expression and DNA methylation of TEs in fibroblast cells that were serially transduced with hTERT, SV40, and HRASR24C to immortalize and then transform them, modeling the different steps of the tumorigenesis process. RNA sequencing and whole-genome bisulfite sequencing were performed at each stage of transformation. TE expression significantly increased as cells progressed through transformation, with the largest increase in expression after the final stage of transformation, consistent with data from human tumors. The upregulated TEs were dominated by endogenous retroviruses [long terminal repeats (LTR)]. Most differentially methylated regions (DMR) in all stages were hypomethylated, with the greatest hypomethylation in the final stage of transformation. A majority of the DMRs overlapped TEs from the RepeatMasker database, indicating that TEs are preferentially demethylated. Many hypomethylated TEs displayed a concordant increase in expression. Demethylation began during immortalization and continued into transformation, while upregulation of TE transcription occurred in transformation. Numerous LTR elements upregulated in the model were also identified in The Cancer Genome Atlas datasets of breast, colon, and prostate cancer. Overall, these findings indicate that TEs, specifically endogenous retroviruses, are demethylated and transcribed during transformation. SIGNIFICANCE: Analysis of epigenetic and transcriptional changes in a transformation model reveals that transposable element expression and methylation are dysregulated during oncogenic transformation.

Epigenetic therapy inhibits metastases by disrupting premetastatic niches.

Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.

Defining UHRF1 Domains that Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties.

UHRF1 facilitates the establishment and maintenance of DNA methylation patterns in mammalian cells. The establishment domains are defined, including E3 ligase function, but the maintenance domains are poorly characterized. Here, we demonstrate that UHRF1 histone- and hemimethylated DNA binding functions, but not E3 ligase activity, maintain cancer-specific DNA methylation in human colorectal cancer (CRC) cells. Disrupting either chromatin reader activity reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes (TSGs), and reduces CRC oncogenic properties. Moreover, an inverse correlation between high UHRF1 and low TSG expression tracks with CRC progression and reduced patient survival. Defining critical UHRF1 domain functions and its relationship with CRC prognosis suggests directions for, and value of, targeting this protein to develop therapeutic DNA demethylating agents.

Pingwei San ameliorates dextran sulfate sodium-induced chronic colitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ping weisan (PWS), a famous traditional Chinese medicinal, is published in the Prescriptions of Taiping Benevolent Dispensary. PWS has been proven to be effective for many diseases, especially chronic diseases. AIM OF THE STUDY: The purpose of this study was to investigate the effect and potential mechanism of PWS on chronic colitis in mice. MATERIALS AND METHODS: Chronic colitis was induced in mice using 2.5% DSS for two cycles of 5 days, and different doses of PWS (2, 4, 8 g/kg) were administered throughout the experiment. The disease activity index (DAI), length of colon and pathological changes were measured. Cytokine levels in vivo and in vitro were detected by ELISA. The protein levels of TLR4, PPARγ and the key proteins in NF-κB pathway and NLRP3 inflammasome were measured by western blot. RESULTS: PWS decreased DSS-induced DAI, colon length shortening and colonic pathological damage. PWS also reduced TNF-α, IL-1ß and IL-12 production. In addition, PWS suppressed NF-κB pathway activation by regulating the expression of TLR4 and PPARγ. Our data also indicated that PWS could inhibit NLRP3 inflammasome activation. CONCLUSIONS: PWS treatment alleviated the degree of colitis caused by DSS, suggesting that PWS might be a novel agent for the treatment of chronic colitis.

Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Stemness, and BrafV600E-Induced Tumorigenesis.

We addressed the precursor role of aging-like spontaneous promoter DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show that promoter hypermethylation spontaneously arises in cells mimicking the human aging-like phenotype. The silenced genes activate the Wnt pathway, causing a stem-like state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by BrafV600E, producing the typical human proximal BRAFV600E-driven colon adenocarcinomas characterized by extensive, abnormal gene-promoter CpG-island methylation, or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitizes to BrafV600E-induced transformation. Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis.

Metastasis-related methyltransferase 1 (Merm1) represses the methyltransferase activity of Dnmt3a and facilitates RNA polymerase I transcriptional elongation.

Stimulatory regulators for DNA methyltransferase activity, such as Dnmt3L and some Dnmt3b isoforms, affect DNA methylation patterns, thereby maintaining gene body methylation and maternal methylation imprinting, as well as the methylation landscape of pluripotent cells. Here we show that metastasis-related methyltransferase 1 (Merm1), a protein deleted in individuals with Williams-Beuren syndrome, acts as a repressive regulator of Dnmt3a. Merm1 interacts with Dnmt3a and represses its methyltransferase activity with the requirement of the binding motif for S-adenosyl-L-methionine. Functional analysis of gene regulation revealed that Merm1 is capable of maintaining hypomethylated rRNA gene bodies and co-localizes with RNA polymerase I in the nucleolus. Dnmt3a recruits Merm1, and in return, Merm1 ensures the binding of Dnmt3a to hypomethylated gene bodies. Such interplay between Dnmt3a and Merm1 facilitates transcriptional elongation by RNA polymerase I. Our findings reveal a repressive factor for Dnmt3a and uncover a molecular mechanism underlying transcriptional elongation of rRNA genes.

DNA Methylation Patterns Separate Senescence from Transformation Potential and Indicate Cancer Risk.

Overall shared DNA methylation patterns between senescence (Sen) and cancers have led to the model that tumor-promoting epigenetic patterns arise through senescence. We show that transformation-associated methylation changes arise stochastically and independently of programmatic changes during senescence. Promoter hypermethylation events in transformation involve primarily pro-survival and developmental genes, similarly modified in primary tumors. Senescence-associated hypermethylation mainly involves metabolic regulators and appears early in proliferating "near-senescent" cells, which can be immortalized but are refractory to transformation. Importantly, a subset of transformation-associated hypermethylated developmental genes exhibits highest methylation gains at all age-associated cancer risk states across tissue types. These epigenetic changes favoring cell self-renewal and survival, arising during tissue aging, are fundamentally important for stratifying cancer risk and concepts for cancer prevention.

Biochemical Studies and Molecular Dynamic Simulations Reveal the Molecular Basis of Conformational Changes in DNA Methyltransferase-1.

DNA methyltransferase-1 (DNMT1) plays a crucial role in the maintenance of genomic methylation patterns. The crystal structure of DNMT1 was determined in two different states in which the helix that follows the catalytic loop was either kinked (designated helix-kinked) or well folded (designated helix-straight state). Here, we show that the proper structural transition between these two states is required for DNMT1 activity. The mutations of N1248A and R1279D, which did not affect interactions between DNMT1 and substrates or cofactors, allosterically reduced enzymatic activities in vitro by decreasing kcat/ Km for AdoMet. The crystallographic data combined with molecular dynamic (MD) simulations indicated that the N1248A and R1279D mutants bias the catalytic helix to either the kinked or straight conformation. In addition, genetic complementation assays for the two mutants suggested that disturbing the conformational transition reduced DNMT1 activity in cells, which could act additively with existing DNMT inhibitors to decrease DNA methylation. Collectively, our studies provide molecular insights into conformational changes of the catalytic helix, which is essential for DNMT1 catalytic activity, and thus aid in better understanding the relationship between DNMT1 dynamic switching and enzymatic activity.

Loss of Barx1 promotes hepatocellular carcinoma metastasis through up-regulating MGAT5 and MMP9 expression and indicates poor prognosis.

Metastasis is the major dominant reason for poor prognosis of hepatocellular carcinoma (HCC) after surgical treatment. However, the molecular mechanism of metastasis has not been well characterzied. Here, we report a novel function of Barx homeobox1 (Barx1) in inhibiting HCC invasion and metastasis. Barx1 expression is significantly decreased in human HCC tissues than in adjacent non-tumorous tissues and normal liver tissues. Low Barx1 expression is correlated with higher tumor-nodule-metastasis stage and indicates poor prognosis. Down-regulation of Barx1 promotes HCC migration, invasion and metastasis, whereas up-regulation of Barx1 inhibits HCC migration, invasion and metastasis. Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase 5 (MGAT5) and matrix metallopeptidase 9 (MMP9) are direct target genes of Barx1. Knockdown of Barx1 up-regulates MGAT5 and MMP9 expression in HCC cells with low metastatic capability, whereas over-expression of Barx1 suppresses their expression in HCC cells with high metastatic capability. Knockdown of both MGAT5 and MMP9 significantly decreases the invasion and metastasis abilities induced by Barx1 knockdown. Barx1 expression is negatively correlated with MGAT5 and MMP9 expression in human HCC tissues. Patients with low expression of Barx1 and high expression of MGAT5 or MMP9 are associated with poorer prognosis. Thus, loss of Barx1 represents a prognostic biomarker in human HCC patients.

CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes.

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing. CHD4 is recruited by the excision repair protein OGG1 for oxidative damage to interact with the damage-induced base 8-hydroxydeoxyguanosine (8-OHdG), while ZMYND8 recruits it to double-strand breaks. CHD4 knockdown activates silenced TSGs, revealing their role for blunting colorectal cancer cell proliferation, invasion, and metastases. High CHD4 and 8-OHdG levels plus low expression of TSGs strongly correlates with early disease recurrence and decreased overall survival.

Acetylation Enhances TET2 Function in Protecting against Abnormal DNA Methylation during Oxidative Stress.

TET proteins, by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are hypothesized, but not directly shown, to protect promoter CpG islands (CGIs) against abnormal DNA methylation (DNAm) in cancer. We define such a protective role linked to DNA damage from oxidative stress (OS) known to induce this abnormality. TET2 removes aberrant DNAm during OS through interacting with DNA methyltransferases (DNMTs) in a "Yin-Yang" complex targeted to chromatin and enhanced by p300 mediated TET2 acetylation. Abnormal gains of DNAm and 5hmC occur simultaneously in OS, and knocking down TET2 dynamically alters this balance by enhancing 5mC and reducing 5hmC. TET2 reduction results in hypermethylation of promoter CGIs and enhancers in loci largely overlapping with those induced by OS. Thus, TET2 indeed may protect against abnormal, cancer DNAm in a manner linked to DNA damage.

DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells.

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila.

Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

[Simultaneous determination of gallic acid and hesperidin contained in Xiaogu capsule by HPLC].

OBJECTIVE: To develop an HPLC method for simultaneous determination of gallic acid and hesperidin in Xiaogu capsule, in order to provide a simple, rapid and accurate method for quality control of the compound preparation of traditional Chinese medicine. METHOD: Xiaogu capsule was extracted with methanol heating reflux method. Synergi 4 mu Hydro-RP 80A (4.6 mm x 250 mm, 5 microm) was adopted as the chromatographic column, with acetonitrile--0.04 mol x L(-1) phosphate monobasic sodium solution (20: 80) as the mobile phase. The flow rate was 1.0 mL x min(-1), the detection wavelength was 283 nm, and the column temperature was 25 degrees C. RESULT: Under the conditions, gallic acid and hesperidin reached the baseline resolved peak, with a good linearity within the range of 21.6-216.0 mg x L(-1) (r = 0.999 93) for gallic acid, and 4.5-45.0 mg x L(-1) (r = 0.999 95) for hesperidin, respectively. Their average recoveries (n = 9) were 101.5% (RSD 3.7%) and 94.7% (RSD 2.7%), respectively. The average contents of gallic acid and hesperidin contained in Xiaogu capsule were detected to 5.10% and 0.091 1%, respectively. CONCLUSION: The method established in this study can determine the content of gallic acid and hesperidin contained in Xiaogu capsule in a rapid and accurate manner, which provided reference for quality evaluation of the medicine.

CHD4/NuRD maintains demethylation state of rDNA promoters through inhibiting the expression of the rDNA methyltransferase recruiter TIP5.

Despite the well-established fact that NuRD (nucleosome remodeling and histone deacetylase) is incapable of actively demethylating DNA, the complex is surprisingly showed to be required for the establishment of unmethylated state at promoters of ribosomal genes. But the molecular mechanism underlying how NuRD mediates unmethylation at rDNA promoters remains obscure. Here we show that NuRD directly binds to the promoter of rDNA transcription silencer TIP5 (TTF-I interacting protein 5), one of the components of nucleolar remodeling complex NoRC that silences rRNA genes by recruiting DNA methyltransferase to rDNA promoters and increasing DNA methylation. NuRD negatively regulates TIP5 expression, thereby inhibiting rDNA methylation and maintaining demethylation state of rDNA promoters. The deficiency of NuRD components in reprogrammed cells activates TIP5 expression, resulting in the increased fraction of heterochromatic rRNA genes and transcriptional silencing. Thus, NuRD is able to control methylation status of rDNA promoters through crosstalking with NoRC complex.

The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c-Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming.

The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

[Speciation of arsenic(III) and arsenic(V) in water by hydride generation atomic fluorescence spectrometry].

A method for arsenic(III) and arsenic(V) speciation in water samples using HG-AFS was established. Without any preseparation technique, the speciation was simply accomplished by just adjustment of the acidity for hydride generation. For a sensitive, reproducible and accurate determination, other conditions for hydride generation, such as the concentration of NaBH4 and the added amount of KI as the reductant for arsenic(V), were also optimized, and the interference experiment was carried out for concomitant elements. As a result, a detection limit of 0.026 microg x L(-1), a relative standard deviation of 2%, and a recovery of 97. 5%-103. 0% were obtained, indicating the robustness of the method.