Promoter Histone Deacetylation Research Articles

Gain of pancreatic beta cell-specific SCD1 improves glucose homeostasis by maintaining functional beta cell mass under metabolic stress.

The key pancreatic beta cell transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA) is critical for the maintenance of mature beta cell function and phenotype. The expression levels and/or activities of MafA are reduced when beta cells are chronically exposed to diabetogenic stress, such as hyperglycaemia (i.e. glucotoxicity). Interventional targets and adjuvant therapies to abate MafA loss in beta cells may provide evidence to support the effective treatment of diabetes. In this study, we aimed to investigate the function of stearoyl-CoA desaturase 1 (SCD1) in the stabilisation of MafA expression and activity in order to maintain functional beta cell mass, with a view to suppressing the development of type 2 diabetes. SCD1 expression levels were analysed in islets obtained from humans with type 2 diabetes, hyperglycaemic db/db mice, and a high-fat diet (HFD)-induced mouse model of diabetes. Pancreatic beta cell-specific Scd1 knockin (βSCD1KI) mice were generated to study the role of SCD1 in beta cell function and identity. The protein-to-protein interactions between SCD1 and MafA were detected in MIN6 and HEK293A cells. We used experiments including chromatin immunoprecipitation, cell-based ubiquitination assay and fatty acid composition analysis to investigate the specific molecular mechanism underlying the effect of SCD1 on the restoration of MafA and beta cell function under glucotoxic conditions. SCD1 expression was reduced in beta cells of humans with type 2 diabetes and in HFD-fed and db/db mice compared with healthy controls, which was attributed to glucotoxicity-induced Scd1 promoter histone deacetylation. Gain-of-function of SCD1 in beta cells improved insulin deficiency, glucose intolerance and beta cell dedifferentiation/transdifferentiation in the HFD-induced mouse model of diabetes. Mechanistically, SCD1 directly bound to the E3 ubiquitin ligase HMG-CoA reductase degradation 1 (HRD1) and stabilised nuclear MafA through interrupting MafA-HRD1 interactions in mouse islets and MIN6 cells, which inhibited the ubiquitination-mediated degradation of MafA. Moreover, the products of SCD enzyme reactions (mainly oleic acid) also alleviated glucotoxicity-mediated oxidative stress in MIN6 cells. Our findings indicate that SCD1 stabilises beta cell MafA both in desaturase-dependent and -independent manners, thus improving glucose homeostasis under metabolic stress. This provides a potential novel target for precision medicine for the treatment of diabetes.

VvMYBA1 and VvMYB3 form an activator–repressor system to regulate anthocyanin biosynthesis in grape

Anthocyanins are important metabolites that provide a red or blue–purple hue to plants. The biosynthesis of these metabolites is mainly activated by the MYB-bHLH-WD40 (MBW) complex and repressed by a wide variety of proteins. Studies have shown that MYB activators activate MYB repressors to balance anthocyanin biosynthesis. However, there is a scarcity of studies investigating this mechanism in grapes. To explore the transcription factors involved in the regulation of anthocyanin biosynthesis, we reanalyzed the RNA-seq database for different developmental stages of ‘Muscat Hamburg’ berries, and the R2R3-MYB gene, annotated as VvMYB3, was screened. Our study revealed the anthocyanin content of the grape cultivar ‘Y73’ was higher than that of its parental cultivar MH, and the putative repressor VvMYB3 was found to be highly expressed in ‘Y73’ by qRT-PCR. The calli transgenic assays demonstrated that the repressive activity of VvMYB3 was conferred by the bHLH-binding motif, as well as by the C1 and C2 motifs. Yeast hybridization and chip-PCR assays revealed that VvMYB3 could repress anthocyanin biosynthesis by competing with VvMYBA1 to bind to VvMYC1 and promoting histone deacetylation of VvUFGT via the C2 motif. However, the expression of VvMYB3 was activated by VvMYBA1, which forms a negative feedback regulatory loop to modulate anthocyanin accumulation. In addition, we found a 408-bp repeat tandem sequence insertion in the VvMYBA1 promoter region of ‘Y73’ by sequencing. The GUS activity analysis showed that this sequence enhanced the expression of VvMYBA1 and led to an excessive accumulation of anthocyanins. Overall, our results provide insights into the anthocyanin activator–repressor system in grapes that prevents overaccumulation of anthocyanins.

SAHA/5-AZA Enhances Acetylation and Degradation of mutp53, Upregulates p21 and Downregulates c-Myc and BRCA-1 in Pancreatic Cancer Cells.

Epigenetic changes are common in cancer and include aberrant DNA methylation and histone modifications, including both acetylation or methylation. DNA methylation in the promoter regions and histone deacetylation are usually accompanied by gene silencing, and may lead to the suppression of tumor suppressors in cancer cells. An interaction between epigenetic pathways has been reported that could be exploited to more efficiently target aggressive cancer cells, particularly those against which current treatments usually fail, such as pancreatic cancer. In this study, we explored the possibility to combine the DNA demethylating agent 5-AZA with HDAC inhibitor SAHA to treat pancreatic cancer cell lines, focusing on the acetylation of mutp53 and the consequences on its stability, as well as on the interaction of this protein with c-myc and BRCA-1, key molecules in cancer survival. The results obtained suggest that SAHA/5-AZA combination was more effective than single treatments to promote the degradation of mutp53, to upregulate p21 and downregulate c-Myc and BRCA-1, thus increasing DNA damage and cytotoxicity in pancreatic cancer cells.

The GATAD2B-NuRD complex drives DNA:RNA hybrid-dependent chromatin boundary formation upon DNA damage

Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B–NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B–NuRD complex leads to chromatin hyperrelaxation and extended DNA end resection, resulting in homologous recombination (HR) repair failure. Our results suggest that the GATAD2B–NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.

Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

Glucocorticoids promote steroid-induced osteonecrosis of the femoral head by down-regulating serum alpha-2-macroglobulin to induce oxidative stress and facilitate SIRT2-mediated BMP2 deacetylation

Our study investigated the possible molecular mechanism of glucocorticoid in steroid-induced osteonecrosis of the femoral head (SINFH) through regulating serum alpha-2-macroglobulin and SIRT2-mediated BMP2 deacetylation. Essential genes involved in glucocorticoid-induced SINFH were screened by transcriptome sequencing and analyzed by bioinformatics, followed by identifying downstream regulatory targets. Rat bone marrow mesenchymal stem cells were isolated and treated with methylprednisolone (MP) for in vitro cell experiments. Besides, a glucocorticoid-induced rat ONFH was established using the treatment of MP and LPS. ChIP-PCR detected the enrichment of SIRT2 in the promoter region of BMP2, and the deacetylation modification of SIRT2 on BMP2 was determined. Bioinformatics analysis revealed that glucocorticoids may induce ONFH through the SIRT2/BMP2 axis. In vitro cell experiments showed that glucocorticoids up-regulated SIRT2 expression in BMSCs by inducing oxidative stress, thereby promoting cell apoptosis. The up-regulation of SIRT2 expression may be due to the decreased ability of α2 macroglobulin to inhibit oxidative stress, and the addition of NOX protein inhibitor DPI could significantly inhibit SIRT2 expression. SIRT2 could promote histone deacetylation of the BMP2 promoter and inhibit its expression. In vitro cell experiments further indicated that knocking down SIRT2 could protect BMSC from oxidative stress and cell apoptosis induced by glucocorticoids by promoting BMP2 expression. In addition, animal experiments conducted also demonstrated that the knockdown of SIRT2 could improve glucocorticoid-induced ONFH through up-regulating BMP2 expression. Glucocorticoids could induce oxidative stress by down-regulating serum α2M to promote SIRT2-mediated BMP2 deacetylation, leading to ONFH.

CircAGO2 promotes colorectal cancer progression by inhibiting heat shock protein family B (small) member 8 via miR-1-3p/retinoblastoma binding protein 4 axis.

This paper was to uncover the mechanism of circular RNA Argonaute 2 (circAGO2) in colorectal cancer (CRC) progression. The expression of circAGO2 was detected in CRC cells and tissues, and the relationship between clinicopathological features of CRC and circAGO2 level was evaluated. The growth and invasion of CRC cells and subcutaneous xenograft of nude mice were measured to evaluate the effect of circAGO2 on CRC development. Bioinformatics databases were applied to analyze levels of retinoblastoma binding protein 4 (RBBP4) and heat shock protein family B 8 (HSPB8) in cancer tissues. The relevance of circAGO2 and RBBP4 expression and the relationship between RBBP4 and HSPB8 during histone acetylation were assessed. The targeting relationship between miR-1-3p and circAGO2 or RBBP4 was predicted and confirmed. The effects of miR-1-3p and RBBP4 on biological functions of CRC cells were also verified. CircAGO2 was upregulated in CRC. CircAGO2 promoted the growth and invasion of CRC cells. CircAGO2 competitively bound to miR-1-3p and regulated RBBP4 expression, thus inhibiting HSPB8 transcription by promoting histone deacetylation. Silencing circAGO2 enhanced miR-1-3p expression and reduced RBBP4 expression, while suppression of miR-1-3p downgraded levels of miR-1-3p, up-regulated RBBP4, and facilitated cell proliferation and invasion in the presence of silencing circAGO2. RBBP4 silencing decreased RBBP4 expression and reduced proliferation and invasion of cells where circAGO2 and miR-1-3p were silenced. CircAGO2 overexpression decoyed miR-1-3p to increase RBBP4 expression, which inhibited HSPB8 transcription via histone deacetylation in HSPB8 promoter region, promoting proliferation and invasion of CRC cells.

MIR497HG-Derived miR-195 and miR-497 Mediate Tamoxifen Resistance via PI3K/AKT Signaling in Breast Cancer.

Tamoxifen is commonly used for the treatment of patients with estrogen receptor-positive (ER+) breast cancer, but the acquired resistance to tamoxifen presents a critical challenge of breast cancer therapeutics. Recently, long noncoding RNA MIR497HG and its embedded miR-497 and miR-195 are proved to play significant roles in many types of human cancers, but their roles in tamoxifen-resistant breast cancer remain unknown. The results indicate that MIR497HG deficiency induces breast cancer progression and tamoxifen resistance by inducing downregulation of miR-497/195. miR-497/195 coordinately represses five positive PI3K-AKT regulators (MAP2K1, AKT3, BCL2, RAF1, and CCND1), resulting in inhibition of PI3K-AKT signaling, and PI3K-AKT inhibition in tamoxifen-resistant cells restored tamoxifen responsiveness. Furthermore, ER α binds the MIR497HG promoter to activate its transcription in an estrogen-dependent manner. ZEB1 interacts with HDAC1/2 and DNMT3B at the MIR497HG promoter, resulting in promoter hypermethylation and histone deacetylation. The findings reveal that ZEB1-induced MIR497HG depletion contributes to breast cancer progression and tamoxifen resistance through PI3K-AKT signaling. MIR497HG can be used as a biomarker for predicting tamoxifen sensitivity in patients with ER+ breast cancer.

Selective CDK9 Inhibition by Natural Compound Toyocamycin in Cancer Cells.

Simple SummaryBy combining drug screens, transcriptomic studies, and in vitro assays, our study identified the natural product toyocamycin as a potent and selective CDK9 inhibitor. Thus, toyocamycin can be used as a new small molecule to modulate CDK9 activity in preclinical research. Through docking simulations, we identified its specific binding sites, which could spark some interest to design novel small molecule CDK9 inhibitors.Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning.

The pregnane X receptor (PXR, NR1I2) is a nuclear receptor which exerts its regulatory function by heterodimerization with the retinoid-X-receptor α (RXRα, NR2B1) and binding to the promoter and enhancer regions of diverse target genes. PXR is involved in the regulation of drug metabolism and excretion, metabolic and immunological functions and cancer pathogenesis. PXR activity is strongly regulated by the association with coactivator and corepressor proteins. Coactivator proteins exhibit histone acetyltransferase or histone methyltransferase activity or associate with proteins having one of these activities, thus promoting chromatin decondensation and activation of the gene expression. On the contrary, corepressor proteins promote histone deacetylation and therefore favor chromatin condensation and repression of the gene expression. Several studies pointed to clear cell- and ligand-specific differences in the activation of PXR. In this article, we will review the critical role of coactivator and corepressor proteins as molecular determinants of the specificity of PXR-mediated effects. As already known for other nuclear receptors, understanding the complex mechanism of PXR activation in each cell type and under particular physiological and pathophysiological conditions may lead to the development of selective modulators with therapeutic potential.

BRMS1L inhibits bone metastasis of breast cancer cells through epigenetic silence of CXCR4.

e13002 Background: We previously demonstrated that BRMS1L (breast cancer metastasis suppressor 1 like) suppresses breast cancer metastasis through HDAC1 recruitment and histone H3K9 deacetylation at the promoter of FZD10, a receptor for Wnt signaling. It is still unclear whether BRMS1L regulates organ-specific metastases, such as bone metastasis, the most prevalent metastatic site of breast cancer. Methods: Examination of the expression of BRMS1L in primary tumors, bone metastatic and other metastatic tissues from breast cancer patients was implemented using qRT-PCR and immunohistochemistry staining. To investigate the mechanism by which BRMS1L drives breast cancer bone metastasis, we tested the mRNA expression by qRT-PCR of a set of potential bone related genes (BRGs) based on PubMed database in MDA-MB-231 cells over expressing BRMS1L and MCF-7 cells knocking-down BRMS1L, and detected the expression of CXCR4 in these established cells by western blot. Transwell assays were performed to assess the migration abilities of breast cancer cells towards osteoblasts. ChIP (Chromatin Immuno-Precipitation) were employed to test the interaction between BRMS1L and CXCR4. Results: At both mRNA and protein levels, the expression of BRMS1L was significantly lower in bone metastatic sites than that in primary cancer tissues and other metastatic sites of breast cancer patients. CXCR4 was screened out in a set of BRGs and negatively correlated with the expression of BRMS1L in breast cancer cell lines. BRMS1L inhibited the migration of breast cancer cells towards osteoblasts through CXCL12/CXCR4 axis. In the presence of TSA treatment, breast cancer cell lines showed an increased expression of CXCR4 in a TSA concentration-dependent manner. In addition, ChIP assays verified that BRMS1L directly bound to the promoter region of CXCR4 and inhibited its transcription through promoter histone deacetylation. Conclusions: BRMS1L mediates the migration abilities of breast cancer cells to bone microenvironment via targeting CXCR4 and contributes to bone metastasis of breast cancer cells. Thus, BRMS1L may be a potential biomarker for predicting bone metastasis in breast cancer.

Investigation of the Effect of 5-Aza-2'-Deoxycytidine in Comparison to and in Combination with Trichostatin A on p16INK4a, p14ARF, p15INK4b Gene Expression, Cell Growth Inhibition and Apoptosis Induction in Colon Cancer Caco-2 Cell Line.

Background:The cell cycle is divided into four phases, G1, G2, S, and M phase. The mammalian cell cycle is controlled and governed by the kinase complexes including cyclin and the cyclin-dependent kinase (CDK), cyclin-CDK complexes. The activity of the complexes is regulated by cyclin-dependent kinase inhibitors (CDKIs), the INK4, and the CDK interacting protein/kinase inhibitory protein (CIP/KIP) families. Promoter hypermethylation and histone deacetylation of CDKIs have been reported in several cancers. These changes can be reversed by DNA demethylating agents, such as decitabine, 5-Aza-2′-deoxycytidine (5-Aza-CdR), and histone deacetylase inhibitors (HDACIs), such as trichostatin A. Previously, we reported the effect of 5-Aza-CdR and trichostatin A (TSA) on hepatocellular carcinoma (HCC). The present study aimed to investigate the effect of 5-Aza-CdR in comparison to and in combination with trichostatin A on p16INK4a, p14ARF, p15INK4b genes expression, cell growth inhibition and apoptosis induction in colon cancer Caco-2 cell line.Methods:The Caco-2 cells were cultured and treated with 5-Aza-CdR and TSA (alone and combined). The cell viability, apoptosis, and relative gene expression were determined by MTT assay, flow cytometry, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively.Results:Both compounds inhibited cell growth, induced apoptosis, and up-regulated the p16INK4a, p14ARF, p15INK4b gene significantly. The TSA had a more significant effect in comparison to 5-Aza-CdR. Furthermore, maximal apoptosis and up-regulation were observed with combined treatment.Conclusions:our finding indicated that 5-Aza-CdR and TSA can epigenetically re-activate the p16INK4a, p14ARF, p15INK4b gene resulting in cell growth inhibition and apoptosis induction in colon cancer.

The inhibition of microRNA-326 by SP1/HDAC1 contributes to proliferation and metastasis of osteosarcoma through promoting SMO expression.

Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA‐326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound‐healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR‐326 expression. Human OS tissues showed lowly expressed miR‐326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR‐326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR‐326 gene. miR‐326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR‐326 gene by promoting histone deacetylation; miR‐326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.

Methylation-mediated silencing of the LIM homeobox 6 (LHX6) gene promotes cell proliferation in human pancreatic cancer

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched for using the methyl-CpG targeted transcriptional activation (MeTA) method, and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as a strong candidate gene. LHX6 was downregulated in most of the pancreatic cancer cell lines (83%, 10/12), mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was methylated in primary pancreatic cancer specimens (57%, 16/28) in a tumor-specific manner. Re-expression of LHX6 inhibited colony formation and proliferation in LHX6 low-expressing pancreatic cancer cell lines, PK-1 and PK-9. In contrast, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines, PCI-35 and MIA PaCa-2. In order to analyze LHX6 downstream genes, we performed microarray analyses using LHX6 inducible PK-1 and PK-9 and found that LHX6 induction upregulated several genes that had tumor suppressive functions. Among these, we focused on TFPI2 (Tissue factor pathway inhibitor-2) and found that TFPI2 was greatly downregulated in all twelve pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation through aberrant transcriptional regulation of several cancer-related genes.

Protein C-ets-2 epigenetically suppresses TLRs-induced interleukin 6 production in macrophages

Interleukin 6 (IL-6) is a major proinflammatory cytokine involved in several aspects of the immune response. Excessive IL-6 production and dysregulated IL-6 receptor signaling lead to multiple inflammatory and autoimmune diseases, such as asthma, even cancer. Thus, its precise regulatory mechanisms need to be fully addressed. Here we found that knockdown of protein C-ets-2 (Ets2) resulted in higher IL-6 production after TLRs activation in macrophages. Mechanistically, Ets2 associated with an epigenetic modifier histone deacetylase 1 (HDAC1) and promoted its recruitment to the Il6 promoter after TLRs activation. Subsequentially, it enhanced histone deacetylation and inhibited Il6 mRNA transcription. Thus, Ets2 epigenetically suppresses TLRs-induced IL-6 production in both human and murine macrophages via promoting histone deacetylation of the Il6 promoter, serving as a new potential therapeutic target in inflammatory diseases therapy.

RBBP8/CtIP suppresses P21 expression by interacting with CtBP and BRCA1 in gastric cancer.

RB Binding Protein 8 (RBBP8) was previously reported being involved in DNA double-strand break (DSB) repair in cancers. However, there is no systematic study about the specific functions and related mechanisms of RBBP8 in gastric carcinogenesis. Through immunohistochemistry staining of paired gastric cancer (GC) tissues, adjacent high-grade intraepithelial neoplasia (HGIEN) tissues, and non-cancerous tissues, we found RBBP8 expression was upregulated in both HGIEN and GC tissues. Functional experiments showed the knockdown of RBBP8 inhibited cell proliferation and colony formation ability. This is mainly achieved through the role of RBBP8 in facilitating G1/S transition and promoting Cyclin D1 and CDK4 level. Then the interaction between RBBP8, BRCA1, and CtBP was revealed by co-immunoprecipitation (co-IP) and immunofluorescence confocal imaging. Moreover, we found RBBP8 acted as an adapter in this complex and RBBP8 overexpression enhanced the nucleus location of BRCA1. RBBP8 overexpression could inhibit P21 expression and HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) eliminated this effect. The HDAC activity of CtBP-RBBP8-BRCA1 complex was also further verified by HDAC activity assay. Through Chromatin immunoprecipitation (ChIP), we found RBBP8 could induce P21 promoter histone deacetylation and inhibit P21 transcription. In conclusion, we found RBBP8 could promote the G1/S transition of GC cells by inhibiting P21 level. Moreover, we revealed the chromatin modification role of RBBP8, which could suppress the histone acetylation level of P21 promoter by recruiting CtBP co-repressor complex to BRCA1 binding site.

GADD45G ACTS AS A NOVEL TUMOR SUPPRESSOR IN AML

State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China (People's Republic) Acute myeloid leukaemia (AML) is an aggressive hematologic neoplasm with distinct molecular and genetic features which is characterized by differentiation blockade and uncontrolled proliferation of malignant clones of immature myeloid cells. The growth arrest and DNA-damage induced 45 (GADD45) family protein GADD45g, has been shown to mediate DNA repair, cell cycle, senescence and apoptosis in response to various physiological and environmental stressors. Altered expression of GADD45g has been observed in multiple types of solid tumors, the function of GADD45g in hematopoietic malignancies remains almost completely unknown. Here, we found that GADD45g expression was preferentially silenced in AML, especially in AML with MLL rearrangements and FLT3-ITD mutations, and reduced expression of GADD45g correlates with poor prognosis in AML patients. Ectopic expression of GADD45g suppresses leukemic growth, induced apoptosis, senescence and differentiation of AML cells in vitro and in vivo. What's more, GADD45g caused downregulation of DNA repair proteins, resulting in inhibition of DNA double-strand break repair pathways and leading to DNA damage accumulation in AML cells. Moreover, GADD45g is epigenetically silenced in AML by both promoter DNA methylation and histone deacetylation, while the HDACi is more powerful. Exogenous expression of FLT3-ITD and MLL-AF9 repress GADD45G expression in AML, and the expression of GADD45G could be significantly induced by the small-molecule inhibitors FLT3 TKI or BRD4i target FLT3 or MLL respectively. Furthermore, the combination of HDACi and FLT3 TKI or BRD4i exerted a synergistic effect against FLT3-ITD+ or MLL-rearranged AML, respectively. These findings suggest that GADD45G may act as a tumor suppressor in AML and provide a novel and promising therapeutic target for AML.

CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway

BackgroundIn colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration.MethodsCDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation.ResultsSixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988–0.997)) as well as BRAFV600E, mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2- to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2.ConclusionCDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.

Rpd3L HDAC links H3K4me3 to transcriptional repression memory.

Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays ‘memory’ of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of ∼540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory (TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is critical to promote histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at active promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.

LACTB, a novel epigenetic silenced tumor suppressor, inhibits colorectal cancer progression by attenuating MDM2-mediated p53 ubiquitination and degradation.

Colorectal cancer (CRC) is one of the most common aggressive malignancies. Like other solid tumors, inactivation of tumor suppressor genes and activation of oncogenes occur during CRC development and progression. Recently, a novel tumor suppressor, LACTB, was proposed to inhibit tumor progression, but the functional and clinical significance of this tumor suppressor in CRC remains unexplored. Herein, we found LACTB was significantly downregulated in CRC due to promoter methylation and histone deacetylation, which was associated with metastasis and advanced clinical stage. CRC patients with low LACTB expression had poorer overall survival and LACTB also determined to be an independent prognostic factor for poorer outcome. Ectopic expression of LACTB suppressed CRC cells proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and inhibited CRC growth and metastasis in vivo, while knockout of LACTB by CRISPR/Cas9 gene editing technique resulted in an opposite phenotype. Interestingly, LACTB could exert antitumorigenic effect only in HCT116 and HCT8 cells harboring wild-type TP53, but not in HT29 and SW480 cells harboring mutant TP53 or HCT116 p53-/- cells. Mechanistic studies demonstrated that LACTB could directly bind to the C terminus of p53 to inhibit p53 degradation by preventing MDM2 from interacting with p53. Moreover, ablation of p53 attenuated the antitumorigenic effects of LACTB overexpression in CRC. Collectively, our findings successfully demonstrate for the first time that LACTB is a novel epigenetic silenced tumor suppressor through modulating the stability of p53, supporting the pursuit of LACTB as a potential therapeutic target for CRC.