Cell-free methylation markers with diagnostic and prognostic potential in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with poor prognosis and high mortality. There is a dearth of effective early diagnostic tools, so liver resection surgery and liver transplantation are the only effective medical treatments. The most commonly used marker for HCC detection is serum alpha fetoprotein (AFP), which has low sensitivity and specificity. Because aberrant DNA methylation of genes and miRNAs occurs early in most cancers, we explored whether circulating methylation markers could be promising clinical tools for HCC diagnosis. Using a whole-genome approach, we identified many hyper-methylated miRNAs in HCC. Furthermore, three abnormally methylated genes and one miRNA were combined to establish a methylation predictive model and tested for its diagnostic and prognostic potential in HCC. Using plasma samples, the predictive model exhibited high sensitivity and specificity (> 80%) for HBV-related HCC. Most importantly, nearly 75% of patients who could not be diagnosed with AFP at 20 ng/mL were detected by this model. Further, the predictive model exhibited an exceedingly high ability to predict 5-year overall survival in HCC patients. These data demonstrate the high diagnostic and prognostic potential of methylation markers in the plasma of HCC patients.

ZMYND8 Reads the Dual Histone Mark H3K4me1-H3K14ac to Antagonize the Expression of Metastasis-Linked Genes.

Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating functions of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a metastasis-suppressive epigenetic mechanism in which ZMYND8's PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.

JARID1D Is a Suppressor and Prognostic Marker of Prostate Cancer Invasion and Metastasis.

Entire or partial deletions of the male-specific Y chromosome are associated with tumorigenesis, but whether any male-specific genes located on this chromosome play a tumor-suppressive role is unknown. Here, we report that the histone H3 lysine 4 (H3K4) demethylase JARID1D (also called KDM5D and SMCY), a male-specific protein, represses gene expression programs associated with cell invasiveness and suppresses the invasion of prostate cancer cells in vitro and in vivo. We found that JARID1D specifically repressed the invasion-associated genes MMP1, MMP2, MMP3, MMP7, and Slug by demethylating trimethyl H3K4, a gene-activating mark, at their promoters. Our additional results demonstrated that JARID1D levels were highly downregulated in metastatic prostate tumors compared with normal prostate tissues and primary prostate tumors. Furthermore, the JARID1D gene was frequently deleted in metastatic prostate tumors, and low JARID1D levels were associated with poor prognosis in prostate cancer patients. Taken together, these findings provide the first evidence that an epigenetic modifier expressed on the Y chromosome functions as an anti-invasion factor to suppress the progression of prostate cancer. Our results also highlight a preclinical rationale for using JARID1D as a prognostic marker in advanced prostate cancer.

Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4.

Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation.

Recognition of histone H3K4 trimethylation by the plant homeodomain of PHF2 modulates histone demethylation.

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications.

The H4 tail domain participates in intra- and internucleosome interactions with protein and DNA during folding and oligomerization of nucleosome arrays.

The condensation of nucleosome arrays into higher-order secondary and tertiary chromatin structures likely involves long-range internucleosomal interactions mediated by the core histone tail domains. We have characterized interarray interactions mediated by the H4 tail domain, known to play a predominant role in the formation of such structures. We find that the N-terminal end of the H4 tail mediates interarray contacts with DNA during self-association of oligonucleosome arrays similar to that found previously for the H3 tail domain. However, a site near the histone fold domain of H4 participates in a distinct set of interactions, contacting both DNA and H2A in condensed structures. Moreover, we also find that H4-H2A interactions occur via an intra- as well as an internucleosomal fashion, supporting an additional intranucleosomal function for the tail. Interestingly, acetylation of the H4 tail has little effect on interarray interactions by itself but overrides the strong stimulation of interarray interactions induced by linker histones. Our results indicate that the H4 tail facilitates secondary and tertiary chromatin structure formation via a complex array of potentially exclusive interactions that are distinct from those of the H3 tail domain.

Detection of interactions between nucleosome arrays mediated by specific core histone tail domains.

The core histone tail domains play important roles in different stages of chromatin condensation. The tails are required for folding nucleosome arrays into secondary chromatin structures such as the approximately 30 nm diameter chromatin fiber and for mediating fiber-fiber interactions important for formation of tertiary chromatin structures. Crosslinking studies have demonstrated that inter-nucleosomal tail-DNA contacts appear in conjunction with salt-induced folding of nucleosome arrays into in higher order chromatin structures. However, since both folding of nucleosome arrays and fiber-fiber interactions take place simultaneously in >2-3 mM MgCl(2) such inter-nucleosome interactions may reflect short range (intra-array) or longer range (inter-array) interactions. Here, we describe a novel technique to specifically identify inter-array interactions mediated by the histone tail domains. In addition, we describe a new method for the preparation of H3/H4 tetramers.

The H3 tail domain participates in multiple interactions during folding and self-association of nucleosome arrays.

The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the molecular details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl(2)-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed cross-linking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray cross-linking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl(2) concentrations. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no reduction in interarray cross-linking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to "acetylated" H3 tails. Collectively these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct molecular interactions that can be differentially regulated by acetylation or binding of linker histones.

Interleukin-6 differentially regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells.

The effects of IL-6 on prostate cancer cells are well documented yet remain controversial. Some reports suggested that IL-6 could promote prostate cancer cell growth, while others showed that IL-6 could repress prostate cancer cell growth. Here, we systemically examined various IL-6 signaling pathways in prostate cancer cells and found that IL-6 could go through at least three distinct pathways to modulate the functions of androgen receptor (AR), a key transcriptional factor to control the prostate cancer growth. Our results show that IL-6 can enhance AR transactivation via either the STAT3 or MAPK pathways. In contrast, IL-6 can suppress AR transactivation via the PI3K-Akt pathway. Co-existence of these various signaling pathways may result in either additive or conflicting effects on AR transactivation. Together, our results indicate that the balance of these various pathways may then determine the overall effect of IL-6 on AR transactivation.

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells.

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.