Se Kye Kim

Transcriptomic Profiling and H3K27me3 Distribution Reveal Both Demethylase-Dependent and Independent Regulation of Developmental Gene Transcription in Cell Differentiation

The removal of histone H3 trimethylation at lysine residue 27 (H3K27me3) plays a critical role in the transcriptional initiation of developmental genes. The H3K27me3-specific KDM6 demethylases JMJD3 and UTX are responsible for the transcriptional initiation of various developmental genes, but some genes are expressed in a KDM6 demethylase-independent manner. To address the role of H3K27me3 in the retinoic acid (RA)-induced differentiation of the human carcinoma NCCIT cell line, we inhibited JMJD3 and UTX using the H3K27me3 demethylase inhibitor GSK-J4. The commitment of JMJD3/UTX-inhibited cells to a specific fate was delayed, and transcriptome profiling also revealed the differential expression of genes related to cell fate specification in demethylase-inactivated cells; the expression levels of RA metabolism and HOX family genes significantly decreased. We observed a weak correlation between H3K27me3 enrichment and transcriptional repression in the control and JMJD/UTX-inhibited cells, except for a few sets of developmental genes that are indispensable for cell fate specification. Taken together, these results provide the H3K27me3 landscape of a differentiating cell line and suggest that both demethylase-dependent and demethylase-independent transcriptional regulation play a role in early differentiation and developmental gene expression activated by H3K27me3 demethylation.

Genetic populations of Bacillus anthracis isolates from Korea

Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics. In the present study, we employed molecular approaches including multiple-locus variable-number tandem repeat analysis (MLVA) and canonical single-nucleotide polymorphism (canSNP) analysis to perform molecular typing of B. anthracis strains isolated in Korea. According to the MLVA, 17 B. anthracis isolates were classified into A3a, A3b, and B1 clusters. The canSNP analyses subdivided the B. anthracis isolates into two of the three previously recognized major lineages (A and B). B. anthracis isolates from Korea were found to belong to four canSNP sub-groups (B.Br.001/2, A.Br.005/006, A.Br.001/002, and A.Br.Ames). The A.Br.001/002 and A.Br.Ames sub-lineages are closely related genotypes frequently found in central Asia and most isolates were. On the other hand, B. anthracis CH isolates were analyzed that belonged to the B.Br.001/002 sub-group which found in southern Africa, Europe and California (USA). B.Br.001/002 genotype is new lineage of B. anthracis in Korea that was not found before. This discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in Korea.

Effects of the activated mitogen-activated protein kinase pathway via the c-ros receptor tyrosine kinase on the T47D breast cancer cell line following alcohol exposure

Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase 1 (MSK1) through the mitogen-activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone 3 serine 10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via ERK1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation.

Effects of pargyline on cellular proliferation in human breast cancer cells

Monoamine oxidase (MAO) inhibitors have been tested for the purpose of treating many types of cancers including breast cancer. Pargyline, an MAO inhibitor, prevents the activity of lysine-specific demethylase 1 (LSD1), which is excessively expressed in breast cancer. The purpose of this study is to investigate the effect of pargyline on cellular proliferation in human breast ductal carcinoma (T47D) cells. After exposing T47D cells to pargyline, we examined cell proliferation, cell cycle, apoptosis, and the expression levels of apoptosis-related proteins and LSD1. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner. The treatment of 2 mM pargyline exhibited an increase of the cell proportion at the G1 phase, while the major decrease of the cell proportion was at the S phase compared to the controls. In addition, pargyline induced an increase in the cleaved PARP expression. However, we did not find a change in the LSD1 expression in the cells exposed to pargyline. Based on our results, it is believed that pargyline has pharmaceutical potential as an anticancer drug for the treatment of human breast cancer.

Late-Exponential Gene Expression in codY-Deficient Bacillus anthracis in a Host-Like Environment

CodY is a pleiotropic regulator commonly found in Gram-positive bacteria and regulates various biological processes during the stringent response in a nutrient-limiting environment. CodY also participates in virulence factor expression in many low G+C Gram-positive pathogens, as observed in Bacillus anthracis. However, the mechanism by which B. anthracis CodY regulates metabolism and virulence factors in response to environmental changes is unclear. Here, we attempted to identify the link between CodY and B. anthracis regulation with codY-deficient and codY-overexpressing mutants using high-throughput transcriptional analysis. Growth pattern analyses of codY mutants in both rich and minimal media showed defects in early cell proliferation, with opposite patterns in the early stationary phase: CodY overexpression prolonged bacterial growth, whereas deletion inhibited growth. RNA sequencing of codY-deficient B. anthracis showed both positive and negative changes in the gene expression of proteases and virulence factors as well as genes related to stringent response-related metabolism and biosynthetic processing. We also found that changes in codY expression could alter virulence gene expression of B. anthracis, suggesting modes of regulation in its virulence in a CodY concentration-dependent manner. Collectively, we conclude from these results that CodY can both positively and negatively regulate its regulon via direct and/or indirect approaches, and that its mode of regulation may be concentration dependent.

Changes in Bacillus anthracis CodY regulation under host-specific environmental factor deprived conditions.

BackgroundHost-specific environmental factors induce changes in Bacillus anthracis gene transcription during infection. A global transcription regulator, CodY, plays a pivotal role in regulating central metabolism, biosynthesis, and virulence in B. anthracis. In this study, we utilized RNA-sequencing to assess changes in the transcriptional patterns of CodY-regulated B. anthracis genes in response to three conditions of environmental starvation: iron, CO2, or glucose deprivation. In addition, we performed chromatin immunoprecipitation on newly identified CodY-mediated genes.ResultsEnvironmental deprivation induced transcriptional changes in CodY-regulated genes in both wild-type and codY null strains, and both CodY-specific and environment-specific patterns were observed. In the iron-depleted condition, overexpression of iron homeostasis genes was observed independent of codY deletion; however, transcription of siderophore and amino acid biosynthesis genes was CodY dependent. Although CodY has a significant regulatory role in central metabolism and the carbon overflow pathway, metabolism-associated genes exhibited CodY-independent expression patterns under glucose starvation. Genes that were differentially expressed in response to CO2 availability showed CodY-dependent regulation, though their maximal expression did require a supply of CO2/bicarbonate.ConclusionsWe speculate that CodY regulates the expression of environmental-responsive genes in a hierarchical manner and is likely associated with other transcription regulators that are specific for a particular environmental change.

Complete Genome Sequence of Bacillus anthracis HYU01, Isolated from Soil Samples in the Korean Peninsula

ABSTRACT Bacillus anthracis is a Gram-positive endospore-forming bacterium that causes the zoonotic disease anthrax. We report a complete genome sequence of B. anthracis strain HYU01, isolated from Changnyung, which belongs to the B branch (B.Br.) 001/002 canonical single nucleotide polymorphism (canSNP) group.

Identification of stringent response-related and potential serological proteins released from Bacillus anthracis overexpressing the RelA/SpoT homolog, Rsh Bant.

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of RshBant, a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-RshBant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of RshBant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of RshBant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-RshBant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.

Genetic diversity of Korean Bacillus anthracis isolates from soil evaluated with a single nucleotide repeat analysis.

Bacillus (B.) anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, classification of isolates of this bacterium requires methods with high discriminatory power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat assay that evaluates regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and the Korean Center for Disease Control and Prevention. The SNR analysis was able to detect 13 subgenotypes, which allowed a detailed evaluation of the Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate between strains with the same multiple-locus variable-number tandem repeat analysis genotypes. In summary, we obtained SNR results for four SNR marker loci of newly acquired strains from Korea. Our findings will be helpful for creating marker systems and help identify markers that could be used for future forensic studies.

Deletion of a putative NlpC/P60 endopeptidase BAS1812 affects germination, long-term survival and endospore formation in Bacillus anthracis

Bacillus anthracis, an aetiologic agent of the zoonotic disease anthrax, encodes a putative NlpC/P60 endopeptidase BAS1812. It harbours a signal peptide, three bacterial SH3 domains and an NlpC/P60 family domain. Previous studies showed that BAS1812 is immunogenic in infected hosts and is a potential biomarker for anthrax treatment. To date, however, little information is known about its function and involvement in anthrax pathogenesis. Here we describe the phenotypic effect of BAS1812 deletion in B. anthracis Sterne strain. Transcriptional analysis showed that BAS1812 expression in a host-like environment was enhanced at the end of log phase, started to diminish after entry to stationary phase and increased again late in stationary phase. The constructed BAS1812 mutant showed impaired long-term survival in the stationary growth phase, less resilience to detergent, lesser endospore formation and delayed germination. The mutant also showed diminished ability to degrade peptidoglycan, but its ability to produce anthrax exotoxins was not affected. We hypothesize that BAS1812 is a cell wall hydrolase involved in biological activities related to maintaining cell wall integrity, sporulation and spore germination.