Journal: Nucleic Acids Research

Article Title: TELP, a sensitive and versatile library construction method for next-generation sequencing

doi: 10.1093/nar/gku818

Figure Lengend Snippet: Highly sensitive preparation of a sequencing library from 25 pg of ChIP DNA using TELP. ( A ) Validation of the sequencing libraries by qPCR. Histone H3K4me3 levels at nine genomic regions were determined through qPCR for sequencing libraries prepared using either the standard Illumina protocol with 10 ng ChIP DNA (Illumina 10 ng) or using TELP with the indicated amounts of ChIP DNA. The data were normalized to the promoter of the Cebpa gene (positive control, PC_1). The details of the qPCR primers are given in the Materials and Methods section. These results are the averages of three independent qPCR experiments, and the error bars indicate standard deviations. ( B ) A comparison of the H3K4me3 ChIP-seq signals from 10 ng ChIP DNA using the standard Illumina library construction method with TELP ChIP-seq analyses from 1 ng, 100 pg and 25 pg ChIP DNA. The data from a similar ChIP experiment using rabbit IgG were included as a negative control. ( C ) Scatter plots showing the comparison of the H3K4me3 levels (averaged RPKM signal intensities at the called H3K4me3 peaks, based on Illumina 10 ng) between those generated by the standard Illumina protocol and those generated by TELP using various amount of ChIP DNA. Pearson correlation coefficients, R , are indicated. ( D ) Venn diagrams showing overlaps between H3K4me3-marked regions identified by Illumina ChIP-seq (10 ng DNA) and TELP ChIP-seq (1 ng, 100 pg and 25 pg DNA). As significantly more peaks were called from the 25 pg DNA library due to elevated background noise ( n = 28,314), we selected those strong H3K4me3 peaks ( n = 10,733) by setting an additional selection criteria (Z-score normalized RPKM > 0) for this analysis.

Article Snippet: In parallel, 10 ng of the same ChIP DNA was used as starting material for the standard Illumina protocol for comparison.

Techniques: Sequencing, Biomarker Discovery, Positive Control, Comparison, ChIP-sequencing, Negative Control, Generated, Selection

Journal: The Journal of Biological Chemistry

Article Title: Histone deacetylase–mediated regulation of endolysosomal pH

doi: 10.1074/jbc.RA118.002025

Figure Lengend Snippet: Regulation of NHE6 expression is mediated by a histone deacetylase. A, schematic to draw parallels between regulation of eNHE in yeast (top) and mammalian cells (bottom). CREB transcription factor regulates response to nutrient deprivation and glucose availability in mammalian cells, analogous to the Abf1 transcription factor in yeast. Both class I (HDAC1) and class II (HDAC4) HDACs directly interact with CREB and negatively regulate its function. Based on this predictive model, we hypothesized NHE6 as a potential CREB target. B and C, ChIP-on-chip (ChIP with DNA microarray) data analyzed using the recommended cutoff for ChIP-positive genes (binding ratio ≥1.5 and log10 p value ≥3) revealed NHE6 as a potential CREB target. Note that the ChIP binding data for NHE6 were similar to those of DUSP1, a known CREB target, whereas no CREB occupancy was detected for the related NHE7 isoform. D, qPCR analysis demonstrating prominent ∼5.2-fold increase in NHE6 expression (****, p < 0.0001; n = 3; Student's t test) and modest increases in NHE9 (∼1.4-fold; **, p = 0.0011; n = 3; Student's t test) with TSA treatment of HEK293 cells. E and F, luciferase assay to confirm TSA-induced activation of CREB using a CRE that drives a firefly luciferase reporter gene and is measured luminometrically (E). Renilla luciferase driven by a constitutively active SV40 promoter was used to normalize for variation of cell number and transfection efficiency. Significant activation of the CRE was seen with TSA treatment (10 μm; 12 h; F) in HEK293 cells, relative to vehicle control (**, p = 0.0064; n = 3; Student's t test). G, qPCR analysis to determine the potential of forskolin to augment the expression of NHE6 in HEK293 cells. Note the significant increase in NHE6 transcript levels with forskolin treatment (***, p = 0.0005; n = 3; Student's t test), which was blocked by expression of a constitutively active/nuclear HDAC4 mutant (S246A/S467A/S632A) (p = 0.116; n = 3; Student's t test). NS, not significant. See related Fig. S6. Error bars, S.D.

Article Snippet: Forskolin (3828) was purchased from Cell Signaling Technology.

Techniques: Expressing, Histone Deacetylase Assay, ChIP-chip, Microarray, Binding Assay, Luciferase, Activation Assay, Transfection, Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Histone deacetylase–mediated regulation of endolysosomal pH

doi: 10.1074/jbc.RA118.002025

Figure Lengend Snippet: Control of NHE6 expression by pharmacological targeting of CREB pathway. A, extracellular signals induce adenylyl cyclase, which elevates cAMP levels and activates the cAMP-dependent protein kinase A (PKA), which in turn induces CREB phosphorylation at serine 133. Active phospho-CREB translocates to the nucleus and activates transcription of NHE6, which contains CRE in its promoter. At least three different pharmacological approaches could be used to activate CREB and enhance NHE6 transcription. First, HDAC inhibitors (HDACi) activate CREB by attenuating HDAC-mediated repression. Second, forskolin (FOR), an agonist of adenylyl cyclase, stimulates production of cAMP and activates CREB. Third, rolipram (ROL), a PDE4 inhibitor, inhibits hydrolysis of cAMP and activates CREB. B, qPCR analysis to confirm CREB-mediated NHE6 regulation in apoE3 astrocytes using the CREB inhibitor (KG501). Note the dose-dependent down-regulation of NHE6 transcript levels with KG501 treatment (****, p < 0.0001; n = 3; Student's t test). C, qPCR analysis demonstrating dose-dependent increase in CREB1 transcript levels with rolipram treatment in apoE4 astrocytes. D, qPCR analysis to demonstrate dose-dependent increase in NHE6 transcript levels with rolipram treatment (1.25–10 μm) in apoE4 astrocytes (****, p < 0.0001; n = 3; Student's t test). E, fluorescence-based assay to monitor clearance of Aβ peptides by apoE4 astrocytes. F, quantitation of Aβ clearance from FACS analysis of 10,000 cells in biological triplicates confirmed restoration of Aβ clearance in apoE4 astrocytes with rolipram treatment (****, p < 0.0001; n = 3; Student's t test). G and H, representative micrographs (G) and quantification (H) showing a ∼2.66-fold increase in cell-associated Aβ in apoE4 astrocytes with rolipram treatment (****, p < 0.0001; n = 160/condition; Student's t test). Error bars, S.D. See related Fig. S7.

Article Snippet: Forskolin (3828) was purchased from Cell Signaling Technology.

Techniques: Control, Expressing, Phospho-proteomics, Fluorescence, Quantitation Assay