1. Volume 2, Issue 4, Pages 722-731 (October 2012)
DSIF Restricts NF-κB Signaling by Coordinating Elongation with mRNA Processing of Negative Feedback Genes 
Gil Diamant, Liat Amir-Zilberstein, Yuki Yamaguchi, Hiroshi Handa, Rivka Dikstein 
Cell Reports 
Volume 2, Issue 4, Pages (October 2012)
DOI: /j.celrep
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2. Cell Reports 2012 2, 722-731DOI: (10.1016/j.celrep.2012.08.041)
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3. Figure 1
Downregulation of DSIF Differentially Affects mRNA and Protein Levels of NF-κB Target Genes
(A) HeLa cells were transfected with a mix of two Spt5 RNAi vectors or with the same amount of the parental vector pSuper. After 48 hr, cells were treated with TNF-α for 1 hr, and the expression of A20, IκBα, JunB, CXCL1, and GAPDH mRNA was analyzed by RT-PCR. The graphs represent the levels of A20, IκBα, JunB, and CXCL1 mRNAs relative to GAPDH of three independent experiments (average ± standard error of the mean [SEM]).
(B) HeLa cells expressing Spt5 RNAi were treated with TNF-α for the indicated times. Levels of β-tubulin, Spt5, IκBα, A20, and JunB proteins were analyzed by western blot. Levels of CXCL1 protein in medium were analyzed by ELISA and presented in the graph.
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4. Figure 2
Downregulation of Spt5 Causes Retention of NF-κB in the Nucleus
(A) HeLa cells were transfected with Spt5 RNAi vectors or with parental vector pSuper. Cells were than treated with TNF-α for the indicated times, followed by preparation of either nuclear or whole-cell extracts, which were then subjected to western blot with the indicated antibodies.
(B) HeLa cells were transfected either with pSuper, Spt5 RNAi, or Spt5 RNAi and Spt5 expression plasmid, then induced by TNF-α and analyzed by western blot as in (A).
(C) HeLa cells were transfected with expression plasmid of either the Spt5 subunit or the parental vector for 48 hr, induced by TNF-α, and analyzed by western blot.
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5. Figure 3
DSIF Differentially Affects mRNA Capping of NF-κB Target Genes
(A) Analysis of 5′-capping of NF-κB-induced transcripts. HeLa cells were transfected with a mix of Spt5 RNAi vectors or parental pSuper and treated with TNF-α for 1 hr. Five micrograms of total RNA was subjected to immunoprecipitation with anti-2,2,7-Trimethylguanosine (m7G) antibody conjugated to agarose beads, as illustrated in the upper panel. Levels of A20, IκBα, JunB, CXCL1, and GAPDH transcripts were analyzed by RT-PCR. The graphs present the A20, IκBα, JunB, and CXCL1 of the capped enriched fractions of three independent experiments (average ± SEM).
(B) HeLa cells were transfected with either control (pSuper) or Spt5 RNAi vectors for 48 hr, treated with TNF-α for 30 min, and then subjected to chromatin immunoprecipitation (ChIP) with anti-HCE antibodies followed by PCR analysis with the primers from the indicated positions of the A20, IκBα, JunB, and CXCL1 genes. Quantified results, normalized to the input, from two to three independent experiments are shown.
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6. Figure 4
DSIF Is Selectively Required for Efficient Processing of A20 and IκBα mRNAs
(A) The phosphorylation state of Pol II on the A20, IκBα, JunB, and CXCL1 genes in HeLa cells. Cells were treated with TNF-α for 30 min and then subjected to ChIP with control IgG or two different Pol II antibodies: 8WG16, which preferentially recognizes the nonphosphorylated CTD, or H5 directed against the serine 2-phosphorylated form of Pol II. The immunoprecipitated DNAs were subjected to PCR analysis with primers from the middle of A20, IκBα, JunB, and CXCL1 genes. Representative results from two independent experiments are shown.
(B) The effect of Spt5 on mRNA splicing. The upper panel shows a schematic representation of the A20, IκBα, and CXCL1 gene segments from which primers were designed to monitor the levels of mature and immature transcripts as indicated. Spt5 knocked down HeLa cells treated with TNF-α for 1 hr. RNA was prepared, and RT was performed with gene-specific primers for A20, IκBα, CXCL1, and GAPDH followed by PCR amplification with primers specific to the mature and immature transcripts. The graphs present the spliced and unspliced levels of A20, IκBα, and CXCL1 relative to GAPDH of three independent experiments (average ± SEM). Control reactions without reverse transcriptase were carried out for each sample to ensure lack of contaminating DNA. Spt5 depletion was validated by western blot for each experiment (data not shown).
(C) DSIF facilitates nuclear export of A20 and IκBα mRNAs. Spt5 knockdown or control HeLa cells were treated with TNF-α followed by RNA extraction from cytosolic and nuclear fractions. Levels of A20, IκBα, JunB, CXCL1, and GAPDH were monitored by RT-PCR. The graphs present quantified results of A20, IκBα, JunB, and CXCL1 levels relative to GAPDH in the different fractions from three independent experiments (average ± SEM).
(D) Control and Spt5 knockdown HeLa cells were treated with TNF-α for 30 min and then subjected to ChIP with anti-THOC1 antibodies followed by PCR analysis with the primers from the indicated positions. Quantified results, normalized to the input, from two to three independent experiments are shown.
(E) HeLa cells transfected with Spt5 RNAi were subjected to western blot with antibodies against major mRNA-processing factors as indicated.
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7. Figure 5
The CTR Domain of Spt5 Is Dispensable for Regulation of A20 and IκBα Expression
(A) A schematic representation of Spt5 domains and mutants.
(B) HeLa cells were transfected with either pSuper, Spt5 RNAi, or Spt5 RNAi together with expression plasmid of Spt5 protein variants shown in (A) and then induced by TNF-α for 1 hr and analyzed by western blot for the levels of exogenous Spt5 proteins (α-flag), β-tubulin, IκBα, and A20.
(C) The effect of DSIF depletion on TFIIF levels. Spt5 knockdown cells were treated with TNF-α and subjected to ChIP assay with anti-Rap74 antibodies followed by PCR analysis with primers from the middle of the A20, IκBα, and JunB genes. Quantified results, normalized to the input, from three independent experiments are shown.
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8. Figure S1
Recruitment of RBP1 and Spt5 to the A20 and IκBα Promoters in HeLa Cells, Related to Figure 1
Cells were transfected with pSuper or Spt5 RNAi vectors for 48 hr, treated with TNF-α for 30 min, and subjected to ChIP with anti-RBP1, anti-Spt5, or control antibodies. PCR analysis was performed using primers from the 5′ regions of A20 and IκBα.
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9. Figure S2
Effect of Spt5 Knockdown on the mRNA and Protein Expression of A20 and IκBα in MCF7 Cells, Related to Figure 1
(A) MCF7 cells expressing DSIF RNAi or the parental vector pSuper were treated with TNF-α for 1 hr, and the expression of A20 and IκBα and GAPDH transcripts was analyzed by quantitative PCR. The results present the relative amounts of A20 and IκBα.
(B) MCF7 cells expressing DSIF RNAi or the parental vector pSuper were treated with TNF-α for the indicated times followed by whole-cell extract preparation and western blot with the indicated antibodies.
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10. Figure S3
DSIF Effect on Polyadenylation of A20 and IκBα mRNAs, Related to Figure 4
Spt5 knockdown or control HeLa cells were treated with TNF-α for 1 hr. RNA was purified and RT was performed using a poly-dT primer with an added adaptor sequence. PCR was performed with primers from last exon of each gene and the adaptor sequence. Polyadenylation patterns of A20, IκBα, and GAPDH were analyzed by semiquantitative RT-PCR (Upper panel shows a schematic description of the analysis).
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11. Figure S4
DSIF Effect on Nuclear Export of A20 and IκBα mRNAs, Related to Figure 4
Spt5 knockdown or control HeLa cells were treated with TNF-α for 1 hr. RNA was then extracted from whole-cell lysate or from cytosolic and nuclear fractions. Levels of A20, IκBα, JunB, CXCL1, and GAPDH were monitored by semiquantitative RT-PCR. Representative images of PCR products are shown. To validate effective fractionation of cytosol and nuclear RNA, gene specific RT-PCR was performed for unspliced A20 transcript.
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12. Figure S5 Spt5 Knockdown Effect on Rap74 Levels, Related to Figure 5C
HeLa cells were subjected to Spt5 knockdown as described, and cell lyssates were subjected to western blotting using indicated antibodies (Rap74 being the larger subunit of TFIIF).
Cell Reports 2012 2, DOI: ( /j.celrep )
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