Atypical CTSK Transcripts and ARNT Transcription Read-Through Into CTSK

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usual CTSK transcription start site is less than 1.4 kb downstream of the end of the longest reported ARNT transcript. By generating an RT-PCR product that overlaps both the 3′ end of ARNT and the 5′ end of CTSK, we show that ARNT transcripts may extend through the ARNT–CTSK intergenic region and progress into the CTSK gene. Furthermore, by using quantitative RT-PCR from several tissues to detect the ARNT expression signature in CTSK introns, we show that ARNT transcripts can read through into CTSK as far as CTSK intron 3, extending approximately 3.7 kb downstream of the end of the longest previously described ARNT mRNA. Given that ARNT and CTSK are expressed in an overlapping range of tissues, ARNT read-through may have a negative impact on CTSK transcript levels by interfering with CTSK expression. We also present evidence for novel CTSK transcripts following sequence analysis of CTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the known CTSK promoter and possibly in the 3′ UTR of ARNT

Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation

The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (TRAP) in osteoclasts. Several lines of evidence were consistent with this model. The combination of MITF and PU.1 synergistically activated the TRAP promoter in transient assays. This activation was dependent on intact binding sites for both factors in the TRAP promoter. MITF and PU.1 physically interacted when coexpressed in COS cells or in vitro when purified recombinant proteins were studied. The minimal regions of MITF and PU.1 required for the interaction were the basic-helix-loop-helix zipper domain and the Ets DNA binding domain, respectively. Significantly, mice heterozygous for both the mutant mi allele and a PU.1 null allele developed osteopetrosis early in life which resolved with age. The size and number of osteoclasts were not altered in the double heterozygous mutant mice, indicating that the defect lies in mature osteoclast function. Taken in total, the results afford an example of how lineage-specific gene regulation can be achieved by the combinatorial action of two broadly expressed transcription factors

The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor

Microphthalmia transcription factor (MITF) regulates osteoclast function by controling the expression of genes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to receptor activator of nuclear factor-kappa B ligand (RANKL)-induced signaling. To identify novel MITF target genes, we have overexpressed MITF in the murine macrophage cell line RAW264.7 subclone 4 (RAW/C4) and examined the gene expression profile after sRANKL-stimulated osteoclastogenesis. Microarray analysis identified a set of genes superinduced by MITF overexpression, including Clcn7 (chloride channel 7) and Ostm1 (osteopetrosis-associated transmembrane protein 1). Using electrophoretic mobility shift assays, we identified two MITF-binding sites (M-boxes) in the Clcn7 promoter and a single M-box in the Ostm1 promoter. An anti-MITF antibody supershifted DNA-protein complexes for promoter sites in both genes, whereas MITF binding was abolished by mutation of these sites. The Clcn7 promoter was transactivated by coexpression of MITF in reporter gene assays. Mutation of one Clcn7 M-box prevented MITF transactivation, but mutation of the second MITF-binding site only reduced basal activity. Chromatin immunoprecipitation assays confirmed that the two Clcn7 MITF binding and responsive regions in vitro bind MITF in genomic DNA. The expression of Clcn7 is repressed in the dominant negative mutant Mitf mouse, mi/mi, indicating that the dysregulated bone resorption seen in these mice can be attributed in part to transcriptional repression of Clcn7. MITF regulation of the TRAP, cathepsin K, Clcn7, and Ostm1 genes, which are critical for osteoclast resorption, suggests that the role of MITF is more significant than previously perceived and that MITF may be a master regulator of osteoclast function and bone resorption

Increased genomic instability following treatment with direct acting anti-hepatitis C virus drugs

Mixed Cryoglobulinemic Vasculitis (MCV) is a prominent extra-hepatic manifestation of Hepatitis C virus (HCV) infection. HCV has been reported to cause B-cell disorders and genomic instability. Here, we investigated B-cell activation and genome stability in HCV-MCV patients receiving the direct antiviral agent, Sofosbuvir, at multiple centers in Egypt. Clinical manifestations in HCV-MCV patients were improved at the end of treatment (EOT), such as purpura (100%), articular manifestations (75%) and neuropathy (68%). Eighteen patients (56%) showed vasculitis relapse after EOT. BAFF and APRIL were higher at EOT and continued to increase one year following treatment onset. Chromosomal breaks were elevated at EOT compared to baseline levels and were sustained at 3 and 6 months post treatment. We report increased expression of DNA genome stability transcripts such as topoisomerase 1 and TDP1 in HCV-MCV patients after treatment, which continued to increase at 12 months from treatment onset. This data suggest that B-cell activation and DNA damage are important determinants of HCV-MCV treatment outcomes

The MITF family of transcription factors: Role in endolysosomal biogenesis, Wnt signaling, and oncogenesis

Canonical Wnt signaling influences cellular fate and proliferation through inhibition of Glycogen Synthase Kinase (GSK3) and the subsequent stabilization of its many substrates, most notably β-Catenin, a transcriptional co-activator. MITF, a melanoma oncogene member of the microphthalmia family of transcription factors (MiT), was recently found to contain novel GSK3 phosphorylation sites and to be stabilized by Wnt. Other MiT members, TFEB and TFE3, are known to play important roles in cellular clearance pathways by transcriptionally regulating the biogenesis of lysosomes and autophagosomes via activation of CLEAR elements in gene promoters of target genes. Recent studies suggest that MITF can also upregulate many lysosomal genes. MiT family members are dysregulated in cancer and are considered oncogenes, but the underlying oncogenic mechanisms remain unclear. Here we review the role of MiT members, including MITF, in lysosomal biogenesis, and how cancers overexpressing MITF, TFEB or TFE3 could rewire the lysosomal pathway, inhibit cellular senescence, and activate Wnt signaling by increasing sequestration of negative regulators of Wnt signaling in multivesicular bodies (MVBs). Microarray studies suggest that MITF expression inhibits macroautophagy. In melanoma the MITF-driven increase in MVBs generates a positive feedback loop between MITF, Wnt, and MVBs

Locality issues in reliable multicasting

Reliable multicasting based distributed simulations and conferences require safe data packet delivery in a reasonably short time. Such highquality service demands substantial network resources. As multicast applications grow, scalability is an important issue. One way to reach scalability is through clustering. The overall load is distributed among clusters, and large groups avoid overloading the network. To make clustering as natural as possible, local groups should be kept together instead of split into artificial clusters. We propose an algorithm to create a multi-level cluster structure for constructing local groups. We also simulated the cluster-building algorithm, compared it to that proposed in [9], and found that it generated more optimal clusters.Godkänd; 2000; 20061128 (ysko)</p

Bandwidth-constrained clustering in ad hoc networks

We present a survey of the basic mechanisms and properties of existing clustering algorithms for wireless ad hoc networks. Based on this evaluation, we then propose a new algorithm with improved stability and a lower communication overhead. This is partly achieved by using a maintenance function that modifies the existing clustering structure rather than building a new one from scratch. Preliminary simulations seem to indicate that the algorithm produces clusters of about the same size and stability as a comparable existing algorithm, while sending significantly fewer messages.Godkänd; 2004; 20061121 (tomasjo)</p

Loop-free link-state routing

Improving the robustness of today's intra-domain link-state networks is of increasing importance. This is because it is becoming commonplace for such networks to carry telephony, on-demand video and other kinds of real-time traffic. Since user requirements on real-time traffic are exacting, it is essential that the network is able to respond rapidly to network errors. A way of reaching fast reaction times in response to network errors is to precompute fail-over paths that can be utilized immediately when an error is detected. The fail-over path is thereafter in use until the network has converged, handling traffic that would otherwise have been lost due to the error. During convergence (when new paths are calculated) there is a caveat that needs to be avoided -the formation of temporary loops. Temporary loops can form naturally during convergence in link-state networks and can unfortunately prevent traffic from reaching a fail-over path. Traffic can therefore be lost even though a fail-over path is in place. We present an algorithm that can be used in conjunction with link-state routing, to ensure that temporary loops do not form during convergence. The algorithm improves on previous loop-free algorithms for link-state routing by reducing the number of state transitions necessary before a router can update its routing table to a new, guaranteed to exist, path for a wide variety of topological configurations.Validerad; 2007; 20071022 (ysko)</p

On clustering in ad hoc networks

This paper presents the reasons for clustering algorithms in ad hoc networks, as well as a short survey of the basic ideas and priorities of existing clustering algorithms. A new algorithm is presented. It makes it possible to define a limit for the maximum size of the clusters as well as the maximum number of hops between a node and its clusterhead.Godkänd; 2003; 20071121 (ysko)</p