PHD Domains Research Articles

Mechanistic Study of Macranthoside B Effects on Apoptotic Cell Death in Human Cervical Adenocarcinoma Cells

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides, a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcys­teine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ring-finger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis-inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

Identifying a novel IRF3/circUHRF1/miR-1306-5p/ARL4C axis in pancreatic ductal adenocarcinoma progression

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is considered one most aggressive and lethal cancer types worldwide. While its underlying mechanisms are still poorly understood. CircRNAs play essential roles in various biological progression, including PDAC. Here, our results found that circUHRF1 was highly expressed in PDAC tumor tissues compared with normal tissues. Next, Cell or animal models were constructed, CCK-8, cell colony, EdU, flow cytometry assay, transwell migration, and Western blot assays were applied. CircUHRF1 knockdown influenced PDAC cell proliferation, apoptosis, migration and EMT level in vitro, and tumor growth in vivo. Subsequently, bioinformatics analysis, AGO2-RIP, RNA pull-down, and dual-luciferase reporter assays were used to explore the downstream targets in PDAC progression. Our findings suggest that circUHRF1 regulated ARL4C expression to promote PDAC progression through sponging miR-1306-5p. The role of miR-1306-5p in PDAC cellular progression has been elucidated, and the expression association between miR-1306-5p and circUHRF1 or ARL4C in PDAC tissues was analyzed. Furthermore, circUHRF1 expression in PDAC cells could be transcriptionally regulated by IRF3. Collectively, our study demonstrated the role of IRF3/circUHRF1/miR-1306-5p/ARL4C axis in PDAC progression. Our results suggest that circUHRF1 is one promising diagnosis or therapeutic target for PDAC management. Abbreviations : CircRNA; Circular RNAPDAC; pancreatic ductal adenocarcinomaUHRF1; Ubiquitin-like with PHD and RING finger domain 1ARL4C; ADP Ribosylation Factor Like GTPase 4CRIP; RNA immunoprecipitationEDU; 5-Ethynyl-2ʹ-deoxyuridineEMT; epithelial to mesenchymal transitionAGO2; Argonaute RISC Catalytic Component 2CCK8; Cell counting Kit-8IRF3; Interferon Regulatory Factor 3

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

PHF6 Positively Regulates Transcription of Myeloid Differentiation Genes By Binding at Enhancer Regions

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled division, and differentiation arrest, of hematopoietic stem cells (HSCs) and myeloid progenitors. AML is a genetically heterogeneous disease, with mutations in genes belonging to multiple functional groups. Of these, chromatin regulators and transcriptional factors (TFs) are an important functional group to study because of a lack of targeted AML therapies against these factors. PHD Finger Protein 6 (PHF6) is one such chromatin-associated protein with a yet-unknown molecular mechanism of action. It is mutated in 3-5% of MDS, CMML, and AMLs and 20% of T- ALLs, and is considered a leukemia suppressor.To assess the effects of PHF6 on the AML transcriptome, we generated CRISPR-mediated knockout (KO) clones in THP-1 monocytic AML cell line, and integrated a Dox-inducible PHF6 construct into a PHF6 KO clone, enabling us to conditionally rescue PHF6 expression to parental levels (Fig A & B). RNA-Seq analysis of these knockout and rescue systems revealed that PHF6 expression upregulates genes related to myeloid differentiation and downregulates genes related to hematopoietic stem cells and cell division in myeloid cells. Additionally, loss of PHF6 increased THP-1 proliferation, and restoring PHF6 expression decreased proliferation. We also performed ChIP-Seq for PHF6 in the THP-1 cells and found that PHF6 binds to open and active enhancer regions. Unbiased motif analysis showed that PHF6-bound enhancers (compared to all enhancers genome-wide) were enriched for RUNX1, PU.1, and IRF8 motifs. Metagene plotting of PHF6 ChIP-Seq signal against ChIP-Seq for these TFs showed striking concordance in their binding patterns, indicating that PHF6 co-occupies chromatin with key hematopoietic transcription factors. (Fig C).PHF6 has two extended PHD (ePHD) domains with a similar structure to canonical PHD domains, but with unknown functions. Based on leukemia genome sequencing results from COSMIC, we observed that while nonsense and frameshift mutations of PHF6 (accounting for 2/3 rd of PHF6 mutations, and expected to produce no protein) are distributed throughout the gene body, missense mutations (accounting for 1/3 rd of PHF6 mutations and expected to produce a full-length protein with single amino acid substitution), are concentrated in the second ePHD domain (ePHD2). To assess the functional consequence of mutations in the ePHD2 domain, we generated from a PHF6 KO clone a clone expressing Dox-inducible PHF6 R274Q, the most common missense mutation of PHF6 seen in leukemia. RNA-Seq showed that PHF6 R274Q induction has no downstream effects on the cellular transcriptome, in striking contrast to the effects of wildtype PHF6 induction (Fig D). Additionally, the expression of PHF6 R274Q has no effect on cell growth. These results indicate that the ePHD2-domain mutant PHF6 R274Q is functionally dead (fully or partially), and the ePHD2 domain is critical for PHF6 function.Our results support an important transcriptional role of PHF6 in the myeloid system, involving co-occupancy with TFs at enhancers to promote the transcription of myeloid differentiation genes. This loss of this transcriptional regulation, either through complete PHF6 protein loss or point mutation of its ePHD2 domain, dysregulates myeloid differentiation and contributes to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evolution of CG Methylation Maintenance Machinery in Plants.

Cytosine methylation is an epigenetic mark present in most eukaryotic genomes that contributes to the regulation of gene expression and the maintenance of genome stability. DNA methylation mostly occurs at CG sequences, where it is initially deposited by de novo DNA methyltransferases and propagated by maintenance DNA methyltransferases (DNMT) during DNA replication. In this review, we first summarize the mechanisms maintaining CG methylation in mammals that involve the DNA Methyltransferase 1 (DNMT1) enzyme and its cofactor, UHRF1 (Ubiquitin-like with PHD and RING Finger domain 1). We then discuss the evolutionary conservation and diversification of these two core factors in the plant kingdom and speculate on potential functions of novel homologues typically observed in land plants but not in mammals.

Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation.

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.

UHRF1 Suppresses HIV-1 Transcription and Promotes HIV-1 Latency by Competing with p-TEFb for Ubiquitination-Proteasomal Degradation of Tat.

ABSTRACTHIV-1 remains incurable due to viral reservoirs, which lead to durably latent HIV infection. Identifying novel host factors and deciphering the molecular mechanisms involved in the establishment and maintenance of latency are critical to discover new targets for the development of novel anti-HIV agents. Here, we show that ubiquitin-like with PHD and RING finger domain 1 (UHRF1) modulates HIV-1 5′-long terminal repeat (LTR)-driven transcription of the viral genome as a novel HIV-1 restriction factor. Correspondingly, UHRF1 depletion reversed the latency of HIV-1 proviruses. Mechanistically, UHRF1 competed with positive transcription factor b (p-TEFb) for the binding to the cysteine-rich motifs of HIV-1 Tat via its TTD, PHD, and RING finger domains. Furthermore, UHRF1 mediated K48-linked ubiquitination and proteasomal degradation of Tat in RING-dependent ways, leading to the disruption of Tat/cyclin T1/CDK9 complex and consequential impediment of transcription elongation. In summary, our findings revealed that UHRF1 is an important mediator of HIV-1 latency by controlling Tat-mediated transcriptional activation, providing novel insights on host-pathogen interaction for modulating HIV-1 latency, beneficial for the development of anti-AIDS therapies.

Screening of hub genes and evaluation of the growth regulatory role of CD44 in metastatic prostate cancer.

Prostate cancer (PCa) is the most common cancer type in men worldwide. Currently, the management of metastatic PCa (mPCa) remains a challenge to urologists. The analysis of hub genes and pathways may facilitate the understanding of the molecular mechanism of PCa. In the present study, to identify the hub genes in the mPCa, the three datasets GSE3325, GSE6919 and GSE38241 were downloaded from the platform of the Gene Expression Omnibus and function enrichment analysis of differentially expressed genes (DEGs) was performed. A total of 168 DEGs were obtained and the DEGs were significantly enriched in ‘cell junction’ and ‘cell adhesion’, among others. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that DEGs were enriched in three pathways including ‘focal adhesion’, ‘renal cell carcinoma’ and ‘Hippo signaling pathway’. The results of the protein-protein interaction network revealed that the hub genes in mPCa were separately PTEN, Rac GTPase-activating protein 1, protein regulator of cytokinesis 1, PDZ binding kinase, centromere-associated protein E, NUF2 component of NDC80 kinetochore complex, TPX2 microtubule nucleation factor, SOX2, CD44 and ubiquitin-like with PHD and ring finger domains 1. As a hub gene, CD44 was differentially expressed in PCa, as determined by Oncomine analysis. Further experiments in vivo demonstrated that SB-3CT, a selective matrix metalloproteinase inhibitor that has been reported to block CD44 cleavage and inhibit the downstream signaling pathway, suppressed the tumorigenicity of PCa cells by decreasing the expression levels of pyruvate dehydrogenase kinase 1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4. Moreover, the combination therapy with SB-3CT and docetaxel was more effective in inhibiting PCa compared with monotherapy. In conclusion, the identification of DEGs and the in vivo experimental results helped to elucidate the molecular mechanisms of PCa and provided a potential strategy for the treatment of PCa.

Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1.

Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.

The phosphoinositide code is read by a plethora of protein domains

Introduction The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. Areas covered Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestin, GAT and VHS modules, membrane-perturbing annexin, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids and assembled bacterial and viral particles. Expert opinion The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses, and lipidons as lipid “codons” recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Functional Impairment of Hematopoietic Stem/Progenitor Cells By Loss of Uhrf2 in Mice

A growing body of evidence has underlined the involvement of epigenetic regulations that include histone modification in hematopoiesis. We previously identified Ubiquitin Like with PHD and Ring Finger Domains 2 (UHRF2) as a crucial multi-domain E3 ubiquitin ligase that facilitates coordination of the cell-cycle machinery and the epigenetic regulations via interaction with cyclins, TP53, DNA methyltransferases, and G9A H3K9 methyltransferase in non-hematopoietic cells (Mori et al, FEBS Letters, 2012). In fact, H3K9 trimethylation (H3K9me3) is reduced in Uhrf2-/- embryonic stem (ES) cells (Pichler et al, J Cell Biochem, 2011). Recently, Lu et al. have also reported that UHRF2 is highly expressed in common lymphoid progenitors and mature lymphocytes rather than myeloid cells, and expression of UHRF2 is reduced in some acute myeloid leukemia (AML) cells and a variety of cancers (Oncotarget, 2016). However, the association of UHRF2 with hematopoiesis has not yet been studied. Here, to clarify the role of UHRF2 in hematopoiesis, we investigated hematopoietic cells and organs in Uhrf2-/- mice.We did not find a significant difference in peripheral blood leukocyte and platelet counts and hemoglobin values, and spleen sizes between wild-type (WT) and Uhrf2-/- mice at young (12 weeks) and old (50 weeks) ages. The total cell counts and proportions of hematopoietic stem/progenitor cell (HSPC)-enriched lineage-Sca1+Kit+ (LSK) cells, common myeloid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in bone marrow (BM) were also similar between WT and Uhrf2-/- mice. We next performed competitive repopulation assay with BM transplants (BMT) by infusing the same numbers of Ly5.1+ WT BM cells and Ly5.2+Uhrf2-/- BM cells into lethally irradiated Ly5.1+ WT recipients (n = 4). Of note, at 12 weeks after the first BMT, the proportions of Ly5.2+Uhrf2-/- cells were significantly less in all lineages compared with control BMT that used Ly5.1+ WT BM cells and Ly5.2+Uhrf2+/+ BM cells. After the second BMT, Uhrf2-/- cells were almost rejected in neutrophils while 20-30% Uhrf2-/- T cells and B cells were still detected. After the third BMT, however, no Uhrf2-/- myeloid or lymphoid cells were found in peripheral blood of recipients. These findings indicate functional impairment of Uhrf2-/- HSPCs with defective self-renewal capacity.We then investigated the mechanism of impairment in the function of HSPCs in Uhrf2-/- mice. As reported in Uhrf2-/- ES cells, we found reduced H3K9me3 in Uhrf2-/- BM cells. We also performed RNA sequencing for LSK cells, which showed downregulations of the genes that are crucial for maintenance and proliferation of HSPCs, such as Flt3 (2.2-fold) and Stat5a (2.3-fold). GSEA (gene set enrichment analysis) revealed significant enrichment of targets of H3K9me3 (FDRq value = 0.045). Furthermore, the pathway analysis identified global reductions in the canonical immunological pathways associated with differentiation of B cells, including Role of NFAT in Regulation of the Immune Response (Z score = -3.258, P = 0.009), B Cell Receptor Signaling (Z score = -2.197, P = 0.021), and PI3K Signaling in B Lymphocytes (Z score = -2.041, P = 0.024) in Uhrf2-/- LSK cells. In agreement, it has been recently reported that reduction of H3K9me3 in HSPCs results in impairments of their abilities to differentiate and generate B cells (Ugarte et al, Stem Cell Reports, 2015; Djeghloul et al, Stem Cell Reports, 2016).Collectively, Uhrf2 possibly play some roles in self-renewal and differentiation of HSPCs in part through the trimethylation of H3K9. Defect of Uhrf2 might contribute to the impairment of differentiation rather than growth advantage in some cancer or AML cells. DisclosuresNo relevant conflicts of interest to declare.

ASXL1 Mutation Change the Migration Ability in JAK2V617F Positive Cell Lines through SDF-1/CXCR4 Axis

ASXL1 plays a vital role in regulation of the histone methylation and acts as a tumor suppressor. Majority of ASXL1 mutations are frame-shift and nonsense mutations. The most frequent mutation is C.1934dupG (P.Gly646TrpfsX12) with a loss of PHD domain and truncated protein formed. Reduced normal ASXL1 protein expression promotes myeloid transformation. ASXL1 mutations change the transcription of certain oncogenes and result in dysfunction of tumor stem cells.We found that ASXL1 mutations were identified in 29.1% (32/110) patients with primary myelofibrosis (PMF). Moreover, JAK2V617F mutated patients with ASXL1 mutations were more likely to present splenomegaly (median LCM: 10 vs. 5 cm, P=0.023), while in JAK2V617F negative patients, the association was gone. There is evidence that SDF-1α/CXCR4 axis plays an important role in the adhesion of leukemic cells and bone marrow stromal cells. Abnormal CXCR4 expression may contribute to constitutive migration of CD34+ cells in PMF. Accordingly, we supposed that on the basis of JAK2V617F mutation, mutated ASXL1 acts on the SDF-1α/CXCR4 axis leading to the stem cell mobilization and splenomagly.To further investigate the molecular mechanism of ASXL1 mutation in patients with JAK2V617F, we performed short hairpin RNA (shRNA)-mediated stable knockdown of ASXL1 in JAK2V617F mutated UKE1 and SET2 cell lines and JAK2V617F negative HL-60 cell line. In JAK2V617F mutated UKE1 and SET2 cells, ASXL1 knockdown accompanied with the significant elevation of CXCR4 mRNA and protein levels (Figure 1A); while in HL-60 cell, ASXL1 knockdown accompanied with the significant decreased expression of CXCR4 (Figure 1A). Next, we tested the effect of ASXL1 expression on cell migration ability by using transwell migration assay. Our observation showed that ASXL1 knockdown cells showed obvious promotion in migration in JAK2V617F mutated UKE1 and SET2 cells (P=0.028 and P<0.001 ), and CXCR4 antagonist AMD3100 could disrupt this effect (Figure 1B), and down regulation of ASXL1 expression accompanied with declined migration ability in HL-60 cells (P=0.0025, Figure 1B). Flow cytometry also confirmed the increased membrane CXCR4 expression in JAK2V617F mutated UKE1 and SET2 cells. In order to further explore the mechanism of ASXL1 effect on cell migration, we performed mRNA expression microarrary and the results showed that the function of cell adhesion ability, cell-matrix adhesion and leukocyte migration ability were up regulated in ASXL1 knockdown SET2 cells (Figure 1C).In summary, our data demonstrated that ASXL1 knockdown in JAK2V617F mutated UKE1 and SET2 cells resulted in increased expression of CXCR4 and enhanced cell migration abilities, which may indicated that ASXL1 mutation may act on SDF-1/CXCR4 axis. We speculate that under the JAK2V617F mutated background, ASXL1 knockdown may promote the migration ability of tumor stem cells and formation of tumor microenvironment through acting on SDF-1/CXCR4 axis and result in splenomegaly and extramedullary hematopoiesis in PMF patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions.

The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including (1) removal of the initiator methionine residue of H3 to prevent steric interference, (2) a groove where arginine-2 binds, and (3) an aromatic cage that engages methylated lysine-4. We hypothesize that some PHDs might have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ∼3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.

The PHD transcription factor Cti6 is involved in the fungal colonization and aflatoxin B1 biological synthesis of Aspergillus flavus

Aspergillus flavus and its main secondary metabolite AFB1 pose a serious threat to several important crops worldwide. Recently, it has been reported that some PHD family transcription factors are involved in the morphogenesis and AFB1 biological synthesis in A. flavus, but the role of Cti6, a PHD domain containing protein in A. flavus, is totally unknown. The study was designed to reveal the biological function of Cti6 in the fungus by deletion of cti6, and its two domains (PHD and Atrophin-1) through homologous recombination, respectively. The results showed that Cti6 might up-regulate the mycelium growth, conidiation, sclerotia formation and AFB1 biological synthesis of A. flavus by its PHD domain, while Atrophin-1 also improved the conidiation of the fungus. The qRT-PCR analysis showed that Cti6 increased the conidiation of the fungus through AbaA and BrlA mediated conidiation pathway, triggered the formation of sclerotia by orthodox sclerotia formation pathway, and improved the production of AFB1 by orthodox AFB1 synthesis pathway. Crops models analysis showed that A. flavus Cti6 plays vital role in colonization and the production of AFB1 on the host grains mainly via PHD domain. Bioinformatics analysis showed Cti6 is conservative in Aspergillus spp., and mCherry mediated subcellular localization showed that most Cti6 accumulated in the nuclei, which reflected that Cti6 performed its important biological function in the nuclei in Aspergillus spp.. The results of the current study elucidate the roles of PHD domain containing proteins in the mechanism of the infection of crops by A. flavus, and provided a novel target for effectively controlling the contamination of Aspergillus spp. to crops.

UV-induced activation of ATR is mediated by UHRF2.

UHRF1 (Ubiquitin-like with PHD and ring finger domains 1) regulates DNA methylation and histone modifications and plays a key role in cell proliferation and the DNA damage response. However, the function of UHRF2, a paralog of UHRF1, in the DNA damage response remains largely unknown. Here, we show that UHRF2 is essential for maintaining cell viability after UV irradiation, as well as for the proliferation of cancer cells. UHRF2 was found to physically interact with ATR in a DNA damage-dependent manner through UHRF2's TTD domain. In addition, phosphorylation of threonine at position 1989, which is required for UV-induced activation of ATR, was impaired in cells depleted of UHRF2, suggesting that UHRF2 is essential in ATR activation. In conclusion, these results suggest a new regulatory mechanism of ATR activation mediated by UHRF2.

The DNA cytosine-5-methyltransferase 3 (DNMT3) involved in regulation of CgIL-17 expression in the immune response of oyster Crassostrea gigas

DNA methyltransferase, a key enzyme mediating DNA methylation, is involved in numerous processes including genomic imprinting, X chromosome inactivation, transposable element suppression, and immune defense in vertebrates. In the present study, a DNA cytosine-5-methyltransferase 3 was identified from oyster Crassostrea gigas (designed as CgDNMT3). There were a PWWP domain, a PHD domain and a DNA-methylase domain in the deduced amino acid sequences of CgDNMT3, and the conserved motifs I, IV, VI, Ⅷ, IX and X were identified in its C-terminal catalytic DNA-methylase domain. The mRNA transcripts of CgDNMT3 were detected in haemocytes, mantle, gill, adductor muscle, digestive gland and labial palp, with higher expression level in haemocytes (6.54 folds of those in gill, p < 0.01). The expression level of CgDNMT3 mRNA in haemocytes increased significantly after LPS primed (2.87 folds of that in control group, p < 0.05) in vitro or Vibrio splendidus challenging (1.94 folds of that in control group, p < 0.05) in vivo. Immunocytochemical analysis revealed that CgDNMT3 protein was distributed mainly in cytoplasm and partial in nucleus of oyster haemocytes. After CgDNMT3 was transfected and expressed in HEK293T cells, the DNA 5-methylcytosine (5-mc) level in the transfected group was significantly increased, which was 1.22 folds (p < 0.05) of the pcDNA-3.1 group. The expressions of oyster CgIL17-1, CgIL17-2 and CgIL17-5 in haemocytes increased (13.05 folds, 4.78 folds and 9.41 folds of that in control group, respectively) at 12 h after V. splendidus challenging, but the increase were significantly inhibited when the oysters were pre-treated with DNA methyltransferase inhibitor 5-Azacytidine, which were 9 folds, 1.93 folds and 3.22 folds of that in control group, respectively. These results collectively suggested that CgDNMT3 was a conserved member of DNA methyltransferase 3 family in oyster, and participated in regulating the expression of cytokines during immune response.

Converging genetic and epigenetic drivers of paediatric acute lymphoblastic leukaemia identified by an information-theoretic analysis.

In cancer, linking epigenetic alterations to drivers of transformation has been difficult, in part because DNA-methylation analyses must capture epigenetic variability, which is central to tumour heterogeneity and tumour plasticity. Here, by conducting a comprehensive analysis, based on information theory, of differences in methylation stochasticity in samples from patients with paediatric acute lymphoblastic leukaemia (ALL), we show that ALL epigenomes are stochastic, and marked by increased methylation entropy at specific regulatory regions and genes. By integrating data methylation and single-cell gene-expression data, we arrived at a relationship between methylation entropy and gene-expression variability, and found that epigenetic changes in ALL converge on a shared set of genes that overlap with genetic drivers involved in chromosomal translocations across the disease spectrum. Our findings suggest that an epigenetically driven gene- regulation network, with UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a central node, links genetic drivers and epigenetic mediators in ALL.

A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1.

Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.

UHRF1 Induces Methylation of the TXNIP Promoter and Down-Regulates Gene Expression in Cervical Cancer

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.