1. Volume 29, Issue 2, Pages 271-278 (February 2008)
A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo 
Christian Kroun Damgaard, Søren Kahns, Søren Lykke-Andersen, Anders Lade Nielsen, Torben Heick Jensen, Jørgen Kjems 
Molecular Cell 
Volume 29, Issue 2, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Transcription Inhibition by a 5′ss Point Mutation
(A) HIV-1 env expression cassettes. The 5′ss sequences of WT, 5′ss-m1, 5′ss-m2, and 5′ss-m3 constructs as well as the U1 snRNA base-pairing potential of 5′ss-WT and 5′ss-m3 are shown. Positions of probes/amplicons used in northern blotting (B), NRO (C), ChIP (named by distance from center of amplicon to TATA-box) (D), and RNA FISH (E) are indicated as black bars below the constructs.
(B) Northern blotting analysis of RNA isolated from 5′ss-m1 or WT cells at the indicated time points after induction. Position of unspliced and spliced RNA is indicated.
(C) NRO analysis. DNA oligonucleotide probes complementary to the CMV promoter region (CMV), exon I (EI), exon II (EII), lac Z (negative control), and 18S (positive control) were used. The intensity of EI and EII signals, normalized to 18S signal, from 5′ss-m1 nuclei are given below the image as percentage of the WT signal.
(D) RNAPII ChIP assay using the WT and 5′ss-m1 cell lines as indicated. Positions of amplicons are shown in (A). Amplicon “-300” is upstream of the transcription start site, and amplicon “TATA” is overlapping the TATA box region. GAPDH and IFRG28 serve as transcriptionally active and repressed controls, respectively. IP efficiencies were normalized to GAPDH. Error bars are given as standard deviation (n = 4).
(E) HIV-1 RNA-FISH analysis of fixed 5′ss-WT or 5′ss-m1 cells. White arrows point to transcription dots, and white square shows magnified representative signal. Quantification of dots from three independent experiments is shown to the right of the images (error bars indicate standard deviation [n = 3]). Dot intensities (dark gray bars) were averaged from ≥20 cells by using Openlab software and normalized to mean 5′ss-WT signal (set to 100%). The frequency of detectable dot signal (light gray bars) was plotted as percentage of 5′ss-WT (n = 60).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
Transcription Activity Correlates with U1 snRNA-5′ss Base-Pairing Potential
(A) qRT-PCR quantification of splicing levels of HIV-1 WT and 5′ss-m1 RNAs. Total (“amplicon 270”), unspliced (“amplicon 2200”), and background (“amplicon -300”) RNA levels are shown. Note the nonlinear nature of the y axis.
(B) Northern blot analysis using total RNA isolated from 5′ss-WT (lane 1), 5′ss-m1 (lane 2), 5′ss-m2 (lane 3), 5′ss-m3 (lane 4), GAR (lane 5), 5′ss-WTΔi (lane 6), or 5′ss-m1Δi (lane 7).
(C) Left panel, quantification of RNA levels from northern blot (B) relative to 5′ss-WT. Right panel, splicing levels quantified as the ratio of spliced to total RNA signal and normalized to GAPDH internal control. Error bars indicate standard deviation (n = 3).
(D) Representative RNA-FISH analysis using the indicated cell lines.
(E) Quantification of transcription dot signals (see Figure 1 for details).
(F) RNAPII ChIP assay using the 5′ss-WT and 5′ss-WTΔi cell lines (see Figure 1 for details).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
5′ss Mutant-Triggered Transcriptional Defect in β-Globin mRNA Context
(A) β-globin constructs containing either 3 (β-GWT) or 2 exons (β-GΔi2WT) were used as “WT” templates for mutation. Splice donor 1 of β-GWT and β-GΔi2WT was mutated by insertion of the 5′ss-m1 sequence (4 nt substitution) from the HIV-1 env expression cassettes, yielding constructs β-Gm1 and β-GΔi2m1.
(B) Northern blot analysis of 5 μg total RNA isolated from cell lines; left panel, β-GWT (lane 1), β-Gm1 (lane 2), β-GΔi2WT (lane 3), or β-GΔi2m1 (lane 4). Right panel, long exposure of northern analysis of 20 μg total RNA from β-GΔi2WT (lane 1) or β-GΔi2m1 (lane 2). Levels of hnRNP A1 or GAPDH mRNA were used as internal standards.
(C) β-globin RNA-FISH analysis of induced and fixed β-GWT, β-Gm1, GΔi2WT, and β-GΔi2m1 cell lines (see Figure 1 legend for further details).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4 A Proximal 5′ss Directly Recruits TBP and TFIIH
(A) HIV-1 gene with positions of Q-PCR amplicons indicated. Numbers indicate distance from TATA box.
(B) An antibody against acetylated H3K9 was used in ChIP analysis. GAPDH and IFRG serve as controls. IP efficiencies at the GAPDH gene were used to normalize WT and 5′ss-m1 samples.
(C) Same as (B) using antibody against H3K4me3.
(D) Same as (B) using antibody against heterochromatin mark (H3K9me3).
(E) Same as (B) using antibody against unmodified H3.
(F) High-resolution GTF ChIP using antibodies against TBP, p89 (TFIIH), and p33/TFIIB. Values for IP efficiency (mean percentage of input) were normalized to GAPDH TATA signals between 5′ss-WT and 5′ss-m1 cells. To enable a direct comparison of GTF distributions, individual GTF TATA signals were set to 100%. Relative IP efficiencies obtained for TBP (light blue), TFIIH (red), and TFIIB (tan) from 5′ss-WT (bar graphs) and 5′ss-m1 (line graphs) cells were plotted according to their distance from the TATA box. Error bars indicate standard deviations (n = 3). Inset shows enlarged view of the five amplicons spanning the 5′ss region and further downstream. Black lines below graph illustrate regions from which ChIP signals likely originate based on the distribution of TFIIB.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions