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Gene target specificity of the Super Elongation Complex (SEC) family: how HIV-1 Tat employs selected SEC members to activate viral transcription.

Lu H, Li Z, Zhang W, Schulze-Gahmen U, Xue Y, Zhou Q - Nucleic Acids Res. (2015)

Bottom Line: Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities.The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency.Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions.

View Article: PubMed Central - PubMed

Affiliation: Innovation Center of Cell Signaling Network, School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, Fujian, China Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

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Genes regulated by AFF1- and AFF4-containing SECs are largely non-overlapping. (A) Total RNAs isolated from HeLa cells expressing the indicated shRNAs were analyzed by qRT-PCR to determine the levels of AFF1 (left) and AFF4 (right) mRNA relative to those of GAPDH, which was used as an internal control. (B) RNAs purified from the AFF1, AFF4 or the control GFP KD cells were analyzed by RNA-seq, and the numbers of differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) and displayed in the Venn diagram. (C) Gene ontology (GO) enrichment analysis of the AFF1 KD- or AFF4 KD-induced DEGs was performed using DAVID. Only the top four gene sets of the AFF KD DEGs and top five gene sets of the AFF4 KD DEGs are shown. (D,E) The AFF1 KD- (D) and AFF4 KD-induced DEGs (E) were mapped to a curated human functional protein interaction network and representative modules are shown. Red: genes whose expression was up-regulated by the KD; Green: genes down-regulated by KD.
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Figure 6: Genes regulated by AFF1- and AFF4-containing SECs are largely non-overlapping. (A) Total RNAs isolated from HeLa cells expressing the indicated shRNAs were analyzed by qRT-PCR to determine the levels of AFF1 (left) and AFF4 (right) mRNA relative to those of GAPDH, which was used as an internal control. (B) RNAs purified from the AFF1, AFF4 or the control GFP KD cells were analyzed by RNA-seq, and the numbers of differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) and displayed in the Venn diagram. (C) Gene ontology (GO) enrichment analysis of the AFF1 KD- or AFF4 KD-induced DEGs was performed using DAVID. Only the top four gene sets of the AFF KD DEGs and top five gene sets of the AFF4 KD DEGs are shown. (D,E) The AFF1 KD- (D) and AFF4 KD-induced DEGs (E) were mapped to a curated human functional protein interaction network and representative modules are shown. Red: genes whose expression was up-regulated by the KD; Green: genes down-regulated by KD.

Mentions: To determine how the AFF1- and AFF4-SEC may function in a gene/activator-specific manner on a genome-wide scale, we expressed shAFF1 or shAFF4 in HeLa cells and performed RNA-seq to determine the downstream target genes regulated by the two types of SEC. The efficiency of KD as determined by qRT-PCR was high, with ∼80% of AFF1 and ∼90% of AFF4 depleted, respectively (Figure 6A). RNAs purified from the AFF1, AFF4 or the control GFP KD cells were prepared for single-end, high-throughput sequencing. The differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) (21). In total, 1517 and 1602 genes were differentially expressed in response to AFF1 and AFF4 KD, respectively (Figure 6B). Importantly, the majority of them (61.8% of the AFF1 KD- and 63.9% of the AFF4 KD-induced DEGs) were only responsive to AFF1 or AFF4 depletion, suggesting that to a large extent the AFF1- and AFF4-SEC control distinct subsets of target genes in vivo.


Gene target specificity of the Super Elongation Complex (SEC) family: how HIV-1 Tat employs selected SEC members to activate viral transcription.

Lu H, Li Z, Zhang W, Schulze-Gahmen U, Xue Y, Zhou Q - Nucleic Acids Res. (2015)

Genes regulated by AFF1- and AFF4-containing SECs are largely non-overlapping. (A) Total RNAs isolated from HeLa cells expressing the indicated shRNAs were analyzed by qRT-PCR to determine the levels of AFF1 (left) and AFF4 (right) mRNA relative to those of GAPDH, which was used as an internal control. (B) RNAs purified from the AFF1, AFF4 or the control GFP KD cells were analyzed by RNA-seq, and the numbers of differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) and displayed in the Venn diagram. (C) Gene ontology (GO) enrichment analysis of the AFF1 KD- or AFF4 KD-induced DEGs was performed using DAVID. Only the top four gene sets of the AFF KD DEGs and top five gene sets of the AFF4 KD DEGs are shown. (D,E) The AFF1 KD- (D) and AFF4 KD-induced DEGs (E) were mapped to a curated human functional protein interaction network and representative modules are shown. Red: genes whose expression was up-regulated by the KD; Green: genes down-regulated by KD.
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Figure 6: Genes regulated by AFF1- and AFF4-containing SECs are largely non-overlapping. (A) Total RNAs isolated from HeLa cells expressing the indicated shRNAs were analyzed by qRT-PCR to determine the levels of AFF1 (left) and AFF4 (right) mRNA relative to those of GAPDH, which was used as an internal control. (B) RNAs purified from the AFF1, AFF4 or the control GFP KD cells were analyzed by RNA-seq, and the numbers of differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) and displayed in the Venn diagram. (C) Gene ontology (GO) enrichment analysis of the AFF1 KD- or AFF4 KD-induced DEGs was performed using DAVID. Only the top four gene sets of the AFF KD DEGs and top five gene sets of the AFF4 KD DEGs are shown. (D,E) The AFF1 KD- (D) and AFF4 KD-induced DEGs (E) were mapped to a curated human functional protein interaction network and representative modules are shown. Red: genes whose expression was up-regulated by the KD; Green: genes down-regulated by KD.
Mentions: To determine how the AFF1- and AFF4-SEC may function in a gene/activator-specific manner on a genome-wide scale, we expressed shAFF1 or shAFF4 in HeLa cells and performed RNA-seq to determine the downstream target genes regulated by the two types of SEC. The efficiency of KD as determined by qRT-PCR was high, with ∼80% of AFF1 and ∼90% of AFF4 depleted, respectively (Figure 6A). RNAs purified from the AFF1, AFF4 or the control GFP KD cells were prepared for single-end, high-throughput sequencing. The differentially expressed genes (DEGs) in response to AFF1 or AFF4 KD were identified by RankProd (P-value ≤ 0.05) (21). In total, 1517 and 1602 genes were differentially expressed in response to AFF1 and AFF4 KD, respectively (Figure 6B). Importantly, the majority of them (61.8% of the AFF1 KD- and 63.9% of the AFF4 KD-induced DEGs) were only responsive to AFF1 or AFF4 depletion, suggesting that to a large extent the AFF1- and AFF4-SEC control distinct subsets of target genes in vivo.

Bottom Line: Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities.The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency.Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions.

View Article: PubMed Central - PubMed

Affiliation: Innovation Center of Cell Signaling Network, School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, Fujian, China Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

Show MeSH
Related in: MedlinePlus