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E47 and Id2 Regulation of Pol III Transcription: Implications for tRNA Genes

This study investigates the role of E47 and Id2 proteins in the regulation of Pol III transcription and their impact on tRNA gene expression. The results demonstrate that E47 negatively affects Pol III transcript levels, while Id2 positively regulates Pol III gene transcription. Furthermore, E47 knockdown enhances the recruitment of Brf1 and RPC155 at tRNA genes, while Id2 knockdown diminishes their recruitment. This research sheds light on the molecular mechanisms underlying tRNA gene expression regulation.

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E47 and Id2 Regulation of Pol III Transcription: Implications for tRNA Genes

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  1. Figure 1: E47 is present on tRNA genes 1A IP/ Input IP 1B Input TFIIB IgG Brf1 E47 WB: Brf1 A) IP/Input enrichment of E47 at 6 active tRNA genes and one inactive tRNA gene (tRNA81Leu-TAA) measured via Chromatin IP. An IP with SL1 antibody is used as control. ETC19 (Extra TFIIIC binding site) and ApoE gene regions are used as no enrichment control. B) Protein immunoprecipitation performed with the shown antibodies or IgG as a control. Brf1 could be precipitated by an E47 antibody more than a TFIIB or IgG pull-downs. 10% input is used in the first lane.

  2. Fig 2 E47 negatively affects pol III transcript levels 2A 2B Fold over Control siRNA Fold over Control siRNA 4 3 Fold over Control siRNA 5S rRNA tRNA-Leu 2D 2 tRNA-Thre 2C tRNA-iMet 1 Fold over Control siRNA 0 Hdac1 Hdac2 Gcn5 cMyc p300 Legend in next page

  3. E47 siRNA knockdown induces 5sRNA and tRNA levels monitored via qPCR. Levels of 3 active tRNAs measured and 5sRNA are depicted • relative to a control siRNA treatment. A value of 1 would mean no change • B) E47 siRNA knockdown enhances recruitment of Brf1 and RPC155 at tRNA genes. Chromatin IP to determine recruitment of Brf1 and RPC155 at 3 tRNA genes upon knockdown of E47 protein levels. Antibodies are used for proteins depicted on the x-axis. Presence of Brf1 and RPC155 is shown relative to a control siRNA treatment. A value of 1 would mean no change. • C) E47 siRNA knockdown affects recruitment of chromatin remodelers at tRNA genes. Chromatin IP to determine recruitment of HDAC and HAT complexes at 3 tRNA genes and 5sRNA upon knockdown of E47 protein levels. Antibodies are used for proteins depicted on the x-axis. Presence of HDAC and HAT complexes are shown relative to a control siRNA treatment. A value of 1 would mean no change. • D) E47 siRNA knockdown enhances presence of permissive chromatin marks and reduces presence of repressive marks at tRNA genes. Chromatin IP to determine recruitment of chromatin marks at tRNA genes upon knockdown of E47 protein levels. Antibodies are used for the appropriate histone modifications are depicted on the x-axis. Presence of acetylated or methylated histone forms is shown relative to a control siRNA treatment. A value of 1 would mean no change.

  4. Fig 3: Id2 protein is detected on tRNA genes 3A 3B ChIP after E47siRNA IP/ Input Fold over Control siRNA 3C IP A) IP/Input enrichment of Id2 at 6 active tRNA genes measured via Chromatin IP. An IP with SL1 antibody is used as control. ETC19 (Extra TFIIIC binding site),ApoE gene and an inactive tRNA, tRNA81-Leu gene regions are used as no enrichment control. B) E47 knockdown affects recruitment of Id2 at tRNA genes. Chromatin IP to determine recruitment of Id2 at 5S rRNA and RNA genes upon knockdown of E47 protein levels. H3 antibody was used a control. Id2 absence at tRNA genes is shown relative to a control siRNA treatment. A value of 1 would mean no change. C) Protein immunoprecipitation performed with the shown antibodies or protein A/G beads. Brf1 could be precipitated by an Id2 antibody more than a TFIIB or just beads pull-downs. 10% input is used in the first lane. Brf1 IgG Input TFIIB Id2 WB: Brf1

  5. Fig 4: Id2 positively regulates pol III gene transcription 4A 4B Vector Ad-Id2 Ad-Id2 Vector Id2 mRNA Id2 Fold over Control siRNA 5S rRNA Actin tRNA Western Blot ARPP P0 4C RT-PCR A) Id2 siRNA knockdown reduces 5sRNA and tRNA expression levels as monitored via qPCR. Levels of 3 active tRNAs measured and 5sRNA are depicted relative to a control siRNA treatment. A value of 1 would mean no change. B) Overexpression of Id2 using an adenoviral vector upregulates 5S rRNA and tRNA expression. Western blot on the left show increased levels of Id2 protein. Semi-quantitative Reverse Transcriptase PCR on the right shows increased Id2 mRNA levels as well as induced 5S rRNA and tRNA levels. ARPP0 gene was used as a control. C) Id2 siRNA knockdown diminishes recruitment of Brf1 and RPC155 at tRNA genes. Chromatin IP to determine recruitment of Brf1 and RPC155 at 3 tRNA genes upon knockdown of Id2 protein levels. Presence of Brf1 and RPC155 is shown relative to a control siRNA treatment. A value of 1 would mean no change. Fold over Control siRNA

  6. Supplemental Fig 1: Id2 and E47 form in vitro and in vivo complexes with pol III transcription machinery s1A s1B 35S-Brf1 35S-Luciferase A) GST tagged and purified E47 and Id2 proteins immunoprecipitate 35S labelled in vitro transcribed Brf1. A purified protein extract from GST-Vector expressing cells, and 35S labelled in vitro transcribed Luciferase were used as a negative control. B) Protein immunoprecipitation performed with the antibodies or protein A/G beads as shown above each lane. RPF155, TFIIIC 220 and TBP could be precipitated by an Id2 and E47 antibody more than just beads pull-down. 10% input is used in the first lane.

  7. Sup Fig 2: Knock down of E2A transcripts induces Pol III transcribed genes Control siRNA E2A siRNA Control siRNA E2A siRNA Knockdown of E47 using siRNA upregulates 5S rRNA and tRNA expression. Western blot on the left shows decreased levels of E2A proteins (upper band E47, lower band E12). Semi-quantitative Reverse Transcriptase PCR on the right shows reduced E2A transcript levels as well as induced 5S rRNA and tRNA levels. ARPP0 gene was used as a control. E47 mRNA E47 E12 5S rRNA Actin tRNA Western Blot ARPP P0 RT-PCR

  8. Control siRNA Id2 siRNA Id mRNA Id2 5S rRNA Actin tRNA Control siRNA ARPP P0 mRNA Id2 siRNA RT-PCR Sup Fig 3 :Id2 depletion negatively affects pol III transcribed genes Knockdown of Id2 using siRNA downregulates 5S rRNA and tRNA expression. Western blot on the left shows decreased levels of Id2 proteins. Semi-quantitative Reverse Transcriptase PCR on the right shows reduced Id2 transcript levels as well as decreased 5S rRNA and tRNA levels. ARPP0 gene was used as a control. Western Blot

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