[Show abstract][Hide abstract] ABSTRACT: Heterochromatin protein 1 (HP1) is an evolutionar-ily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hall-mark of heterochromatin. Although HP1 phospho-rylation has been described in several organisms, the biological implications of this modification re-main largely elusive. Here we show that HP1's phos-phorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation as-says and conventional chromatography, we demon-strated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1␣. Pull-down assays using in vitro-reconstituted nucleosomes showed that un-modified HP1␣ bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affin-ity, whereas CK2-phosphorylated HP1␣ showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1␣'s intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phos-phorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.
Nucleic Acids Research 10/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing HDAC complex, and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two HDAC complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the C. elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes.
[Show abstract][Hide abstract] ABSTRACT: Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin, and plays a crucial role in forming higher-order chromatin structures. HP1 has an N-terminal chromodomain and a C-terminal chromo shadow domain, linked by an unstructured hinge region. Although biochemical and structural studies have revealed each domain's properties, little is known about the mechanisms by which these domains cooperate to carry out HP1's function in forming higher-order chromatin structures. In this review, we summarize HP1's biochemical and structural properties and highlight the latest findings regarding HP1's interactions with nucleosomes.
Journal of Biochemistry 05/2014; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fission yeast Schizosaccharomyces pombe has been widely used as a model eukaryote to study a diverse range of biological processes. However, population genetic studies of this species have been limited to date, and we know very little about the evolutionary processes and selective pressures that are shaping its genome. Here, we sequenced the genomes of 32 worldwide S. pombe strains and examined the pattern of polymorphisms across their genomes. In addition to introns and untranslated regions (UTRs), intergenic regions also exhibited lower levels of nucleotide diversity than synonymous sites, suggesting that a considerable amount of noncoding DNA is under selective constraint and thus likely to be functional. A number of genomic regions showed a reduction of nucleotide diversity probably caused by selective sweeps. We also identified a region close to the end of chromosome 3 where an extremely high level of divergence was observed between 5 of the 32 strains and the remain 27, possibly due to introgression, strong positive selection, or that region being responsible for reproductive isolation. Our study should serve as an important starting point in using a population genomics approach to further elucidate the biology of this important model organism.
PLoS ONE 01/2014; 9(8):e104241. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tail of histone H3 is an ideal medium for storing epigenetic information because displacement of histone H3 is heavily restricted during transcription. To maintain the locus-specific modifications of histone H3, histone molecules should be retained locally at the original position through multiple rounds of transcription. Here, we found that fission yeast Spt6, a highly conserved RNA polymerase II-interacting histone H3-H4 chaperone, is essential for the maintenance of Lys-4 and Lys-9 methylation of histone H3 in euchromatin and heterochromatin, respectively. In euchromatin, loss of Lys-4 methylated histone H3 and deposition of newly synthesized Lys-56 acetylated histone H3 induced by Spt6 inactivation were coupled with transcription. While in heterochromatin, Spt6 prevents histone turnover and cryptic transcription in parallel with Clr3 histone deacetylase. We propose that Spt6 retains posttranslationally modified histone H3 during transcription to maintain epigenome integrity.
[Show abstract][Hide abstract] ABSTRACT: The budding yeast Saccharomyces cerevisiae contains active and inactive chromatin separated by boundary domains. Previously, we used genome-wide screening to identify 55 boundary-related genes. Here, we focus on Sgf73, a boundary protein that is a component of the Spt-Ada-Gcn5 acetyltransferase (SAGA) and SLIK (SAGA-like) complexes. These complexes have histone acetyltransferase (HAT) and histone deubiquitinase activity, and Sgf73 is one of the factors necessary to anchor the deubiquitination module. Domain analysis of Sgf73 was carried out, and the minimum region (373-402 aa) essential for boundary function was identified. This minimum region does not include the domain involved in anchoring the deubiquitination module, suggesting that the histone deubiquitinase activity of Sgf73 is not important for its boundary function. Next, Sgf73-mediated boundary function was analyzed in disruption strains in which different protein subunits of the SAGA/SLIK/ADA complexes were deleted. Deletion of ada2, ada3 or gcn5 (a HAT module component) caused complete loss of the boundary function of Sgf73. The importance of SAGA or SLIK complex binding to the boundary function of Sgf73 was also analyzed. Western blot analysis detected both the full-length and truncated forms of Spt7, suggesting that SAGA and SLIK complex formation is important for the boundary function of Sgf73.
[Show abstract][Hide abstract] ABSTRACT: Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity.
[Show abstract][Hide abstract] ABSTRACT: In fission yeast, siRNA is generated from pericentromeric noncoding RNA by the RNAi machinery. siRNA synthesis and heterochromatin formation are interdependent, forming a self-reinforcing loop on chromatin. In this system, siRNA is amplified by the RNA-dependent RNA polymerase complex (RDRC) and the endoribonuclease Dcr1, which synthesizes dsRNA and processes the dsRNA, respectively. The amplification is essential for stable heterochromatin formation. Here, a novel gene, dsh1(+) (defect of the gene silencing at centromeric heterochromatin), is identified as an essential component of RNAi-directed heterochromatin assembly. Loss of dsh1(+) abolishes normal RNAi function and heterochromatic gene silencing at pericentromeres. Dsh1 interacts with Dcr1 and RDRC and couples the reactions of both proteins to the effective production of siRNA in vivo. Dsh1 binds to heterochromatin in the absence of RDRC, while RDRC requires Dsh1 for its chromatin-binding activity, suggesting that Dsh1 recruits RDRC to chromatin. Immunofluorescence analysis shows that Dsh1 forms foci at the nuclear periphery, and some Dsh1 foci colocalize with Dcr1 and RDRC. Dsh1 is required for the colocalization of Dcr1 and RDRC. Moreover, loss of the nuclear periphery localization of Dsh1 abolishes Dsh1 function. Taken together, these results suggest that Dsh1 assembles the RNAi machinery on heterochromatin and forms a perinuclear compartment for amplification of heterochromatic siRNA.
Genes & development 08/2012; 26(16):1811-24. · 12.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.
[Show abstract][Hide abstract] ABSTRACT: Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.
Molecular and Cellular Biology 04/2012; 32(12):2279-88. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1(+) mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6(+) reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.
Proceedings of the National Academy of Sciences 04/2012; 109(16):6159-64. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-translational modifications of histones are critical not only for local regulation of gene expression, but also for higher-order structure of the chromosome and genome organization in general. These modifications enable a preset state to be maintained over subsequent generations and thus provide an epigenetic level of regulation. Heterochromatic regions of the genome are epigenetically regulated to maintain a "silent state" and protein coding genes inserted into these regions are subject to the same epigenetic silencing. The fission yeast Schizosaccharomyces pombe has well characterized regions of heterochromatin and has proven to be a powerful model for elucidation of epigenetic silencing mechanisms. Research in S. pombe led to the breakthrough discovery that epigenetic silencing is not solely a chromatin-driven transcriptional repression and that RNA interference of nascent transcripts can guide epigenetic silencing and associated histone modifications. Over the last 10 years, an eloquent integration of genetic and biochemical studies have greatly propelled our understanding of major players and effector complexes for regulation of RNAi-mediated epigenetic silencing in S. pombe. Here, we review recent research related to regulation of the epigenetic state in S. pombe heterochromatin, focusing specifically on the mechanisms by which transcription and RNA processing interact with the chromatin modification machinery to maintain the epigenetically silent state.
[Show abstract][Hide abstract] ABSTRACT: Grc3 is an evolutionarily conserved protein. Genome-wide budding yeast studies suggest that Grc3 is involved in rRNA processing. In the fission yeast Schizosaccharomyces pombe, Grc3 was identified as a factor exhibiting distinct nuclear dot localization, yet its exact physiological function remains unknown. Here, we show that S. pombe Grc3 is required for both rRNA processing and heterochromatic gene silencing. Cytological analysis revealed that Grc3 nuclear dots correspond to heterochromatic regions and that some Grc3 is also present in the nucleolar peripheral region. Depleting the heterochromatic proteins Swi6 or Clr4 abolished heterochromatic localization of Grc3 and resulted in its preferential accumulation in the perinucleolar region, suggesting its dynamic association with these nuclear compartments. Cells expressing mutant grc3 showed defects in 25 S rRNA maturation and in heterochromatic gene silencing. Protein analysis of Grc3-containing complexes led to the identification of Las1 and components of the IPI complex (Rix1, Ipi1, and Crb3). All of these Grc3-interacting proteins showed a dynamic nuclear localization similar to that observed for Grc3, and those conditional mutants showed defects in both rRNA processing and silencing of centromeric transcripts. Our data suggest that Grc3 functions cooperatively with Las1 and the IPI complex in both ribosome biogenesis and heterochromatin assembly.
Journal of Biological Chemistry 03/2011; 286(17):15391-402. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.
Genes & Genetic Systems 01/2011; 86(5):305-14. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.
Molecular and Cellular Biology 01/2011; 31(6):1186-200. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic gene silencing is one of the fundamental mechanisms for ensuring proper gene expression patterns during cellular differentiation and development. Histone deacetylases (HDACs) are evolutionally conserved enzymes that remove acetyl modifications from histones and play a central role in epigenetic gene silencing. In cells, HDAC forms a multiprotein complex (HDAC complex) in which the associated proteins are believed to help HDAC carry out its cellular functions. Though each HDAC complex contains distinct components, the presence of isoforms for some of the components expands the variety of complexes and the diversity of their cellular roles. Recent studies have also revealed a functional link between HDAC complexes and specific histone demethylases. In this paper, we summarize the distinct and cooperative roles of four class I HDAC complexes, Sin3, NuRD, CoREST, and NCoR/SMRT, with respect to their component diversity and their relationship with specific histone demethylases.
BioMed Research International 01/2011; 2011:129383. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Delta set13 mutant cells. Delta set13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.
Journal of Biological Chemistry 05/2010; 285(29):22448-60. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PALB2 physically and functionally connects the proteins encoded by the BRCA1 and BRCA2 breast and ovarian cancer genes into a DNA-damage-response network. However, it remains unclear how these proteins associate with chromatin that contains damaged DNA. We show here that PALB2 binds directly to a conserved chromodomain protein, MRG15, which is a component of histone acetyltransferase-deacetylase complexes. This interaction was identified by analysis of purified MRG15- and PALB2-containing protein complexes. Furthermore, MRG15 interacts with the entire BRCA complex, which contains BRCA1, PALB2, BRCA2 and RAD51. Interestingly, MRG15-deficient cells, similarly to cells deficient in PALB2 or BRCA2, showed reduced efficiency for homology-directed DNA repair and hypersensitivity to DNA interstrand crosslinking agents. Additionally, knockdown of MRG15 diminished the recruitment of PALB2, BRCA2 and RAD51 to sites of DNA damage and reduced chromatin loading of PALB2 and BRCA2. These results suggest that MRG15 mediates DNA-damage-response functions of the BRCA complex in chromatin.
[Show abstract][Hide abstract] ABSTRACT: The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 degrees C and preferably utilizes both C(10) and C(15) allylic diphosphates to yield mainly the C(60)-C(65) products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C(65)-C(70)) than the wild-type and the substitution at 109 (K109A) was the most effective. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 degrees C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.
Journal of Biotechnology 08/2009; 143(2):151-6. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies have revealed various functions for the small ubiquitin-related modifier (SUMO) in diverse biological phenomena, such as regulation of cell division, DNA repair and transcription, in yeast and animals. In contrast, only a limited number of proteins have been characterized in plants, although plant SUMO proteins are involved in many physiological processes, such as stress responses, regulation of flowering time and defense reactions to pathogen attack. Here, we reconstituted the Arabidopsis thaliana SUMOylation cascade in Escherichia coli. This system is rapid and effective for the evaluation of the SUMOylation of potential SUMO target proteins. We tested the ability of this system to conjugate the Arabidopsis SUMO isoforms, AtSUMO1, 2, 3 and 5, to a model substrate, AtMYB30, which is an Arabidopsis transcription factor. All four SUMO isoforms tested were able to SUMOylate AtMYB30. Furthermore, SUMOylation sites of AtMYB30 were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by mutational analysis in combination with this system. Using this reconstituted SUMOylation system, comparisons of SUMOylation patterns among SUMO isoforms can be made, and will provide insights into the SUMO isoform specificity of target modification. The identification of SUMOylation sites enables us to investigate the direct effects of SUMOylation using SUMOylation-defective mutants. This system will be a powerful tool for elucidation of the role of SUMOylation and of the biochemical and structural features of SUMOylated proteins in plants.
Plant and Cell Physiology 05/2009; 50(6):1049-61. · 4.98 Impact Factor