Naturally occurring antisense RNA of histone H2a in mouse cultured cell lines

BACKGROUND: An antisense transcript of histone H2a that has no significant protein-coding region has been cloned from a mouse full-length cDNA library. In the present study, we evaluated this transcript by using RT-PCR and compared the expression patterns of the sense and antisense transcripts by using quantitative RT-PCR (qRT-PCR). RESULTS: This antisense RNA was expressed in three mouse cell lines. We call it ASH2a. ASH2a includes not only the complementary sequence of the transcript of Hist2h2aa2 (a replication-dependent histone H2a gene), but also that of the promoter of Hist2h2aa2. The upstream genomic sequence of the transcription start site of the ASH2a-coding gene (ASH2a) lacks both CCAAT and TATA boxes. This absence suggests that the regulation of ASH2a is different from that of the replication-dependent histone H2a genes. Findings from qRT-PCR indicated that the expression pattern of ASH2a was different from that of Hist2h2aa2. Expression of Hist2h2aa2 peaked at 2 to 4 h during S-phase, but that of ASH2a peaked at 1 h. CONCLUSION: We showed the existence of ASH2a, a histone H2a antisense RNA, in mouse cultured cells. The expression pattern of ASH2a is different from that of the sense RNA

A novel replication-independent histone H2a gene in mouse

BACKGROUND: An uncharacterized histone H2a-coding transcript (E130307C13) has been cloned from a mouse full-length cDNA library. This transcript is encoded on chromosome 6, approximately 4 kb upstream of a histone H4 gene, Hist4h4. The proteins encoded by this transcript and the human H2afj mRNA isoform-2 have the highest amino acid similarity. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR. RESULTS: Quantitative RT-PCR indicated that the gene that encodes E130307C13 (E130307C13) is regulated in a replication-independent manner, and therefore it is H2afj. Certainly, H2afj transcript lacks a stem-loop structure at the 3'-UTR but contains a poly (A) signal. In addition, its promoter region has a different structure from those of the replication-dependent histone H2a genes. CONCLUSION: The bioinformatics imply that E130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse

Comparative analysis of expression of histone H2a genes in mouse

BACKGROUND: At least 18 replication-dependent histone H2a genes are distributed in 3 Hist gene clusters on different chromosomes of the mouse genome. In this analysis we designed specific PCR primers for each histone H2a transcript and studied the expression levels and patterns using quantitative RT-PCR (qRT-PCR). In addition, we compared histone H3 K9 acetylation levels in the promoter regions of H2a genes by ChIP (chromatin immunoprecipitation) – quantitative PCR (qPCR) analysis. RESULTS: RT-PCR analysis indicated that all 20 histone H2a genes assessed in this study are expressed. The replication-dependent histone H2a genes have different expression levels but similar expression patterns. Among the 20 histone H2a genes, the expression-level of H2afz, a replication-independent gene, was highest, and that of Hist1h2aa, a replication-dependent gene, was lowest. Among 18 replication-dependent H2a genes, the expression level of Hist3h2a was highest. The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in promoter regions of both H2afz and Hist3h2a are clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa is similar to that in the γ-satellite region. CONCLUSION: These results strongly suggest that histone H3 K9 acetylation plays a role in the expression of histone genes

Regulatory interdependence of myeloid transcription factors revealed by Matrix RNAi analysis

The knockdown of 78 transcription factors in differentiating human THP-1 cells using matrix RNAi reveals their interdependenc

Multiplex Polymerase Chain Reaction Assay for Early Diagnosis of Viral Infection

Viral reactivation is one of the most serious complications for immunocompromised patients. Under immunosuppressive conditions, some viruses can be reactivated solely or simultaneously and may thus cause life-threatening infection. Therefore, the prompt and proper diagnosis of viral reactivation is important for the initiation of preemptive therapy. For this purpose, we recently developed a multiplex-virus polymerase chain reaction (PCR) assay. The multiplex PCR assay is designed to qualitatively measure the genomic DNA of 12 viruses at once: cytomegalovirus (CMV), human herpesvirus type 6 (HHV-6), HHV-7, HHV-8, Epstein-Barr virus (EBV), varicella-zoster virus (VZV), BK virus (BKV), JC virus (JCV), parvovirus B19 (ParvoB19), herpes simplex virus type 1 (HSV-1), HSV-2, and hepatitis B virus (HBV). When a specific PCR signal is obtained, the viral load is determined by a quantitative real-time PCR. The qualitative multiplex and quantitative real-time PCR procedures take only 3 hours to complete. With this assay system, we can identify viremia at the early stage and thereby prevent it from progressing to overt and symptomatic viral infection in immunocompromised patients, such as those receiving hematopoietic stem cell transplantation

Genome-wide investigation of in vivo EGR-1 binding sites in monocytic differentiation

A Genome-wide analysis of EGR-1 binding sites reveals co-localization with CpG islands and histone H3 lysine 9 binding. SP-1 binding occupancies near EGR-1 binding sites are dramatically altered

Identification of transcriptional regulatory cascades in retinoic acid-induced growth arrest of HepG2 cells

All-trans retinoic acid (ATRA) is a potent inducer of cell differentiation and growth arrest. Here, we investigated ATRA-induced regulatory cascades associated with growth arrest of the human hepatoma cell line HepG2. ATRA induced >2-fold changes in the expression of 402 genes including 55 linked to cell-cycle regulation, cell growth or apoptosis during 48 h treatment. Computational search predicted that 250 transcriptional regulatory factors (TRFs) could recognize the proximal upstream regions of any of the 55 genes. Expression of 61 TRF genes was significantly changed during ATRA incubation, providing many potential regulatory edges. We focused on six TRFs that could regulate many of the 55 genes and found a total of 160 potential edges in which the expression of each of the genes was changed later than the expression change of the corresponding regulator. RNAi knockdown of the selected TRFs caused perturbation of the respective potential targets. The genes showed an opposite regulation pattern by ATRA and specific siRNA treatments were selected as strong candidates for direct TRF targets. Finally, 36 transcriptional regulatory edges were validated by chromatin immunoprecipitation. These analyses enabled us to depict a part of the transcriptional regulatory cascades closely linked to ATRA-induced cell growth arrest

Building promoter aware transcriptional regulatory networks using siRNA perturbation and deepCAGE

Perturbation and time-course data sets, in combination with computational approaches, can be used to infer transcriptional regulatory networks which ultimately govern the developmental pathways and responses of cells. Here, we individually knocked down the four transcription factors PU.1, IRF8, MYB and SP1 in the human monocyte leukemia THP-1 cell line and profiled the genome-wide transcriptional response of individual transcription starting sites using deep sequencing based Cap Analysis of Gene Expression. From the proximal promoter regions of the responding transcription starting sites, we derived de novo binding-site motifs, characterized their biological function and constructed a network. We found a previously described composite motif for PU.1 and IRF8 that explains the overlapping set of transcriptional responses upon knockdown of either factor

Identification of an inter-transcription factor regulatory network in human hepatoma cells by Matrix RNAi

Transcriptional regulation by transcriptional regulatory factors (TRFs) of their target TRF genes is central to the control of gene expression. To study a static multi-tiered inter-TRF regulatory network in the human hepatoma cells, we have applied a Matrix RNAi approach in which siRNA knockdown and quantitative RT-PCR are used in combination on the same set of TRFs to determine their interdependencies. This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships. These include the cross-talks between hepatocyte nuclear factors (HNFs) and the other TRF groups such as CCAAT/enhancer-binding proteins (CEBPs), retinoic acid receptors (RARs), retinoid receptors (RXRs) and RAR-related orphan receptors (RORs), which play key regulatory functions in human hepatocytes and liver. In addition, various multi-component regulatory motifs, which make up the complex inter-TRF regulatory network, were identified. A large part of the regulatory edges identified by the Matrix RNAi approach could be confirmed by chromatin immunoprecipitation. The resultant significant edges enabled us to depict the inter-TRF TRN forming an apparent regulatory hierarchy of (FOXA1, RXRA) → TCF1 → (HNF4A, ONECUT1) → (RORC, CEBPA) as the main streamline