Mutation Analysis of 2009 Pandemic Influenza A(H1N1) Viruses Collected in Japan during the Peak Phase of the Pandemic

BACKGROUND: Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. METHODOLOGY: A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. RESULTS AND CONCLUSIONS: Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo

A screening system to identify transcription factors that induce binding site-directed DNA demethylation

Abstract Background DNA methylation is a fundamental epigenetic modification that is involved in many biological systems such as differentiation and disease. We and others recently showed that some transcription factors (TFs) are involved in the site-specific determination of DNA demethylation in a binding site-directed manner, although the reports of such TFs are limited. Results Here, we develop a screening system to identify TFs that induce binding site-directed DNA methylation changes. The system involves the ectopic expression of target TFs in model cells followed by DNA methylome analysis and overrepresentation analysis of the corresponding TF binding motif at differentially methylated regions. It successfully identified binding site-directed demethylation of SPI1, which is known to promote DNA demethylation in a binding site-directed manner. We extended our screening system to 15 master TFs involved in cellular differentiation and identified eight novel binding site-directed DNA demethylation-inducing TFs (RUNX3, GATA2, CEBPB, MAFB, NR4A2, MYOD1, CEBPA, and TBX5). Gene ontology and tissue enrichment analysis revealed that these TFs demethylate genomic regions associated with corresponding biological roles. We also describe the characteristics of binding site-directed DNA demethylation induced by these TFs, including the targeting of highly methylated CpGs, local DNA demethylation, and the overlap of demethylated regions between TFs of the same family. Conclusions Our results show the usefulness of the developed screening system for the identification of TFs that induce DNA demethylation in a site-directed manner

RUNX1 induces DNA replication independent active DNA demethylation at SPI1 regulatory regions

Abstract Background SPI1 is an essential transcription factor (TF) for the hematopoietic lineage, in which its expression is tightly controlled through a −17-kb upstream regulatory region and a promoter region. Both regulatory regions are demethylated during hematopoietic development, although how the change of DNA methylation status is performed is still unknown. Results We found that the ectopic overexpression of RUNX1 (another key TF in hematopoiesis) in HEK-293T cells induces almost complete DNA demethylation at the −17-kb upstream regulatory region and partial but significant DNA demethylation at the proximal promoter region. This DNA demethylation occurred in mitomycin-C-treated nonproliferating cells at both regulatory regions, suggesting active DNA demethylation. Furthermore, ectopic RUNX1 expression induced significant endogenous SPI1 expression, although its expression level was much lower than that of natively SPI1-expressing monocyte cells. Conclusions These results suggest the novel role of RUNX1 as an inducer of DNA demethylation at the SPI1 regulatory regions, although the mechanism of RUNX1-induced DNA demethylation remains to be explored

Asymmetric Regulation of Peripheral Genes by Two Transcriptional Regulatory Networks

<div><p>Transcriptional regulatory network (TRN) reconstitution and deconstruction occur simultaneously during reprogramming; however, it remains unclear how the starting and targeting TRNs regulate the induction and suppression of peripheral genes. Here we analyzed the regulation using direct cell reprogramming from human dermal fibroblasts to monocytes as the platform. We simultaneously deconstructed fibroblastic TRN and reconstituted monocytic TRN; monocytic and fibroblastic gene expression were analyzed in comparison with that of fibroblastic TRN deconstruction only or monocytic TRN reconstitution only. Global gene expression analysis showed cross-regulation of TRNs. Detailed analysis revealed that knocking down fibroblastic TRN positively affected half of the upregulated monocytic genes, indicating that intrinsic fibroblastic TRN interfered with the expression of induced genes. In contrast, reconstitution of monocytic TRN showed neutral effects on the majority of fibroblastic gene downregulation. This study provides an explicit example that demonstrates how two networks together regulate gene expression during cell reprogramming processes and contributes to the elaborate exploration of TRNs.</p></div

Microarray analysis of monocytic and fibroblastic gene expression in treated cells.

<p>(A) Principal component analysis of monocyte- and fibroblast-specific gene expression. (B) Heatmap diagram of monocyte- and fibroblast-specific gene expression. (C–E) The enrichment plot in the monocytic gene set of KDOE, OE-only, and KD-only. (F–H) Gene set enrichment analysis in the fibroblastic gene set of KDOE, KD-only, and OE-only.</p

Experimental design and confirmation of knock-down as well as induction of target TF genes.

<p>(A) Flow chart of experimental design. Four fibroblastic TFs were suppressed by using siRNA transfection on day 0, and four monocyte TFs were overexpressed on day 1. The monocyte medium was introduced at day 2, followed by sample collection on day 8. (B) Parallel experiments were performed for comparison. (C and D) Validation of four fibroblastic TF knockdown and four monocytic TF induction, respectively. (E) qPCR analysis of monocyte makers revealed increased gene expression levels in KDOE. Independent experiments were repeated a minimum of three times (**: p <0.05, *: p <0.1, #: p >0.1, t-test, black bar: KDOE, gray bar: OE-only, white bar: KD-only).</p

Active interaction of two TRNs in monocytic gene regulation.

<p>(A) Analysis of KDOE against OE-only shows significant enrichment. (B) Venn diagram of genes upregulated in each treatment. (C) Box-plots of fold-change distribution for all genes upregulated (N = 61, x: 1% and 99%, ☐: mean). (D) Pie charts showing the interactions between two TRNs in regulating monocytic gene expression.</p

Poor regulations of monocytic TRN in fibroblastic gene suppression.

<p>(A) The enrichment was insignificant when we analyzed KDOE against KD-only. (B) Venn diagram showing overlapped gene numbers downregulated in each treatment. (C) The box-plot for fold-change distributions of all genes downregulated (N = 140, x: 1% and 99%, ☐: mean). (D) The interactions of two TRNs affecting fibroblastic gene suppression.</p