nyi Ju

Heterochromatin protein HP1γ promotes colorectal cancer progression and is regulated by miR-30a

Colorectal cancer pathogenesis remains incompletely understood. Here, we report that the heterochromatin protein HP1γ is upregulated commonly in human colorectal cancer, where it promotes cell proliferation in vitro and in vivo. Gene-expression and promoter-binding experiments demonstrated that HP1γ directly regulated CDKN1A (p21(Waf1/Cip1)) in a manner associated with methylation of histone H3K9 on its promoter. We identified miR-30a as a tumor-suppressive microRNA that targets HP1γ in vitro and in vivo to specifically suppress the growth of colorectal cancer in mouse xenograft models. MiR-30a was widely downregulated in primary human colorectal cancer tissues, where its expression correlated inversely with high levels of HP1γ protein. Our results identify a new miR-30a/HP1γ/p21 regulatory axis controlling colorectal cancer development, which may offer prognostic and therapeutic opportunities.

Synergistic Effect of SRY and Its Direct Target, WDR5, on Sox9 Expression

SRY is a sex-determining gene that encodes a transcription factor, which triggers male development in most mammals. The molecular mechanism of SRY action in testis determination is, however, poorly understood. In this study, we demonstrate that WDR5, which encodes a WD-40 repeat protein, is a direct target of SRY. EMSA experiments and ChIP assays showed that SRY could bind to the WDR5 gene promoter directly. Overexpression of SRY in LNCaP cells significantly increased WDR5 expression concurrent with histone H3K4 methylation on the WDR5 promoter. To specifically address whether SRY contributes to WDR5 regulation, we introduced a 4-hydroxy-tamoxifen-inducible SRY allele into LNCaP cells. Conditional SRY expression triggered enrichment of SRY on the WDR5 promoter resulting in induction of WDR5 transcription. We found that WDR5 was self regulating through a positive feedback loop. WDR5 and SRY interacted and were colocalized in cells. In addition, the interaction of WDR5 with SRY resulted in activation of Sox9 while repressing the expression of β-catenin. These results suggest that, in conjunction with SRY, WDR5 plays an important role in sex determination.

Human fetal globin gene expression is regulated by LYAR

Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.

The role of WDR5 in silencing human fetal globin gene expression.

Background Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established. Design and Methods To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP. Results We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program. Conclusions Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

LYAR promotes colorectal cancer cell mobility by activating galectin-1 expression.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. However, the molecular mechanisms of CRC pathogenesis are not fully understood. In this study, we report the characterization of LYAR (Ly-1 antibody reactive clone) as a key regulator of the migration and invasion of human CRC cells. Immunohistochemistry analysis demonstrated that LYAR is expressed at a higher level in metastatic CRC tissues. We found that LYAR promoted the migratory and invasive capabilities of CRC cells. Gene expression profile analysis of CRC cells showed that LGALS1, which encodes the galectin-1 protein, was a potential target of LYAR. The ChIP assay and gene reporter assays indicated that LYAR directly bound to the LGALS1 promoter. The ectopic expression of galectin-1 partially restored the mobile potential of LYAR knocked-down cells, which suggests that galectin-1 contributed to the LYAR-promoted cell migration and invasion of CRC cells. Thus, this study revealed a novel mechanism by which the transcription factor LYAR may promote tumor cell migration and invasion by upregulating galectin-1 gene expression in CRC.

NatD promotes lung cancer progression by preventing histone H4 serine phosphorylation to activate Slug expression

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation (Nt-acetylation) of histone H4 known to be involved in cell growth. Here we report that NatD promotes the migratory and invasive capabilities of lung cancer cells in vitro and in vivo. Depletion of NatD suppresses the epithelial-to-mesenchymal transition (EMT) of lung cancer cells by directly repressing the expression of transcription factor Slug, a key regulator of EMT. We found that Nt-acetylation of histone H4 antagonizes histone H4 serine 1 phosphorylation (H4S1ph), and that downregulation of Nt-acetylation of histone H4 facilitates CK2α binding to histone H4 in lung cancer cells, resulting in increased H4S1ph and epigenetic reprogramming to suppress Slug transcription to inhibit EMT. Importantly, NatD is commonly upregulated in primary human lung cancer tissues where its expression level correlates with Slug expression, enhanced invasiveness, and poor clinical outcomes. These findings indicate that NatD is a crucial epigenetic modulator of cell invasion during lung cancer progression.NatD is an acetyltransferase responsible for N-α-terminal acetylation of the histone H4 and H2A and has been linked to cell growth. Here the authors show that NatD-mediated acetylation of histone H4 serine 1 competes with the phosphorylation by CK2α at the same residue thus leading to the upregulation of Slug and tumor progression.

Heterochromatin Protein 1γ Is a Novel Epigenetic Repressor of Human Embryonic ϵ-Globin Gene Expression

Production of hemoglobin during development is tightly regulated. For example, expression from the human β-globin gene locus, comprising β-, δ-, ϵ-, and γ-globin genes, switches from ϵ-globin to γ-globin during embryonic development and then from γ-globin to β-globin after birth. Expression of human ϵ-globin in mice has been shown to ameliorate anemia caused by β-globin mutations, including those causing β-thalassemia and sickle cell disease, raising the prospect that reactivation of ϵ-globin expression could be used in managing these conditions in humans. Although the human globin genes are known to be regulated by a variety of multiprotein complexes containing enzymes that catalyze epigenetic modifications, the exact mechanisms controlling ϵ-globin gene silencing remain elusive. Here we found that the heterochromatin protein HP1γ, a multifunctional chromatin- and DNA-binding protein with roles in transcriptional activation and elongation, represses ϵ-globin expression by interacting with a histone-modifying enzyme, lysine methyltransferase SUV4–20h2. Silencing of HP1γ expression markedly decreased repressive histone marks and the multimethylation of histone H3 lysine 9 and H4 lysine 20, leading to a significant decrease in DNA methylation at the proximal promoter of the ϵ-globin gene and greatly increased ϵ-globin expression. In addition, using chromatin immunoprecipitation, we showed that SUV4–20h2 facilitates the deposition of HP1γ on the ϵ-globin-proximal promoter. Thus, these data indicate that HP1γ is a novel epigenetic repressor of ϵ-globin gene expression and provide a potential strategy for targeted therapies for β-thalassemia and sickle cell disease.

ε-globin expression is regulated by SUV4-20h1

Research efforts to establish therapeutic modalities for treating sickle cell disease (SCD) and β-thalassemia have been hindered by our incomplete understanding of the developmental regulation of human β-like globin gene expression. It has been demonstrated that increased embryonic globin (e-

Suv4‑20h1 promotes G1 to S phase transition by downregulating p21WAF1/CIP1 expression in chronic myeloid leukemia K562 cells

Methylation of histone H4 lysine 20 (H4K20) has been associated with cancer. However, the functions of the histone methyltransferases that trigger histone H4K20 methylation in cancers, including suppressor of variegation 4–20 homolog 1 (Suv4-20h1), remain elusive. In the present study, it was demonstrated that the knockdown of the histone H4K20 methyltransferase Suv4-20h1 resulted in growth inhibition in chronic myeloid leukemia K562 cells. Disruption of Suv4-20h1 expression induced G1 arrest in the cell cycle and increased expression levels of cyclin dependent kinase inhibitor 1A (p21WAF1/CIP1), an essential cell cycle protein involved in checkpoint regulation. Chromatin immunoprecipitation analysis demonstrated that Suv4-20h1 directly binds to the promoter of the p21 gene and that methylation of histone H4K20 correlates with repression of p21 expression. Thus, these data suggest that Suv4-20h1 is important for the regulation of the cell cycle in K562 cells and may be a potential therapeutic target for leukemia.