The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.     

Gene silencing of EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphate storing mucopolysaccharidoses

Neurological pathology is characteristic of the mucopolysaccharidoses (MPSs) that store heparan sulphate (HS) glycosaminoglycan (gag) and has been proven to be refractory to systemic therapies. Substrate deprivation therapy (SDT) using general inhibitors of gag synthesis improves neurological function in mouse models of MPS, but is not specific to an MPS type. We have investigated RNA interference (RNAi) as a method of targeting SDT to the HS synthesising enzymes, EXTL2 and EXTL3. Multiple shRNA molecules specific to EXTL2 or EXTL3 were designed and validated in a reporter gene assay, with four out of six shRNA constructs reducing expression by over 90%. The three EXTL2-specific shRNA constructs reduced endogenous target gene expression by 68, 32 and 65%, and decreased gag synthesis by 46, 50 and 27%. One EXTL3-specific shRNA construct reduced endogenous target gene expression by 14% and gag synthesis by 39%. Lysosomal gag levels in MPS IIIA and MPS I fibroblasts were also reduced by EXTL2 and EXTL3-specific shRNA. Incorporation of shRNAs into a lentiviral expression system reduced gene expression, and one EXTL2-specific shRNA reduced gag synthesis. These results indicate that deprivation therapy through shRNA-mediated RNAi has potential as a therapy for HS-storing MPSs.     

A charge-based interaction between histone H4 and Dot1 is required for H3K79 methylation and telomere silencing: identification of a new trans-histone pathway

Saccharomyces cerevisiae cells lacking Dot1 exhibit a complete loss of H3K79 methylation and defects in heterochromatin-mediated silencing. To further understand the mechanism of Dot1-mediated methylation, the substrate requirement of Dot1 was determined. This analysis found that Dot1 requires histone H4 for in vitro methyltransferase activity and the histone H4 tail for Dot1-mediated methylation in yeast. Mutational analyses demonstrated that the basic patch residues (R(17)H(18)R(19)) of the histone H4 N-terminal tail are required for Dot1 methyltransferase activity in vitro as well as Dot1-mediated histone H3K79 methylation in vivo. In vitro binding assays show that Dot1 can interact with the H4 N-terminal tail via the basic patch residues. Furthermore, an acidic patch at the C terminus of Dot1 is required for histone H4 tail binding in vitro, histone H3K79 di- and trimethylation in vivo, and proper telomere silencing. Our data suggest a novel trans-histone regulatory pathway whereby charged residues of one histone are required for the modification of another histone. These findings not only provide key insights into the mechanism of Dot1 histone methylation but also illustrate how chromatin-modifying enzymes engage their nucleosomal substrates in vivo.     

Genetic Association and Gene-gene interaction of HAS2, HABP1 and HYAL3 Implicate Hyaluronan Metabolic Genes in Glaucomatous Neurodegeneration

Hyaluronan (HA) plays a significant role in maintaining aqueous humor outflow in trabecular meshwork, the primary ocular tissue involved in glaucoma. We examined potential association of the single nucleotide polymorphisms (SNPs) of the HA synthesizing gene – hyaluronan synthase 2 (HAS2), hyaluronan binding protein 1 (HABP1) and HA catabolic gene hyaluronidase 3 (HYAL3) in the primary open angle glaucoma (POAG) patients in the Indian population. Thirteen tagged SNPs (6 for HAS2, 3 for HABP1 and 4 for HYAL3) were genotyped in 116 high tension (HTG), 321 non-high tension glaucoma (NHTG) samples and 96 unrelated, age-matched, glaucoma-negative, control samples. Allelic and genotypic association were analyzed by PLINK v1.04; haplotypes were identified using PHASE v2.1 and gene-gene interaction was analyzed using multifactor dimensionality reduction (MDR) v2.0. An allelic association (rs6651224; p = 0.03; OR: 0.49; 95% CI: 0.25–0.94) was observed at the second intron (C>G) of HAS2 both for NHTG and HTG. rs1057308 revealed a genotypic association (p = 0.03) at the 5’ UTR of HAS2 with only HTG. TCT haplotype (rs1805429 – rs2472614 – rs8072363) in HABP1 and TTAG and TTGA (rs2285044 – rs3774753 – rs1310073 – rs1076872) in HYAL3 were found to be significantly high (p < 0.05) both for HTG and NHTG compared to controls. Gene-gene interaction revealed HABP1 predominantly interacts with HAS2 in HTG while it associates with both HYAL3 and HAS2 in NHTG. This is the first genetic evidence, albeit from a smaller study, that the natural polymorphisms in the genes involved in hyaluronan metabolism are potentially involved in glaucomatous neurodegeneration.     

Differential expression of multiple genes in association with MADH4/DPC4/SMAD4 inactivation in pancreatic cancer

The Gene Logic Inc. Gene Express(R) tools and Affymetrix GeneChip(R) arrays were utilized to discover genes differentially expressed in pancreatic cancers with MADH4/DPC4/SMAD4 gene inactivation. cDNA was prepared from thirteen pancreas cancer cell lines with known MADH4 status (5 with wild-type MADH4 and 8 with inactivated MADH4) and hybridized to the complete Affymetrix Human Genome U133 GeneChip(R) set (arrays U133 A,B) for simultaneous analysis of 45,000 gene fragments corresponding to 33,000 known genes. 25 known genes were identified as down-regulated at least three fold in the MADH4 mutant cancer cell lines. 9 were decreased in expression at least 5 fold, and 1 in particular (ID3) was decreased 23 fold. Only 2 of the 25 down-regulated genes (ID1 and ID3) have been previously reported as MADH4-dependent targets, and the remaining 23 genes represent potential novel direct or indirect MADH4 downstream targets. Immunolabeling for Id1 and Id3 did not show a relationship with known MADH4 status in pancreatic cancer tissues, suggesting additional regulation of these two genes than activation by MadH4. Further investigations to validate and to determine the significance of these candidate target genes in pancreatic carcinogenesis and progression are warranted.     

Utility of immunohistochemistry with an antibody against MYC at the initial diagnosis of follicular lymphoma,grade 3A,for predicting a more aggressive clinical course: a case report and review of the literature

Follicular lymphoma (FL) is the most common indolent lymphoma, and associated with the chromosomal translocation t(14;18)(q32;q21). While, FL harboring both BCL2 and MYC translocation at diagnosis is very rare. The evaluation of MYC expression in typical FL at presentation using southern blot, G-banded karyotyping or fluorescence in situ hybridization (FISH) analyses has been described so far. However, there are no reports about the use of immunohistochemistry (IHC) to evaluate MYC protein expression in FL at presentation. Here, we present a FL patient who transformed to a B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt’s lymphoma, accompanied by concurrent BCL2, BCL6, and MYC translocations; i.e., triple-hit lymphoma. Paraffin-embedded tissue section-FISH analysis demonstrated that the FL was negative for MYC, but MYC protein expression was subsequently detected in the lymph node specimen obtained at the initial diagnosis using IHC. This case revealed aggressive clinical course and central nervous system involvement. In the literature concerning MYC positive FL five out of 8 patients were dead within 24 months. The detection of MYC protein expression in FL using IHC might be useful to predict more aggressive clinical course.     

Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing.     

A novel locus for episodic ataxia:UBR4 the likely candidate

Episodic ataxias (EAs) are rare neurological channelopathies that are characterized by spells of imbalance and a lack of co-ordination. There are seven clinically recognized EAs and multiple isolated cases. Five disease-causing genes have been identified to date. We describe a novel form of autosomal dominant EA in a large three-generation Irish family. This form of EA presents in early childhood with periods of unsteadiness generalized weakness and slurred speech during an attack, which may be triggered by physical tiredness or stress. Linkage analysis undertaken in 13 related individuals identified a single disease locus (1p36.13-p34.3) with a LOD score of 3.29. Exome sequencing was performed. Following data analysis, which included presence/absence within the linkage peak, two candidate variants were identified. These are located in the HSPG2 and UBR4 genes. UBR4 is an ubiquitin ligase protein that is known to interact with calmodulin, a Ca²⁺ protein, in the cytoplasm. It also co-localizes with ITPR1 a calcium release channel that is a major determinant of mammal co-ordination. Although UBR4 is not an ion channel gene, the potential for disrupted Ca²⁺ control within neuronal cells highlights its potential for a role in this form of EA.     

Pax3:Fkhr interferes with embryonic Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin

To investigate the role of the translocation-associated gene Pax3:Fkhr in alveolar rhabdomyosarcomas, we generated a Cre-mediated conditional knock-in of Pax3:Fkhr into the mouse Pax3 locus. Exploring embryonic tumor cell origins, we replaced a Pax3 allele with Pax3:Fkhr throughout its expression domain, causing dominant-negative effects on Pax3 and paradoxical activation of the Pax3 target gene, c-Met. Ectopic neuroprogenitor cell proliferation also occurs. In contrast, activation later in embryogenesis in cells that express Pax7 results in viable animals with a postnatal growth defect and a moderately decreased Pax7+ muscle satellite cell pool, phenocopying Pax7 deficiency but remarkably not leading to tumors.     

Roles of genetic variants in the PI3K/PTEN pathways in susceptibility to colorectal carcinoma and clinical outcomes treated with FOLFOX regimen

The genetic or abnormal activation of PI3K/PTEN signaling pathway play an important role with regard to disease progression in variety of human malignancies. Experimental and epidemiologic studies indicated that the genetic polymorphisms in the PTEN, PI3K genes are associated with cancer risk, yet little evidence exists for those 2 genes and colorectal cancer (CRC) risk. To address this, we evaluated whether PTEN rs701848, PIK3CA rs2699887 variants are associated with CRC susceptibility, clinicopathological parameters and clinical outcomes in CRC patients treated with FOLFOX (Oxaliplatin, Leucovorin, 5-Fluorouracil) regimen. A case-control study was performed in 780 CRC patients and 764 healthy controls using the TaqMan assay method. A significant increased risk of CRC was observed in patients carrying PTEN rs701848 TC or CC genotype (adjusted OR=1.306, 95% CI=1.030-1.655, P=0.027; adjusted OR=1.543, 95% CI=1.148-2.075, P=0.004, respectively), TC/CC genotype (adjusted OR=1.367, 95% CI=1.090-1.714, P=0.043) in the dominant model, and C allele (adjusted OR=1.229, 95% CI=1.067-1.416, P=0.004). However, no association was detected between rs2699887 in the PIK3CA gene and CRC risk. A significant association was found between pathological grade (Dukes A and B vs. Dukes C and D) and PIK3CA rs2699887 genotypes. Furthermore, Kaplan-Meier analysis revealed that PTEN rs701848 genotypes were significantly associated with the overall survival (OS) of CRC patients treated with FOLFOX regimen (n=780). Individuals carrying PTEN rs701848 TC or TC/CC genotypes showed significantly longer median survival time (MST) than TT genotype and significant hazard ratio (TC: adjusted HR=0.523, 95% CI=0.325-0.840, P=0.007; TC/CC: adjusted HR=0.545, 95% CI=0.351-0.845, P=0.007). Therefore, rs701848 polymorphism in the PTEN gene is associated with susceptibility to CRC, and C allele of rs701848 showed significant independent better prognosis of CRC patients treated with FOLFOX regimen. These results indicate that rs701848 in the PTEN gene might be a candidate pharmacogenomic factor to assess the susceptibility and prognosis in CRC patients.     

Genetic alterations in quadruple malignancies of a patient with multiple sclerosis: their role in malignancy development and response to therapy

Multiple cancers represent 2.42% of all human cancers and are mainly double or triple cancers. Many possible causes of multiple malignancies have been reported such as genetic alterations, exposure to anti-cancer chemotherapy, radiotherapy, immunosuppressive therapy and reduced immunologic response. We report a female patient with multiple sclerosis and quadruple cancers of different embryological origin. Patient was diagnosed with stage III (T3, N1a, MO) medullary thyroid carcinoma (MTC), multicentric micropapillary thyroid carcinoma, scapular and lumbar melanomas (Clark II, Breslow II), and lobular invasive breast carcinoma (T1a, NO, MO). All tumors present in our patient except micropapillary thyroid carcinomas were investigated for gene alterations known to have a key role in cancer promotion and progression. Tumor samples were screened for the p16 alterations (loss of heterozygosity and homozygous deletions), loss of heterozygosity of PTEN, p53 alterations (mutational status and loss of heterozygosity) and mutational status of RET, HRAS and KRAS. Each type of tumor investigated had specific pattern of analyzed genetic alterations. The most prominent genetic changes were mutual alterations in PTEN and p53 tumor suppressors present in breast cancer and two melanomas. These co-alterations could be crucial for promoting development of multiple malignancies. Moreover the insertion in 4^th codon of HRAS gene was common for all tumor types investigated. It represents frameshift mutation introducing stop codon at position 5 which prevents synthesis of a full-length protein. Since the inactivated RAS enhances sensitivity to tamoxifen and radiotherapy this genetic alteration could be considered as a good prognostic factor for this patient.     

Carboxylic acid derivatives of histone deacetylase inhibitors induce full length SMN2 transcripts: a promising target for spinal muscular atrophy therapeutics

Introduction

Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder. It is caused by homozygous absence of the survival motor neuron 1 (SMN1) gene. SMN2, which modulates the severity of the disease, represents a major target for therapy. The aim of this study was to investigate whether SMN2 expression can be increased by caffeic acid, chlorogenic acid and curcumin, which are designed by modifications of the carboxylic acid class of histone deacetylase (HDAC) inhibitors.

Material and methods

Using quantitative real-time PCR, we analysed the levels of full-length SMN2 and Δ7SMN2 mRNA. We performed LDH cytotoxicity assay to analyse whether SMN2 activating concentrations of caffeic acid, chlorogenic acid and curcumin were cytotoxic to fibroblasts.

Results

We found that caffeic acid and curcumin were more efficient than chlorogenic acid and increased full-length SMN2 mRNA levels 1.5 and 1.7-fold, respectively. Δ7SMN2 mRNA levels were measured to investigate alternative splicing of exon 7. We also found that cytotoxicity was not observed at SMN2 activating concentrations.

Conclusions

Our data suggest that carboxylic acid derivatives including phenolic structure and symmetry could be a good candidate for SMA treatment.     

MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline

RNA silencing can be initiated by endogenous or exogenously delivered siRNAs. In Caenorhabditis elegans, RNA silencing guided by primary siRNAs is inefficient and therefore requires an siRNA amplification step involving RNA-dependent RNA polymerases (RdRPs). Many factors involved in RNA silencing localize to protein- and RNA-rich nuclear pore-associated P granules in the germline, where they are thought to surveil mRNAs as they exit the nucleus. Mutator class genes are required for siRNA-mediated RNA silencing in both germline and somatic cells, but their specific roles and relationship to other siRNA factors are unclear. Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. The glutamine/asparagine (Q/N)-rich protein MUT-16 is specifically required for the formation of a protein complex containing the mutator proteins, and in its absence, Mutator foci fail to form at the nuclear periphery. The RdRP RRF-1 colocalizes with MUT-16 at Mutator foci, suggesting a role for Mutator foci in siRNA amplification. Furthermore, we demonstrate that genes that yield high levels of siRNAs, indicative of multiple rounds of siRNA amplification, are disproportionally affected in mut-16 mutants compared with genes that yield low levels of siRNAs. We propose that the mutator proteins and RRF-1 constitute an RNA processing compartment required for siRNA amplification and RNA silencing.     

Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association

The amino-terminal histone tails are subject to covalent post-translational modifications such as acetylation, methylation, and phosphorylation. In the histone code hypothesis, these exposed and unstructured histone tails are accessible to a repertoire of regulatory factors that specifically recognize the various modified histones, thereby generating altered chromatin structures that mediate specific biological responses. Here, we report that lysine (Lys) 79 of histone H3, which resides in the globular domain, is methylated in eukaryotic organisms. In the yeast Saccharomyces cerevisiae, Lys 79 of histone H3 is methylated by Dot1, a protein shown previously to play a role in telomeric silencing. Mutations of Lys 79 of histone H3 and mutations that abolish the catalytic activity of Dot1 impair telomeric silencing, suggesting that Dot1 mediates telomeric silencing largely through methylation of Lys 79. This defect in telomeric silencing might reflect an interaction between Sir proteins and Lys 79, because dot1 and Lys 79 mutations weaken the interaction of Sir2 and Sir3 with the telomeric region in vivo. Our results indicate that histone modifications in the core globular domain have important biological functions.