Identification of novel tumour suppressor genes involved in the development of cutaneous malignant melanoma

Skin cancer is one of the most common forms of adult solid tumour. The incidence is increasing rapidly making skin cancer a major health problem in several countries. Cutaneous Malignant Melanoma (CMM) is the least common but the most life threatening type of skin cancers and is responsible for 90% of all skin malignancy associated deaths. The precise cellular and molecular etiology of malignant melanoma is quite complex and the molecular events directly related to melanoma progression are yet to be elucidated. However, recent advances in molecular biology have resulted in a clearer understanding of the cellular and molecular events of skin cancer development. The best-characterized locus associated with CMM development is the CDKN2A that maps to chromosome 9p21 and encodes for the cell cycle regulator p16 tumour suppressor gene (TSG), and is frequently inactivated in melanoma tumours. In addition to p16, other loci located in 9p21 appear to be important in CMM development and functional evidence for the presence of TSG(s) has been provided (Parris et al., 1999). The aim of our study is to contribute to the understanding of CMM development by isolating and characterising novel TSG(s) at this location. In order to pursue identifying potential TSG(s), we have developed several monochromosome hybrids using microcell mediated chromosome transfer, and evaluated the tumourigenicity of the constructed hybrids by anchorage independent growth in soft agar. For the molecular biology aspects, expression analysis of the genes in the 9p21 region was carried out by reverse transcription PCR. Potential candidate tumour suppressor genes were then carefully evaluated by generating expression profiles via conducting real time PCR. Experimental evidence is provided which supports the candidacy of interferon alpha 1 (IFNA1) as a tumour suppressor gene for melanoma development

A Case of Adult-Onset Still’s Disease Caused by a Novel Splicing Mutation in TNFAIP3 Successfully Treated With Tocilizumab

TNFAIP3 encodes the NF-κB regulatory protein A20. High-penetrance heterozygous mutations in TNFAIP3 cause a haploinsufficiency of A20 (HA20), inadequate inhibition of NF-κB pathway, and an early onset autoinflammatory disorder. However, the clinical phenotype of patients with HA20 varies greatly and clinical diagnoses prior to establishing the genetic cause, included both autoimmune and autoinflammatory conditions. Here, we present the first patient with HA20, who was previously diagnosed with AOSD but was later found to have a novel heterozygous variant in TNFAIP3 and who was successfully treated with anti-IL6 receptor biologic tocilizumab (RoActemra). We discovered a novel heterozygous mutation in TNFAIP3 c.1906C>T, not previously found in ExAC database. Further analysis shows that this single-nucleotide variant at the terminal residue of TNFAIP3 exon 7 produces an alternatively spliced mRNA resulting in p.His636fsTer1. Additional genetic analysis of family members shows that this variant does segregate with the inflammatory clinical phenotypes. Subsequent functional test show that NF-κB activation, measured as intracellular phosphorylation of p65 in CD14 + monocytes, was more enhanced in the patient compared with healthy controls (HC) following stimulation with LPS. This was associated with higher production of inflammatory cytokines by the patients PBMC in response to LPS and ATP and enhanced activation of NLRP3 inflammasome complex. Furthermore, increased activation of NLRP3 inflammasome was evident systemically, since we detected higher levels of ASC specks in patients’ sera compared with HC. Finally, we used population genetics data from GnomAD to construct a map of both genetic conservation and most probable disease-causing variants in TNFAIP3 which might be found in future cases of HA20

Heterochromatin protein 1 gamma and IiB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages

IiB kinase a (IKKa) is part of the cytoplasmic IKK complex regulating nuclear factor-{kappa}B (NF-iB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKa can also be recruited directly to the promoter of NF-iB-dependent genes by NF-iB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKa travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1c) at NF-iBdependent genes in activated macrophages. IKKa binds to and phosphorylates HP1c, which in turn controls IKKa binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKa accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKa inactivation and transcription termination

The −1.9 kb element is a NF-κB-dependent LPS inducible promoter and enhancer in transient transfection.

<p>HD11 macrophages were transfected for 18 hrs with Jetpei 2 µl and 1 µg DNA prior to 7 hrs 1 µg/ml LPS stimulation, black bars, or remained untreated for 7 hrs, grey bars. The constructs are illustrated adjacent to the y-axis. (<b>a</b>) <i>Clys</i> promoter is a black arrow, the −1.9 kb CRE is a dark grey arrow, the firefly luciferase coding region is grey. (<b>b</b>) The −1.9 kb element is cloned in antisense orientation, Position of the NF-κB, C/EBP, X unknown protein and AP1 are indicated next to the Y axis. (<b>a</b> and <b>b</b>) The data are plotted as the mean value of two independent experiments, individual experiments had triplicate samples for each condition. Inter sample variation has been corrected by Renilla normalisation. Positive error bars indicate standard deviations.</p

LPS induces recruitment of NF-κB, IKKα, RNAPII and HP1γ with different kinetics.

<p>(<b>a–d</b>) ChIP performed with primary macrophages treated with LPS for the indicated time points in minutes and the following antibodies (<b>a</b>) anti-p65 (NF-κB), (<b>b</b>) anti-RNAPII CTD, (<b>c</b>) anti-IKKα and (<b>d</b>) anti-HP1γ. Horizontal axis indicates primers used for the Real time PCR (distance in kb from the transcription start site of <i>cLys</i>). Data are normalized versus input. Error bars represent SD from three independent qPCR replicates. These data are representative of at least three independent experiments.</p

A NF-κB-Dependent Dual Promoter-Enhancer Initiates the Lipopolysaccharide-Mediated Transcriptional Activation of the Chicken Lysozyme in Macrophages

<div><p>The transcriptional activation of the chicken lysozyme gene (c<i>Lys</i>) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPβ controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both <i>cLys</i> and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating <i>cLys</i> transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both <i>cLys</i> transcription and NF-κB binding to the CRE. Finally, comparing <i>cLys</i> with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3′ end of the gene.</p> </div

Rapid nucleosome loss at the−1.9 kb CRE in macrophages after LPS treatment.

<p>(<b>a</b>) Illustration of the chicken lysozyme locus including cis-regulatory elements and the approximate location of LINoCR transcript, grey arrow and <i>cLys</i> mRNA, black arrow <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059389#pone.0059389-Lefevre1" target="_blank">[7]</a>. (<b>b</b>) ChIP assay performed with anti-histone H3 antibody at the −1.9 kb element compared to the −6.1 kb enhancer after LPS treatment in macrophages. Horizontal axis represents time after LPS induction in minutes. Data are normalised versus input and then versus a positive control region. Data are representative of at least three independent experiments.</p

<i>In vivo</i> MNase and DNase I mapping of the −1.9 kb element reveal chromatin remodelling in response to LPS.

<p>Southern blot of (<b>a</b>) MNase or (<b>b</b>) DNAse I digested genomic DNA from permeabilised nuclei of unstimulated HD11 (lane 1 and 5) or HD11 LPS (5 µg/ml) stimulated (<b>a</b>) for 30 min, 60 min and 240 min (lanes 2–4) or (<b>b</b>) for 45 min, 120 min and 360 min (lanes 6–8). Inferred nucleosome positions are illustrated in grey. Width of arrows indicates the degree of cleavage and the approximate position relative to the <i>clys</i> transcription start site. These experiments are representative of two independent experiments. Quantification of (<b>c</b>) the MNase, LPS stimulated samples are normalised to unstimulated HD11 and (<b>d</b>) the DNAse I southern blots. Arrows are indicating approximate position of cleavages. DNAse I protection domain within the −1.9 Kb element is illustrated by a black box.</p

RNAPII S2p and IKKα occupancies at <i>cLys</i> locus overlap.

<p>(<b>a–c</b>) ChIP performed with primary macrophages treated with LPS for 30 min and the following antibodies (<b>a</b>) anti-p65 (NF-κB) and anti-IKKα, (<b>b</b>) anti-RNAPII S2p and anti-IKKα (<b>a–b</b>) Position of the −1.9 kb element is indicated by an arrow. Horizontal axis indicates distance from <i>cLys</i> transcription start site. Data are normalized versus input and then versus the average of all IP/Input values. These data are representative of at least three independent experiments.</p