Large Intragenic Deletion in DSTYK Underlies Autosomal-Recessive Complicated Spastic Paraparesis, SPG23.

SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24-q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3' UTR of DSTYK. Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.

Lipodermatosclerosis: a clinicopathologic study of 17 cases and differential diagnosis from erythema nodosum.

BACKGROUND: The clinical manifestations of lipodermatosclerosis (LDS) may mimic cellulitis and various panniculitides. METHODS: To better characterize the histopathologic changes of LDS, we reviewed the clinicopathologic findings of 26 cases with a pathologic diagnosis consistent with LDS. A final diagnosis of LDS was made in 17 cases based on the clinicopathological correlation. As some cases manifested erythema nodosum (EN)-like lesions, 14 specimens of EN were reviewed to identify features for differential diagnosis. RESULTS: Microscopically, the acute LDS lesions were characterized by patchy hemorrhage, ischemic fat necrosis with lipophages or hyalinization in the fat lobules. As the disease progressed to the subacute and chronic stages, lipomembranous or membranocystic fat necrosis, septal fibrosis and background venous stasis in the dermis became more pronounced. In contrast, EN typically displayed minimal venous stasis and membranocystic fat necrosis. CONCLUSIONS: LDS may manifest as EN-like lesions. Therefore LDS should be included in the differential diagnosis of EN. Clinicopathologic correlation is essential for diagnosis. Differentiating the acute LDS from the early EN is more difficult. A constellation of the findings of septal/lobular panniculitis, hemorrhage in the subcutaneous tissue, and lipophages and/or ischemic fat necrosis in the fat lobules favors the diagnosis of acute LDS.

Sgs1 RecQ helicase inhibits survival of Saccharomyces cerevisiae cells lacking telomerase and homologous recombination.

In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.

Genomic distribution and functional analyses of potential G-quadruplex-forming sequences in Saccharomyces cerevisiae.

Although well studied in vitro, the in vivo functions of G-quadruplexes (G4-DNA and G4-RNA) are only beginning to be defined. Recent studies have demonstrated enrichment for sequences with intramolecular G-quadruplex forming potential (QFP) in transcriptional promoters of humans, chickens and bacteria. Here we survey the yeast genome for QFP sequences and similarly find strong enrichment for these sequences in upstream promoter regions, as well as weaker but significant enrichment in open reading frames (ORFs). Further, four findings are consistent with roles for QFP sequences in transcriptional regulation. First, QFP is correlated with upstream promoter regions with low histone occupancy. Second, treatment of cells with N-methyl mesoporphyrin IX (NMM), which binds G-quadruplexes selectively in vitro, causes significant upregulation of loci with QFP-possessing promoters or ORFs. NMM also causes downregulation of loci connected with the function of the ribosomal DNA (rDNA), which itself has high QFP. Third, ORFs with QFP are selectively downregulated in sgs1 mutants that lack the G4-DNA-unwinding helicase Sgs1p. Fourth, a screen for yeast mutants that enhance or suppress growth inhibition by NMM revealed enrichment for chromatin and transcriptional regulators, as well as telomere maintenance factors. These findings raise the possibility that QFP sequences form bona fide G-quadruplexes in vivo and thus regulate transcription.

Evidence that a RecQ helicase slows senescence by resolving recombining telomeres.

RecQ helicases, including Saccharomyces cerevisiae Sgs1p and the human Werner syndrome protein, are important for telomere maintenance in cells lacking telomerase activity. How maintenance is accomplished is only partly understood, although there is evidence that RecQ helicases function in telomere replication and recombination. Here we use two-dimensional gel electrophoresis (2DGE) and telomere sequence analysis to explore why cells lacking telomerase and Sgs1p (tlc1 sgs1 mutants) senesce more rapidly than tlc1 mutants with functional Sgs1p. We find that apparent X-shaped structures accumulate at telomeres in senescing tlc1 sgs1 mutants in a RAD52- and RAD53-dependent fashion. The X-structures are neither Holliday junctions nor convergent replication forks, but instead may be recombination intermediates related to hemicatenanes. Direct sequencing of examples of telomere I-L in senescing cells reveals a reduced recombination frequency in tlc1 sgs1 compared with tlc1 mutants, indicating that Sgs1p is needed for tlc1 mutants to complete telomere recombination. The reduction in recombinants is most prominent at longer telomeres, consistent with a requirement for Sgs1p to generate viable progeny following telomere recombination. We therefore suggest that Sgs1p may be required for efficient resolution of telomere recombination intermediates, and that resolution failure contributes to the premature senescence of tlc1 sgs1 mutants.

Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence.

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.

Histone acetylation and chromatin conformation are regulated separately at the TNF-alpha promoter in monocytes and macrophages.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which participates in a wide range of immunoregulatory activities. It is generally produced at highest levels by cells of the myeloid lineage in response to activation of pathogen recognition receptors such as Toll-like receptors. Impaired production predisposes to infection with intracellular organisms, and overproduction results in systemic or organ-specific inflammation. Control of expression is essential to maintain homeostasis, and this control is mediated via multiple strategies. We examined two separate aspects of chromatin accessibility in this study of the human TNF-alpha promoter. We examined the role of histone acetylation and chromatin remodeling in cell lines and primary cells and identified two individual steps associated with activation of TNF-alpha production. Histone H3 and H4 acetylation was found to be strongly dependent on the developmental stage of human monocytes. It did not appear to be regulated by acute stimuli, and instead, chromatin remodeling was found to occur after acute stimuli in a cell line competent to produce TNF-alpha. These data suggest that there is a hierarchy of controls regulating expression of TNF-alpha. Acetylation of histones is a prerequisite but is insufficient on its own for TNF-alpha production.

A role for PKC-delta and PI 3-kinase in TNF-alpha-mediated antiapoptotic signaling in the human neutrophil.

The proinflammatory cytokine tumor necrosis factor (TNF)-alpha has been implicated in the attenuation of neutrophil spontaneous apoptosis during sepsis. Antiapoptotic signaling is principally mediated through the p60TNF receptor (p60TNFR). In neutrophils, TNF-alpha is an incomplete secretagogue and requires input from a ligated integrin(s) for neutrophil activation. In adherent neutrophils, TNF-alpha triggers association of both protein kinase C (PKC)-delta and phosphatidylinositol (PI) 3-kinase with the p60TNFR. In this study, a role for PKC-delta and PI 3-kinase in TNF-alpha-mediated antiapoptotic signaling was examined. TNF-alpha inhibited spontaneous apoptosis in fibronectin-adherent neutrophils, and this antiapoptotic signaling was blocked by the PKC-delta inhibitor rottlerin, but not by an inhibitor of Ca(2+)-dependent PKC isotypes, Go-6976. Inhibition of PI 3-kinase by LY-294002 also inhibited TNF-alpha-mediated antiapoptotic signaling. Cycloheximide blocked TNF-alpha-mediated antiapoptotic signaling, suggesting protein synthesis is required. Inhibition of either PKC-delta or PI 3-kinase attenuated TNF-alpha-mediated activation of the antiapoptotic transcription factor NFkappaB. Thus both PKC-delta and PI 3-kinase have essential roles in TNF-alpha-mediated antiapoptotic signaling in adherent neutrophils.