RING Finger Domain-containing Protein Research Articles

PHRF1 Promotes Cell Invasion by Modulating SOX4 Expression in Colorectal Cancer HCT116-p53-/- Cells.

PHD and RING finger domain-containing protein 1 (PHRF1) ubiquitinates TGIP (TG-interacting protein) and redistributes cPML (cytoplasmic variant of PML) to the cytoplasm to enhance TGF-β signaling by. It is unclear whether PHRF1 affects invasion and survival when both mutations of the activated oncogene Kras and inactivation of the tumor suppressor p53 are present. We knockout PHRF1 expression using Crispr-Cas9 editing in HCT116-p53-/- (KrasG13D/p53-/-) cells and analyzed the expression profile in HCT116-p53-/-PHRF1-/- cells. In contrast to lung cancer A549 (KrasG12S/p53wt) cells, the expression of Zeb1, a transcription factor for epidermal-mesenchymal transition (EMT), was not affected in PHRF1-knockout HCT116 p53-/- cells. Instead, SOX4 displayed a significant contribution to the impaired invasion in HCT116-p53-/-PHRF1-/- cells. Mechanistically, the C-terminal SRI domain of PHRF1 was required for both transwell invasion and SOX4 expression. The reintroduction of SOX4 into HCT116-p53-/- PHRF1-/- cells partially restored their invasive capability. This study sheds light on the role of PHRF1 in the invasion of colorectal cancer HCT116-p53-/- cells, which harbor the oncogenic KrasG13D mutation and lack p53. These findings provide novel insights regarding the role of PHRF1 in invasion by modulating SOX4 expression in colorectal cancer HCT116-p53-/- cells.

Inhibition of Protein Disulfide Isomerase (PDIA1) Leads to Proteasome-Mediated Degradation of Ubiquitin-like PHD and RING Finger Domain-Containing Protein 1 (UHRF1) and Increased Sensitivity of Glioblastoma Cells to Topoisomerase II Inhibitors.

Glioblastoma (GBM) is the most aggressive brain tumor, and the prognosis remains poor with current available treatments. PDIA1 is considered a promising therapeutic target in GBM. In this study, we demonstrate that targeting PDIA1 results in increased GBM cell death by topoisomerase II (Top-II) inhibitors resulting in proteasome-mediated degradation of the oncogenic protein UHRF1. Combination of the PDIA1 inhibitor, bepristat-2a, produces strong synergy with doxorubicin, etoposide, and mitoxantrone in GBM and other cancer cell lines. Our bioinformatics analysis of multiple datasets revealed downregulation of UHRF1, upon PDIA1 inhibition. In addition, PDIA1 inhibition results in proteasome-mediated degradation of UHRF1 protein. Interestingly, treatment of GBM cells with bepristat-2a results in increased apoptosis and resistance to ferroptosis. Our findings emphasize the importance of PDIA1 as a therapeutic target in GBM and present a promising new therapeutic approach using Top-II inhibitors for GBM treatment.

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1.

Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition.

PHRF1 promotes migration and invasion by modulating ZEB1 expression.

PHRF1 (PHD and RING finger domain-containing protein 1) suppresses acute promyelocytic leukemia (APL) by promoting TGIF (TG-interacting factor) ubiquitination, while the PML-RARα protein interferes with PHRF1-mediated TGIF breakdown to facilitate APL. Beyond its role in APL tumorigenesis, PHRF1 contributes to non-homologous end-joining by linking H3K36 methylation and Nbs1 upon DNA damage insults. However, little is known regarding its function in tumor invasion. Here we highlight the unreported details of PHRF1 in the invasion of lung cancer cells by modulating the transcriptional level of ZEB1, a prominent regulator involved in epithelial-mesenchymal transition. PHRF1 associated with the phosphorylated C-terminal repeat domain of Rpb1, the large subunit of RNA polymerase II, through its C-terminal Set2 Rpb1 Interacting (SRI) domain. Chromatin immunoprecipitation revealed that PHRF1 bound to the proximal region adjacent to the transcription start site of ZEB1. SRI-deleted PHRF1 neither associated with Rpb1 nor increased ZEB1's expression. Collectively, PHRF1 might take the stage at migration and invasion by modulating the expression of ZEB1.

DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.

To accomplish the remarkable task of lifelong infection, Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programs to navigate the B-cell compartment and evade immune responses. The transforming program, comprised of highly immunogenic EBV nuclear antigen (EBNA) and Latent Membrane Proteins (LMP), is expressed in newly infected B-lymphocytes and in post-transplant lymphomas. Upon memory cell differentiation and in most EBV-associated Burkitt lymphomas (BL), all but one viral antigen are repressed for immunoevasion. To gain insights into epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR/Cas9 screen was performed in EBV+ BL cells. Here we show that the ubiquitin ligase UHRF1 and its DNA methyltransferase partner DNMT1 were critical for restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing, and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency program switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal center B-cells, the BL cell of origin, providing a molecular link between B-cell state and EBV latency program. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.

CircFAT1 Suppresses Colorectal Cancer Development Through Regulating miR-520b/UHRF1 Axis or miR-302c-3p/UHRF1 Axis.

Background: It was reported that circular RNAs (circRNAs) exerted important functions in various human cancers. However, the function of circFAT1 was less known. The purpose of this study was to reveal the functional mechanism of circFAT1 in colorectal cancer (CRC). Materials and Methods: Quantitative real-time polymerase chain reaction and Western blot assay were used to detect the levels of genes. Cell proliferation ability was assessed by 3-(4, 5-dimethyl-2-thiazoyl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry was used to investigate cell apoptosis rate. The glucose consumption and lactate production were determined using related kits. Furthermore, the interaction between circFAT1 or ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and miR-520b or miR-302c-3p was predicted by starbase3.0, and then confirmed by the dual-luciferase reporter assay. Besides, xenograft experiment was performed to analyze the effect of circFAT1 on tumor growth in vivo. Results: The levels of circFAT1 and UHRF1 were increased, as well as the levels of miR-520b and miR-302c-3p were decreased in CRC tissues and cells. CircFAT1 knockdown suppressed cell proliferation, cycle, and glycolysis as well as induced apoptosis. Interestingly, circFAT1 was a sponge of miR-520b and miR-302c-3p, and miR-520b and miR-302c-3p could target UHRF1. Both miR-520b overexpression and miR-302c-3p overexpression inhibited CRC cell growth. Furthermore, both miR-520b knockdown and miR-302c-3p depletion weakened the effect of circFAT1 knockdown on the growth of CRC cells. Besides, circFAT1 depletion repressed tumor growth in vivo. Conclusion: The authors' findings suggested that circFAT1 upregulated UHRF1 to affect CRC cell proliferation, apoptosis, and glycolysis through targeting miR-520b and miR-302c-3p, providing theoretical basis for the treatment of CRC.

Overexpression of UHRF1 and its potential role in the development of invasive ductal breast cancer validated by integrative bioinformatics and immunohistochemistry analyses.

BackgroundIncreasing evidence has highlighted the role of ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) in the development of cancers, including hepatocellular carcinoma, pancreatic cancer, and bladder cancer. However, the correlation between UHRF1 and breast cancer remains unclear. The present study aimed to analyze the expression of UHRF1 and its role in the development of invasive ductal breast cancer (IDC) by integrating multilevel expression data and immunohistochemistry analysis.MethodsThe Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to gather UHRF1 expression data on IDC. Additionally, immunohistochemistry analysis was used to investigate the correlations between UHRF1 expression and the clinical characteristics of IDC.ResultsThe GEO and TCGA databases indicated that UHRF1 was up-regulated in IDC. Consistently, the immunohistochemical specimens showed that the significant overexpression of UHRF1 in IDC, and its expression level showed an increasing trend from ductal carcinomas in situ to IDC. Notably, the increased levels of UHRF1 were closely correlated with estrogen receptor expression, pathological grade, and the prognosis of the disease. In addition, patients with a high UHRF1 expression had a poorer prognosis.ConclusionsIn conclusion, our findings suggested that UHRF1 plays a promoting role in breast tumorigenesis, and the over-expression of UHRF1 could serve as a biomarker for the prognosis in invasive ductal carcinomas in breast cancer.

RN181 is a tumour suppressor in gastric cancer by regulation of the ERK/MAPK-cyclin D1/CDK4 pathway.

RN181, a RING finger domain‐containing protein, is an E3 ubiquitin ligase. However, its biological function and clinical significance in cancer biology are obscure. Here, we report that RN181 expression is significantly down‐regulated in 165 tumour tissues of gastric carcinoma (GC) versus adjacent non‐tumour tissues, and inversely associated with tumour differentiation, tumour size, clinical stage, and patient's overall survival. Alterations of RN181 expression in GC cells by retrovirus‐transduced up‐regulation and down‐regulation demonstrated that RN181 functions as a tumour suppressor to inhibit growth of GC in both in vitro culture and in vivo animal models by decreasing tumour cell proliferation and increasing tumour cell apoptosis. Cell cycle analysis revealed that RN181 controls the cell cycle transition from G1 to S phase. Mechanistic studies demonstrated that RN181 inhibits ERK/MAPK signalling, thereby regulating the activity of cyclin D1–CDK4, and consequently controlling progression in the cell cycle from G1 to S phase. Restoring CDK4 in GC cells rescued the inhibitory phenotype produced by RN181 in vitro and in vivo, suggesting a dominant role of CDK4 in control of the tumour growth by RN181. Importantly, RN181 expression is inversely correlated with the expression of cyclin D1 and CDK4 in GC clinical samples, substantiating the role of the RN181–cyclin D1/CDK4 pathway in control of the tumour growth of GC. Our results provide new insights into the pathogenesis and development of GC and rationale for developing novel intervention strategies against GC by disruption of ERK/MAPK–cyclin D1/CDK4 signalling. In addition, RN181 may serve as a novel biomarker for predicting clinical outcome of GC. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster.

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Ubiquitome Analysis Reveals PCNA-Associated Factor 15 (PAF15) as a Specific Ubiquitination Target of UHRF1 in Embryonic Stem Cells

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.

UHRF2 regulates local 5-methylcytosine and suppresses spontaneous seizures

ABSTRACTThe 5-methylcytosine (5mC) modification regulates multiple cellular processes and is faithfully maintained following DNA replication. In addition to DNA methyltransferase (DNMT) family proteins, ubiquitin-like PHD and ring finger domain-containing protein 1 (UHRF1) plays an important role in the maintenance of 5mC levels. Loss of UHRF1 abolishes 5mC in cells and leads to embryonic lethality in mice. Interestingly, UHRF1 has a paralog, UHRF2, that has similar sequence and domain architecture, but its biologic function is not clear. Here, we have generated Uhrf2 knockout mice and characterized the role of UHRF2 in vivo. Uhrf2 knockout mice are viable, but the adult mice develop frequent spontaneous seizures and display abnormal electrical activities in brain. Despite no global DNA methylation changes, 5mC levels are decreased at certain genomic loci in the brains of Uhrf2 knockout mice. Therefore, our study has revealed a unique role of UHRF2 in the maintenance of local 5mC levels in brain that is distinct from that of its paralog UHRF1.

Abstract 3059: UHRF1 deficiency exerts anti-tumor immune responses in non-small cell lung cancer

Abstract Immune surveillance is essential for effective tumor clearance. However, the tumor microenvironment is typically impermissive for proper stimulation of both innate and adaptive immune populations. Epigenetic inhibitors have recently been shown to function as immune sensitizing reagents in the suppressive microenvironment. In our search for novel epigenetic regulators with immunomodulatory functions, we found ubiquitin-like with PHD and ring finger domain-containing protein 1 (UHRF1) being crucial for the suppression of immune activation. Loss of UHRF1 in cancer cells led to significant reduction of tumor masses in a syngeneic lung cancer model. The tumor shrinkage was due to increased infiltration of T cells with the enhanced ability to proliferate and produce IFN-gamma. Mechanistically, lung cancer cells deficient of UHRF1 had elevated expression of endogenous retrovirus genes due to DNA demethylation, which associated with enhanced type I interferon responses. Type I interferons are potent antitumor effector molecules by promoting immune cell infiltration into the tumor microenvironment, yet they can also dampen T cell activation by upregulating PD-L1 expression in tumor cells. Surprisingly, UHRF1 deficient cancer cells failed to upregulate PD-L1 even in the presence of excessive type I interferons, indicative of additional regulatory pathways for PD-L1. We further found that UHRF1 directly interacted with PD-L1 promoter regions and that UHRF1 promoted PD-L1 transcription. Therefore, the inhibition of UHRF1 augmented type I interferon responses while diminished PD-L1 expression, which synergistically contributed to the immune activation phenotype. Moreover, in non-small cell lung cancer (NSCLC) patients, UHRF1 expression was positively correlated with PD-L1 expression as well as poor prognosis. Together, our data suggested that UHRF1 represents a novel immuno-oncology target for NSCLC. Citation Format: Handan Xiang, Junying Chen, Xiao-Fan Wang. UHRF1 deficiency exerts anti-tumor immune responses in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3059. doi:10.1158/1538-7445.AM2017-3059

UHRF1 is required for basal stem cell proliferation in response to airway injury

Cellular senescence is a cell fate characterized by an irreversible cell cycle arrest, but the molecular mechanism underlying this senescence hallmark remains poorly understood. Through an unbiased search for novel senescence regulators in airway basal cells, we discovered that the epigenetic regulator ubiquitin-like with PHD and ring finger domain-containing protein 1 (UHRF1) is critical for regulating cell cycle progression. Upon injury, basal cells in the mouse airway rapidly induce the expression of UHRF1 in order to stimulate stem cell proliferation and tissue repair. Targeted depletion of Uhrf1 specifically in airway basal cells causes a profound defect in cell cycle progression. Consistently, cultured primary human basal cells lacking UHRF1 do not exhibit cell death or differentiation phenotypes but undergo a spontaneous program of senescence. Mechanistically, UHRF1 loss induces G1 cell cycle arrest by abrogating DNA replication factory formation as evidenced by loss of proliferating cell nuclear antigen (PCNA) puncta and an inability to enter the first cell cycle. This proliferation defect is partially mediated by the p15 pathway. Overall, our study provides the first evidence of an indispensable role of UHRF1 in somatic stem cells proliferation during the process of airway regeneration.

MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-\u03baB and negatively regulates inflammatory responses

Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation. Using yeast two-hybrid screening, we sought to isolate PDLIM2-interacting proteins that are critical for suppressing NF-κB signaling. Here we identified MKRN2, a RING finger domain-containing protein that belongs to the makorin ring finger protein gene family, as a novel p65 ubiquitin E3 ligase. MKRN2 bound to p65 and promoted the polyubiquitination and proteasome-dependent degradation of p65 through the MKRN2 RING finger domain, thereby suppressing p65-mediated NF-κB transactivation. Notably, MKRN2 and PDLIM2 synergistically promote polyubiquitination and degradation of p65. Consistently, MKRN2 knockdown in dendritic cells resulted in larger amounts of nuclear p65 and augmented production of proinflammatory cytokines in responses to innate stimuli. These results delineate a novel role of MKRN2 in negatively regulating NF-κB-mediated inflammatory responses, cooperatively with PDLIM2.

Homozygous Mutations in PHRF1 and PPARG in a Patient with Congenital Neutropenia and Profound Monocytosis: Understanding the Pathway Beyond Monocyte Versus Granulocyte Cell-Fate Choice

Myelopoiesis is a strictly regulated process of monocytes and granulocytes production, originating from common myeloid progenitors. Studies on patients with inherited severe congenital neutropenia (CN) could help to better elucidate myelopoiesis regulation. In CN, maturation arrest of granulocytic precursors at the promyelocytic stage leads to a shift towards monopoiesis and an ineffective granulopoiesis. Hereby we report on a particular CN patient with a typical "maturation arrest" of granulopoiesis at the stage of promyelocytes, very low levels of neutrophils but extremely high levels of monocytes in the peripheral blood and bone marrow. Upon G-CSF treatment (5μg/kg/day) this patient produces high levels of monocytes (up to 24.8x103/μL, more than 50 % of total leukocytes) and only up to 1x103/μl neutrophils. Sanger sequencing of DNA from this patient revealed no mutations in CN-related genes such as ELANE, HAX1 and G6PC3. Therefore we performed whole genome sequencing (Complete Genomics. Inc, Mountain View, CA) of DNA from blood of this patient and his mother to discover causative gene mutations. We identified a homozygous deletion in PHRF1 (PHD and Ring Finger Domain-Containing Protein 1) (p.R1015-G1019, NP_065952.2; rs144630030) and a homozygous missense mutation in PPARG (Peroxisome proliferator-activated receptor gamma) (p.P12A, NP_0569553.2; rs1801282). Both mutations are heterozygote in the patient's mother. Population frequency for heterozygote allele of these two variations was reported to be 13% and 7%, respectively, but no homozygote variants were reported till date. PHRF1 functions as an essential component of the TGF-ß tumour suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF (TG-Interacting Factor) and a consequent retinoic acid signalling activation in haematopoiesis and monopoiesis. PPARG interacts with Retinoid X Receptors (RXR) and controls the expansion of macrophages. In order to evaluate the functional role of the detected mutations on disturbed G-CSF-triggered myelopoiesis in reference CN patient, CD33+ bone marrow myeloid progenitor cells of two healthy controls and this patient were treated with G-CSF in vitro and mRNA expression profiles were analysed in an Affymetrix Microarray platform, followed by Ingenuity Pathway Analysis (IPA). We found, that 'TREM1 signalling' was among the top three pathways with most significant differences (p<9x10-7) between this CN patient and healthy individuals. 'Granulocyte adhesion and diapedesis' and 'LXR/RXR pathway' were the next two significantly affected gene sets (p<8x10-6 and p<1.3x10-5, respectively). TREM1 (Triggering Receptor Expressed on Myeloid cells 1) is a chemokine receptor that is expressed by neutrophils and monocytes, however the ligand that activates this receptor is yet unknown. TREM-1 is involved in neutrophil apoptosis and is known to positively regulate monopoiesis by activation of M-CSF synthesis. Intriguingly, M-CSF was 4.2-fold upregulated in myeloid cells of patient, in comparison to healthy individuals. Other known components of TREM 1 signalling were also among the top 10 differentially expressed genes identified by IPA: HSD11B1 (+20 fold), ATP1B2 (+14 fold) and THBS1 (-10 fold). Functional connections between PPARG and TREM1 is known. PPARG mutation could lead to TREM1 signalling activation that consequently lead to M-CSF over-expression (+4.2 fold). In addition to the activation of TREM1 signalling, deletion in the PHFR1 gene could be the causative effect of marked upregulation of ALDH1A2 (+17 fold), which also could lead to an increase in M-CSF levels and in a retinoid acid signalling activation ultimately leading to increased monocyte production. Together, PPARG and PHRF1 mutations could hyper-activate the secretion of M-CSF by myeloid progenitors leading to a strong shift towards monopoiesis upon G-CSF treatment. Disclosures No relevant conflicts of interest to declare.

Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin are poorly defined. Here we show that the linked tandem Tudor and plant homeodomain (PHD) of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1) operates as a functional unit in cells, providing a defined combinatorial readout of a heterochromatin signature within a single histone H3 tail that is essential for UHRF1-directed epigenetic inheritance of DNA methylation. These findings provide critical support for the "histone code" hypothesis, demonstrating that multivalent histone engagement plays a key role in driving a fundamental downstream biological event in chromatin.

The E3 ubiquitin ligases RNF126 and Rabring7 regulate endosomal sorting of the Epidermal Growth Factor Receptor

Activation of the epidermal growth factor receptor (EGFR) results in internalization and ubiquitin-dependent endosomal sorting, leading to lysosomal degradation. Here we describe the role of the RING-finger-domain-containing protein RNF126 and the related protein, Rabring7 in EGFR endosomal sorting. We demonstrate that RNF126 specifies K48-linked chains with UbcH5b and also functions with Ubc13/Uev1a to form K63-linked chains in vitro. RNF126 and Rabring7 associate with the EGFR through a ubiquitin-binding zinc finger domain and both E3 ubiquitin ligases promote ubiquitylation of EGFR. In the absence of c-Cbl or in cells expressing Cbl-70Z, the binding of RNF126 and Rabring7 to the EGFR is reduced, suggesting that RNF126 and Rabring7 function downstream of c-Cbl. In HeLa cells depleted of either RNF126 or Rabring7 the EGFR is retained in a late endocytic compartment and is inefficiently degraded. In addition, depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. Together these data suggest that RNF126 and Rabring7 play a role in the ubiquitin-dependent sorting and downregulation of membrane receptors.

Amplitude Control of Protein Kinase C by RINCK, a Novel E3 Ubiquitin Ligase

Protein kinase C (PKC) isozymes play a central role in cellular signaling. Levels of PKC control the amplitude of agonist-induced signaling and alterations in these levels are associated with disease states, most notably cancer, yet mechanisms that control the turnover of the protein are poorly understood. Here we identify an E3 ligase that catalyzes the ubiquitin-mediated degradation of PKC. Specifically, we identified a RING finger domain-containing protein, RINCK (for RING-finger protein that interacts with C kinase) from a yeast two-hybrid screen using the amino terminus of PKCbeta as bait. RINCK encodes a protein of 581 amino acids that contains a RING finger domain, a B-box, and two coiled-coil regions, the three domains that form the signature motif of the large family of diverse TRIM (tripartite motif) proteins. Co-immunoprecipitation studies using tsA201 cells reveal that RINCK and PKC associate with each other in cells. Studies using fragments of PKCbeta reveal that this interaction is mediated by the C1A domain of PKC. RINCK induces the ubiquitination of PKC both in vitro and in cells. Overexpression of RINCK reduces the levels of PKC in cells, whereas genetic knockdown of endogenous RINCK increases the levels of PKC. This increase was observed for all PKC isozymes examined (including conventional, novel, and atypical). The RINCK-mediated degradation of PKC occurs independently of the classic phorbol ester-mediated down-regulation: genetic depletion of RINCK had no effect on the phorbol ester-mediated down-regulation and, additionally, up-regulated the levels of isozymes that cannot bind phorbol esters. Our data reveal a novel mechanism that provides amplitude control in PKC signaling through ubiquitination catalyzed by RINCK, an E3 ligase that specifically recognizes the C1 domain of PKC isoforms.