FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair

Two simple assays for assessing the seeding activity of proteopathic tau

The regional distribution of neurofibrillary tangles of hyperphosphorylated tau aggregates is associated with the progression of Alzheimer’s disease (AD). Misfolded proteopathic tau recruits naïve tau and templates its misfolding and aggregation in a prion-like fashion, which is believed to be the molecular basis of propagation of tau pathology. A practical way to assess tau seeding activity is to measure its ability to recruit/bind other tau molecules and to induce tau aggregation. Based on the properties of proteopathic tau, here we report the development of two simple assays to assess tau seeding activity ----- capture assay in vitro and seeded-tau aggregation assay in cultured cells. In the capture assay, proteopathic tau was applied onto a nitrocellulose membrane and the membrane was incubated with cell lysate containing HA-tagged tau151-391 (HA-tau151-391). The captured tau on the membrane was determined by immuno-blots developed with anti-HA. For the seeded-tau aggregation assay, HEK-293FT cells transiently expressing HA-tau151-391 were treated with proteopathic tau in the presence of Lipofectamine 2000 and then lysed with RIPA buffer. RIPA-insoluble fraction containing aggregated tau was obtained by ultracentrifugation and analyzed by immuno-blot developed with anti-HA. To validate these two assays, we assessed the seeding activity of tau in the middle frontal gyrus, middle temporal gyrus and basal forebrain of AD and control brains and found that AD, but not control, brain extracts effectively captured and seeded tau151-391 aggregation. Basal forebrain contained less phospho-tau and tau seeding activity. The levels of captured tau or seeded-tau aggregates were positively correlated to the levels of phospho-tau, Braak stages and tangle sores. These two assays are specific and sensitive and can be carried out in a regular biomedical laboratory setting by using routine biochemical techniques

Epistasis between MicroRNAs 155 and 146a during T Cell-Mediated Antitumor Immunity

An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155^(−/−) mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4^+ and CD8^+ T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses

BRIT1/MCPH1 links chromatin remodelling to DNA damage response

To detect and repair damaged DNA, DNA damage response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions1. ATP-dependent chromatin remodeling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair2–3. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA damage response protein that is mutated in human primary microcephaly4–8. We report here a previously unknown function of BRIT1 as a regulator of ATP-dependent chromatin remodeling complex SWI/SNF in DNA repair. Upon DNA damage, BRIT1 increases its interaction with SWI/SNF through the ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase of binding affinity provides a means by which SWI/SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation owing to reduced association of SWI/SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired survival from DNA damage. Our findings, therefore, identify BRIT1 as a key molecule that links chromatin remodeling with DNA damage response in the control of DNA repair, and its dysfunction contributes to human disease

Introduction of risk size in the determination of uncertainty factor UFL in risk assessment

The methodology for using uncertainty factors in health risk assessment has been developed for several decades. A default value is usually applied for the uncertainty factor UF _L , which is used to extrapolate from LOAEL (lowest observed adverse effect level) to NAEL (no adverse effect level). Here, we have developed a new method that establishes a linear relationship between UF _L and the additional risk level at LOAEL based on the dose–response information, which represents a very important factor that should be carefully considered. This linear formula makes it possible to select UF _L properly in the additional risk range from 5.3% to 16.2%. Also the results remind us that the default value 10 may not be conservative enough when the additional risk level at LOAEL exceeds 16.2%. Furthermore, this novel method not only provides a flexible UF _L instead of the traditional default value, but also can ensure a conservative estimation of the UF _L with fewer errors, and avoid the benchmark response selection involved in the benchmark dose method. These advantages can improve the estimation of the extrapolation starting point in the risk assessment

Using two TALEN pairs to delete the entire human miR-155 hairpin sequence.

<p>(A) TALEN pairs targeting miR-155 were designed and constructed (called TALEN C). The upper panel shows a schematic of the TALEN A pair binding sites. The lower panel shows the sequence alignments comparing Wt and TALEN C mutated miR-155. The left and right TALEN binding sites are highlighted in yellow and the miR-155 seed region is boxed in red. (B) Schematic of the binding sites of two TALEN pairs (TALEN A and TALEN C) targeting miR-155. (C–D) Both TALEN A and TALEN C pairs were transfected into 293T cells. The miR-155 locus was amplified by PCR and subjected to TOPO cloning and sequencing. (C) Electrophoresis gel analysis showing deletions in the miR-155 locus. The arrows on the right indicate the two expected PCR products with or without large deletions. (D) Sequence alignments between a Wt clone and two TOPO clones with large deletions. The left and right TALEN binding sites for both TALEN A and TALEN C are highlighted in yellow and the miR-155 hairpin sequence is boxed in red.</p

The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence.

<p>293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.</p

Mutation rate mediated by TALEN pairs in transfected 293T cells.

<p>Mutation rate mediated by TALEN pairs in transfected 293T cells.</p