Tamar Kahan’s research while affiliated with Hebrew University of Jerusalem and other places

In order to strengthen the argument demonstrated in figure 2 we present the cumulative distributions of all bins separated according to CMepG count, CUn-MepG and CMepG percentage in figures S1, S2 and S3 respectively. The same results were obtained also in primary fibroblast cells (Figures S4-S7) and in ES-F cells (Figures S8-S11). The data was also insensitive to the source of the methylome data since we got essentially the same results using a different methylome data (Figures S12-S13). The results were also insensitive to the binning method since using a different binning method gave essentially the same results (Figures S15-S16) Repeating the experiment using expression data from cells containing either WT levels or KD KDM2a (Blackledge et al., 2010) reveals that KDM2a is not responsible for the higher expression in promoters with many CUn-MepG (Figures S17-S18). Figure S19 is similar to Figure 4 using 20 bins instead of 10 bins.

Background: Promoter methylation is associated with gene repression; however, little is known about its mechanism. It was proposed that the repression of methylated genes is achieved through the recruitment of methyl binding proteins (MBPs) that participate in closing the chromatin. An alternative mechanism suggests that methylation interferes with the binding of either site specific activators or more general activators that bind to the CpG dinucleotide. However, the relative contribution of these two mechanisms to gene repression is not known. Results: Bioinformatics analyses of genome-wide transcriptome and methylome data support the latter hypothesis by demonstrating a strong association between transcription and the number of unmethylated CpGs at the promoter of genes lacking CpG islands. Conclusions: Our results suggest that methylation represses gene expression mainly by preventing the binding of CpG binding activators.

The complete genome of Mycoplasma hyorhinisstrain MCLD has been sequenced and annotated. This genome differs by the inversion of a 14.4-kb and a 3.7-kb fragment and the deletion of a 9.9-kb fragment from M. hyorhinisstrain HUB-1, isolated from swine respiratory tract. The genome revealed 778 coding sequences (CDSs), with a limited number of vlpgenes encoding variable surface lipoproteins.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and alpha-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1+, like mutations in silencing genes or overexpression of swi6+, upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1+ and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates approximately 12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.

A large DNA virus, designated koi herpes virus (KHV), carp interstitial nephritis gill necrosis virus (CNGV) and Cyprinid herpes virus-3 (CyHV-3), causes massive mortality of carp. Morphologically, the virus resembles herpes viruses, but it contains a genome of ca 295 kbp, larger than that of any Herpesviridae member. Interestingly, three CyHV-3 genes, thymidylate monophosphate kinase (TmpK), ribonucleotide reductase and thymidine kinase, which are involved in deoxynucleotide tri-phosphate synthesis, resemble those of pox viruses. In addition to the TmpK gene, which is nonexistent in the genome of herpes viruses, CyHV-3 contains a B22R-like gene, exclusively expressed by pox viruses. These results raise questions on the phylogenic origin of CyHV-3.

The heterochromatin domain at the mat locus of Schizosaccharomyces pombe is bounded by the IR-L and IR-R barriers. A genetic screen for mutations that promote silencing beyond IR-L revealed a novel gene named epe1, encoding a conserved nuclear protein with a jmjC domain. Disruption of epe1 promotes continuous spreading of heterochromatin-associated histone modifications and Swi6 binding to chromatin across heterochromatic barriers. It also enhances position effect variegation at heterochromatic domains, suppresses mutations in silencing genes, and stabilizes the repressed epigenetic state at the mat locus. However, it does not enhance silencing establishment. Our analysis suggests that the jmjC domain is essential for Epe1 activity and that Epe1 counteracts transcriptional silencing by negatively affecting heterochromatin stability. Consistent with this proposition, the meiotic stability of established heterochromatin beyond IR-L is diminished by Epe1 activity, and overexpression of Epe1 disrupts heterochromatin through acetylation of H3-K9 and H3-K14 and methylation of H3-K4. Furthermore, overexpression of Epe1 elevates the rate of chromosome loss. We propose that Epe1 helps control chromatin organization by down-regulating the stability of epigenetic marks that govern heterochromatization.

RING finger (C3HC4-type zinc finger) is a variant zinc finger motif present in a large family of functionally distinct proteins. We describe the cloning and characterization of a novel human transcript RNF38 encoding a new member of the RING finger protein family. The complete mRNA consists of about 6.8 kb widely expressed in human tissues as a single transcript, most abundantly in testis. The predicted proline-rich protein consists of 432 amino acid residues with a coiled-coil motif and a RING-H2 motif (C3H2C2) at its carboxy-terminus. High degree homology was found between the human protein and hypothetical peptides from several other species including Rattus norvegicus, Mus musculus, and Drosophila melanogaster, indicating a significant conservation throughout evolution. The RNF38 genomic structure was determined and comprises at least 13 exons extending over more than 65 kb in the genome, 78 kb centromeric to the GNE gene on human chromosome 9p12-p13. The involvement of this chromosomal segment in a large number of human diseases and in particular in various types of malignancies urges the assessment of the potential functional role of RNF38 in these disorders.

A novel human transcript, C9orf19, mapped to the genomic region involved in hereditary inclusion body myopathy (IBM2) at chromosome 9p12-p13, has been cloned and characterized. A single cDNA clone consisting of the full-length 1.9 kb transcript has been isolated from a human placenta cDNA library and further analyzed. Genomic characterization of the C9orf19 gene identified five exons extending over 27.2 kb of genomic DNA, located 12 kb centromeric to the tumor suppressor RECK gene. C9orf19 mRNA is expressed in a wide range of adult tissues as a single transcript, most abundantly in lung and peripheral blood leukocytes. The predicted protein contains the SCP-like extracellular protein signature classified to IPR001283, a family of evolutionary related proteins with extracellular domains, which includes the human glioma pathogenesis-related protein (GliPR), the human testis specific glycoprotein (TPX-1), and several other extracellular proteins from rodents (SCP), insects venom allergens (Ag5, Ag3), plants pathogenesis proteins (PR-1) and yeast hypothetical proteins. Homology searches with the deduced 154 amino acid protein sequence of C9orf19 revealed highly similar proteins in mouse, drosophila, nematode and yeast. Mutation analysis of C9orf19 in IBM2 patients excluded it as the disease causing gene and revealed four single nucleotide polymorphisms within and in the vicinity of the gene, which will certainly be useful tools to study its potential role in several human diseases mapped to chromosome 9p12-p13. Parallel to this study, the gene termed GNE, approximately 50 kb centromeric to C9orf19, was shown to be the disease causing gene in IBM2.

The nicotinic acetylcholine receptor family (nAChR) is a large family of acetylcholine-gated cation channels. Here we characterize the Caenorhabditis elegans DEG-3/DES-2 nAChR, a receptor identified due to its involvement in neuronal degeneration. Pharmacological analysis of a DEG-3/DES-2 receptor expressed in Xenopus oocytes shows that this receptor is preferentially activated by choline. This choline sensitivity of the DEG-3/DES-2 channel can explain its role in neuronal degeneration, as shown by the toxic effects of choline on oocytes expressing the mutant DEG-3/DES-2 channel. We also show that in C. elegans the DEG-3/DES-2 receptor is localized to nonsynaptic regions, including the sensory endings of chemosensory neurons. This localization is in agreement with a role for this receptor in chemosensation of choline, as inferred from a defect in chemotaxis for choline seen in deg-3 mutants. Thus, this work also provides evidence for the diversity of nonsynaptic activities associated with nAChRs.