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Many analytical or cell culture procedures require homogeneous starting cell populations that cannot be obtained directly from organ dissection. Here, we describe two enrichment procedures to achieve this goal and discuss their respective advantages in specific experimental contexts.

Retinoblastoma (RB) is a pediatric ocular malignancy that is initiated mostly by biallelic inactivation of the RB transcriptional corepressor 1 (

Identification of new genetic pathways or molecular targets that sensitize cancer cells to chemotherapeutic drugs may improve the efficacy of current chemotherapy. Here, we report that downmodulation of UHRF1 (ubiquitin‐like with PHD and RING finger domains 1) in retinoblastoma (RB) cells increases the sensitivity to histone deacetylase (HDAC) inhibitors, augmenting apoptotic cell death. We found that UHRF1 depletion downregulates two redox‐responsive genes GSTA4 (glutathione S‐transferase α4) and TXN2 (thioredoxin‐2) in RB cells, and increases the basal level of intracellular oxidative stress. Antioxidant treatment significantly reduced both basal and HDAC inhibitor‐induced DNA damage and apoptosis in UHRF1‐depleted cells. Knockdown of GSTA4 or TXN2 sensitized RB cells to HDAC inhibitors, demonstrating that GSTA4 and TXN2 play key roles in redox homeostasis in RB cells and the susceptibility to HDAC inhibitor treatment upon UHRF1 depletion. In human primary RB, GSTA4 and TXN2 proteins were found to be mostly elevated along with high UHRF1 expression. In addition to augmentation of apoptosis in UHRF1‐depleted RB cells, we also show that UHRF1 downmodulation derepresses the expression of photoreceptor‐specific genes in RB cells in cooperation with a HDAC inhibitor MS‐275 and promotes neuron‐like differentiation. However, further investigation revealed that the enhanced growth‐inhibitory effects of MS‐275 in UHRF1‐depleted cells were still mainly due to robust apoptosis induction rather than differentiation‐mediated growth arrest. Consistent with our findings, UHRF1 depletion in RB cells increased the therapeutic efficacy of MS‐275 in murine orthotopic xenografts. These results provide a novel basis for potential benefits of UHRF1 targeting for RB treatment., This study presents two novel tumor‐promoting functions of UHRF1 in retinoblastoma (RB). UHRF1 regulates reactive oxygen species (ROS)‐detoxifying genes to counteract the accumulation of intracellular ROS and thereby protects the cells from histone deacetylase (HDAC) inhibitor‐induced apoptosis. On the other hand, UHRF1 participates in repression of photoreceptor differentiation as a corepressor in a multiprotein complex containing HDAC to repress photoreceptor‐specific genes in RB cells.

Retinoblastoma (RB) is a pediatric ocular tumor mostly occurring due to the biallelic loss of RB1 gene in the developing retina. Early studies of genomic aberrations in RB have provided a valuable insight into how RB can progress following the tumor‐initiating RB1 mutations and have established a notion that inactivation of RB1 gene is critical to initiate RB but this causative genetic lesion alone is not sufficient for malignant progression. With the advent of high‐throughput sequencing technologies, we now have access to the comprehensive genomic and epigenetic landscape of RB and have come to appreciate that RB tumorigenesis requires both genetic and epigenetic alterations that might be directly or indirectly driven by RB1 loss. This integrative perspective on RB tumorigenesis has inspired research efforts to better understand the types and functions of epigenetic mechanisms contributing to RB development, leading to the identification of multiple epigenetic regulators misregulated in RB in recent years. A complete understanding of the intricate network of genetic and epigenetic factors in modulation of gene expression during RB tumorigenesis remains a major challenge but would be crucial to translate these findings into therapeutic interventions. In this review, we will provide an overview of chromatin regulators identified to be misregulated in human RB among the numerous epigenetic factors implicated in RB development. For a subset of these chromatin regulators, recent findings on their functions in RB development and potential therapeutic applications are discussed., In retinoblastoma (RB) cells, E2F activity remains unconstrained and promotes expression of cell cycle genes and chromatin regulators, leading to uncontrolled proliferation and aberrant alterations in epigenetic landscape and gene expression. These downstream effects of deregulated E2F activity are postulated to drive RB tumorigenesis. In this review, recent findings on the functions of misregulated chromatin regulators in RB are discussed.

// Guangyan Kan 1, 2, * , Heng He 1, * , Qi Zhao 3 , Xiubo Li 1 , Min Li 1 , Huasheng Yang 1 and Jong Kyong Kim 1 1 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China 2 Department of Immunology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510060, China 3 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China * These authors have contributed equally to the work Correspondence to: Jong Kyong Kim, email: kimjongk@mail.sysu.edu.cn Keywords: retinoblastoma, DNA methylation, UHRF1, global hypomethylation, tumorigenesis of murine retinoblastoma Received: January 02, 2017 Accepted: March 21, 2017 Published: April 13, 2017 ABSTRACT UHRF1 (ubiquitin-like with PHD and RING finger domains 1) is a critical regulator for DNA methylation, and its frequent overexpression in human cancers has been associated with tumor-promoting effects. However, whether the overexpressed UHRF1 contributes to the establishment and maintenance of tumor methylomes and whether this process can affect the tumorigenesis remain unclear. In this study, we show that UHRF1 is highly expressed in retinoblastoma, and genomes of human primary retinoblastoma and cell lines have differential DNA methylation patterns compared with those of normal retina, characterized by lower global methylation and higher promoter methylation of tumor suppressors. However, our genome-wide DNA methylation study uncovers that UHRF1 down-modulation in retinoblastoma cells exerts minor effects on the existing methylation patterns at both bulk genome and individual gene loci, suggesting that retinoblastoma methylome is primarily maintained by other mechanisms. Furthermore, using two murine retinoblastoma models, we found that high UHRF1 expression does not alter global methylation levels in both premalignant neonatal retina and retinoblastoma tumors, implying that DNA hypomethylation may not be an early mechanism driving retinoblastoma tumorigenesis unlike what has been proposed for other types of cancer. These results suggest that tumor-promoting functions of UHRF1 in retinoblastoma are largely independent of its role in DNA methylation.

UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is highly expressed in various human cancers including retinoblastoma, and associated with tumor-promoting effects such as inhibition of apoptosis and high proliferation. However, the molecular mechanisms underlying tumor-promoting functions of UHRF1 in retinoblastoma still remain elusive. Here, we show that stable knockdown of UHRF1 renders retinoblastoma cells sensitized to conventional chemotherapeutic drugs such as etoposide and camptothecin, resulting in enhanced DNA damage and apoptotic cell death. We found that UHRF1-depleted retinoblastoma cells can recognize DNA damages normally but have markedly low expression of XRCC4 (X-ray repair cross complementing 4) among the components of nonhomologous end-joining (NHEJ) repair complex. Conversely, overexpression of UHRF1 increased the XRCC4 expression and stable knockdown of XRCC4 sensitized retinoblastoma cells to etoposide treatment, suggesting that XRCC4 is a key mediator for the drug sensitivity upon UHRF1 depletion in retinoblastoma cells. Consistent with the findings, chromatin association of DNA ligase IV in response to acute DNA damage was found to be significantly reduced in UHRF1-depleted retinoblastoma cells and functional complementation for XRCC4 in UHRF1-depleted cells attenuated the drug sensitivity, demonstrating that XRCC4 downregulation in UHRF1-depleted cells impaired DNA repair and consequently induced robust apoptosis upon genotoxic drug treatment. In human primary retinoblastoma, high expression of UHRF1 and XRCC4 could be detected, and elevated XRCC4 expression correlated with reduced apoptosis markers, implying that UHRF1-mediated XRCC4 upregulation under pathophysiological conditions triggered by RB1 gene inactivation may confer protection against endogenous DNA damages that arise during retinoblastoma development. Taken together, these results present a new mechanistic insight into how UHRF1 mediates its tumor-promoting functions in retinoblastoma, and also provide a basis for UHRF1 targeting to improve the efficacy of current chemotherapy for retinoblastoma treatment.

Using Ezh2-deficient iNKT cells, Dobenecker et al. show that loss of H3K27me3 at the bivalently marked PLZF promoter is an essential link between TCR specificity and iNKT development in mice., The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4+ cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

Perturbations in the regulation of the core cell cycle machinery are frequently observed in human cancers. Cyclin D1 which functions as a mitogenic sensor and allosteric activator of CDK4/6, is one of the more frequently altered cell cycle regulators in cancers. Cyclin D1 is frequently overexpressed in cancers and its overexpression can be attributed to many factors including increased transcription, translation, and protein stability. Although cyclin D1 overexpression is clearly implicated in the affected cancers, overexpression of cyclin D1 is not sufficient to drive oncogenic transformation. Rather, emerging evidence suggests that nuclear retention of cyclin D1 resulting from altered nuclear trafficking and proteolysis is critical for the manifestation of its oncogenicity. This review provides a brief overview of current data documenting various mechanisms underlying aberrant cyclin D1 regulation in human cancers and their impact on neoplastic transformation.

Many analytical or cell culture procedures require homogeneous starting cell populations that cannot be obtained directly from organ dissection. Here, we describe two enrichment procedures to achieve this goal and discuss their respective advantages in specific experimental contexts. Notes in this chapter include some tips on how to determine the appropriate level of purity (see Note 1 ).

Angiotensin II (ANGII) has been identified as a proapoptotic and profibrotic factor in experimental lung fibrosis models, and patients with the ID/DD polymorphism of ANG converting enzyme (ACE), which confers higher levels of ACE, are predisposed to lung fibrosis (Hum. Pathol. 32:521-528, 2001). Previous work from this laboratory has shown that human lung myofibroblasts isolated from patients with Idiopathic Pulmonary Fibrosis (IPF) synthesize the ANGII precursor angiotensinogen (AGT) constitutively. In attempts to understand the mechanisms and consequences of constitutive AGT synthesis by myofibroblasts, we studied myofibroblast-rich primary cultures of lung fibroblasts from patients with IPF (HIPF isolates), primary fibroblasts from normal human lung (NLFs), the IMR90 and WI38 human lung fibroblasts cell lines, and paraffin sections of lung biopsies from patients with IPF. Compared to the normal NLF isolates, HIPF primary fibroblast isolates constitutively synthesized more AGT and TGF-beta1 mRNA, and released more AGT protein, ANGII and active TGF-beta1 protein into serum-free conditioned media (both p

Many analytical or cell culture procedures require homogeneous starting cell populations that cannot be obtained directly from organ dissection. Here, we describe two enrichment procedures to achieve this goal and discuss their respective advantages in specific experimental contexts. Notes in this chapter include some tips on how to determine the appropriate level of purity (see Note 1 ).

Transient influx of Ca2+ constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca2+ signaling are largely unknown. Because Ca2+ signals are mediated by Ca2+-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca2+ regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo;OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca2+-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca2+-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca2+/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca2+-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.

Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4+ T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation., Histone post-translational modifications such as the trimethylation of histone H3 at lysine 27 (H3K27Me3), affect DNA accessibility and transcription. Here, the authors show that Jmjd3 and Utx, two H3K27Me3 demethylases, are important for regulating the expression of genes involved in terminal thymocyte differentiation.

Mitogen-activated protein kinases (MAPKs) play important roles in responses to various environmental stresses. In a previous study, we demonstrated that OsBWMK1, which localizes in the nucleus, mediates PR gene expression by activating the OsEREBP1 transcription factor, and that the constitutive expression of OsBWMK1 also enhances resistance against pathogen infections [Y.H. Cheong, B.C. Moon, J.K. Kim, C.Y. Kim, M.C. Kim, I.H. Kim, C.Y. Park, J.C. Kim, B.O. Park, S.C. Koo, H.W. Yoon, W.S. Chung, C.O. Lim, S.Y. Lee, M.J. Cho, BWMK1, rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor, Plant Physiol. 132 (2003) 1961--1972]. Here, we report that OsBWMK1 phosphorylates OsWRKY33, which binds to the W-box element (TTGACCA) in several PR gene promoters, thereby enhancing DNA-binding activity of the factor to its in vitro cognate binding site. Transient coexpression of OsBWMK1 and OsWRKY33 in Arabidopsis protoplasts elevates SA-dependent expression of the GUS-reporter gene driven by the W-box element and the PR1 promoter. Furthermore, the levels of SA and H(2)O(2) are elevated in 35S-OsBWMK1 transgenic plants that show HR-like cell death. Altogether, OsBWMK1 may mediate SA-dependent defense responses by activating the WRKY transcription factor in plants.

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multi-domain protein associated with cellular proliferation and epigenetic regulation. The UHRF1 binds to methylated CpG dinucleotides and recruits transcriptional repressors DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through its distinct domains. However, the molecular basis of UHRF1-mediated transcriptional regulation via chromatin modifications is yet to be fully understood. Here we show that UHRF1 binds histone lysine methyltransferase G9a, and both are co-localized in the nucleus in a cell-cycle-dependent manner. Concurrent with the cell-cycle progression, gradual deposition of UHRF1 and G9a was observed, which mirrored H3K9me2 accumulation on chromatin. Murine Uhrf1-null embryonic stem (ES) cells displayed a reduced amount of G9a and H3K9me2 on chromatin. UHRF1 recruited and cooperated with G9a to inhibit the p21 promoter activity, which correlated with the elevated p21 protein level in both human UHRF1 siRNA-transfected HeLa cells and murine Uhrf1-null ES cells. Furthermore, endogenous p21 promoter remained bound to UHRF1, G9a, DNMT1 and HDAC1, and knockdown of UHRF1 impaired the association of all three chromatin modifiers with the promoter. Thus, our results suggest that UHRF1 may serve as a focal point of transcriptional regulation mediated by G9a and other chromatin modification enzymes.

Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.

Angiotensin II (ANGII) has been identified as a proapoptotic and profibrotic factor in experimental lung fibrosis models, and patients with the ID/DD polymorphism of ANG converting enzyme (ACE), which confers higher levels of ACE, are predisposed to lung fibrosis (Hum. Pathol. 32:521-528, 2001). Previous work from this laboratory has shown that human lung myofibroblasts isolated from patients with Idiopathic Pulmonary Fibrosis (IPF) synthesize the ANGII precursor angiotensinogen (AGT) constitutively. In attempts to understand the mechanisms and consequences of constitutive AGT synthesis by myofibroblasts, we studied myofibroblast-rich primary cultures of lung fibroblasts from patients with IPF (HIPF isolates), primary fibroblasts from normal human lung (NLFs), the IMR90 and WI38 human lung fibroblasts cell lines, and paraffin sections of lung biopsies from patients with IPF. Compared to the normal NLF isolates, HIPF primary fibroblast isolates constitutively synthesized more AGT and TGF-beta1 mRNA, and released more AGT protein, ANGII and active TGF-beta1 protein into serum-free conditioned media (both p0.01). Incubation of HIPF fibrotic isolates with the ANGII receptor antagonist saralasin reduced both TGF-beta1 mRNA and active protein, suggesting that the constitutive expression of AGT drives the higher expression of TGF-beta1 by the HIPF cells. Consistent with this premise, treatment of either the primary NLFs or the WI38 cell line with 10(-7) M ANGII increased both TGF-beta1 mRNA and soluble active TGF-beta1 protein. Moreover, induction of the myofibroblast transition in the IMR90 cell line with 2 ng/ml TGF-beta1 increased steady state AGT mRNA levels by realtime PCR (8-fold, p0.01) and induced expression of an AGT promoter-luciferase reporter construct by over 10-fold (p0.001). Antisense oligonucleotides against TGF-beta1 mRNA or TGF-beta neutralizing antibodies, when applied to the fibrotic HIPF cells in serum-free medium, significantly reduced AGT expression. In lung sections from IPF patient biopsies, immunoreactive AGT/ANGI proteins were detected in myofibroblasts, epithelial cells and presumptive alveolar macrophages. Together, these data support the existence of an angiotensin/TGF-beta1 "autocrine loop" in human lung myofibroblasts and also suggest ANG peptide expression by epithelia and macrophages in the IPF lung. These findings may explain the ability of ACE inhibitors and ANG receptor antagonists to block experimental lung fibrosis in animals, and support the need for evaluation of these agents for potential treatment of human IPF. This manuscript discusses the data described above and their implications regarding IPF pathogenesis.

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli , a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.

Bacillus subtilis contains urease structural genes but lacks the accessory genes typically required for GTP-dependent incorporation of nickel. Nevertheless, B. subtilis was shown to possess a functional urease, and the recombinant enzyme conferred low levels of nickel-dependent activity to Escherichia coli . Additional investigations of the system lead to the suggestion that B. subtilis may use unidentified accessory proteins for in vivo urease activation.

Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the β-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors.

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 co-regulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth including CUL4 repression, CDT1 overexpression, and DNA re-replication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.