Journal: eLife

Article Title: RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry

doi: 10.7554/eLife.67033

Figure Lengend Snippet: ( A ) Western blots were probed with antibodies to detect Ser5p and Ser2p of the CTD of Rpb1 subunit of Pol II. An antibody against the Rpb3 subunit of Pol II was used as a loading control. ( B ) Nascent RNA-seq analysis. ( C ) Pol II ChIP-seq analysis. Heatmaps show k-means clusters of 6030 genes. Genes are linked across the heatmaps. ( D ) Metaplots of ChIP-seq data shown in ( C ) without k-means clustering. Figure 1—source data 1. Gene Ontology analysis of genes transcribed during quiescence exit.

Article Snippet: Antibody , Ser2p (rat monoclonal) , Active Motif , 61083 , WB: (1:1000).

Techniques: Western Blot, Control, RNA Sequencing, ChIP-sequencing

Journal: eLife

Article Title: RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry

doi: 10.7554/eLife.67033

Figure Lengend Snippet:

Article Snippet: Antibody , Ser2p (rat monoclonal) , Active Motif , 61083 , WB: (1:1000).

Techniques: Recombinant, Control

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Pie charts showing that the distribution percentages of AFF1 or AFF4 peaks at different genome locations including TSS, within a gene, and upstream or downstream of the nearest gene. (B) Genome browser track examples for the TBP, AFF1, AFF4 and Pol II ChIP-seq signals. (C) RT-qPCR analyses showing the AFF1 and AFF4 knockdown efficiency as well as the mRNA levels of AFF1 and AFF4 were not affected by each other. Expression levels were normalized to Gapdh .

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: ChIP-sequencing, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Heatmap showing AFF1, AFF4 and Pol II ChIP-seq signals at AFF1 and AFF4 nearest genes from 3 kb upstream of TSS to 3 kb downstream of transcription termination site (TES). (B) Metaplot analysis of AFF1, AFF4 and Pol II occupancy at AFF1 and AFF4 nearest genes from 1 kb upstream to 1 kb downstream of TSS. Grey horizontal dotted line indicates TSS, other three different color horizontal dotted lines indicate AFF1, AFF4 and Pol II peaks respectively. (C) Genome browser track example for the TBP, AFF1, AFF4 and Pol II occupancy profiles. (D) Western blot analyses showing the protein levels change of Pol II, AFF1 or AFF4 in AFF1 or AFF4 knockdown A549 cells. α-Tubulin was used as a loading control. (E) Metaplot of Pol II ChIP-seq signal at AFF1 nearest genes in control and AFF1 depletion cells over regions 3 kb upstream of TSS to 3 kb downstream of TES. (F) Metagene analysis comparing Pol II occupancy at AFF4 nearest genes in control and AFF4 knockdown cells from 3 kb upstream of TSS to 3 kb downstream of TES.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: ChIP-sequencing, Western Blot

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Metaplots of Pol II ChIP-seq read densities within −3 kb to TSS and +3 kb of TES regions in the control and AFF4 knockdown A549 cells in a subset of active genes with strong AFF4 binding peaks. Vertical dotted lines represent Pol II pause site in the termination region. Black arrows indicate the direction of Pol II peak shift. (B) Genome browser track examples of the AFF4 ChIP-seq signals, and Pol II occupancy profiles in AFF4 KD A549 cells. (C) Genome browser track examples of PRO-seq signals in wildtype and AFF4 KD A549 cells. (D) Diagrams showing the positions of pre-mRNA products at PLK2 and UGDH in DRB-qRT-PCR assay. (E) qRT-PCR analysis showing pre-mRNA production in different positions of PLK2 and UGDH at different DRB release times in wildtype and AFF4 knockdown A549 cells.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: ChIP-sequencing, Binding Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Schematic diagrams showing the generation and the degradation of AFF4 degron ES cells. (B) Western blot detecting the AFF4 protein levels in the AFF4 degron ES cells. The degron cells were treated without or with 500 μM Auxin for 2h. α-Tubulin was used as a loading control. (C) Genome browser track examples of the AFF4 ChIP-seq signals in embryonic stem cells, and Pol II occupancy profiles at active genes before and after auxin treatment in AFF4 degron ES cells for 2h.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Western Blot, ChIP-sequencing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Western blot analyses showing the protein levels of AFF1 and Pol II in AFF1 knockout HCT116 cells as well as the protein levels of AFF4 and Pol II in AFF4 knockout HCT116 cells. α-Tubulin was used as a loading control. (B) ChIP-qPCR analysis showing that the Pol II occupancy change at FOS gene in control and AFF1 KO cells. (C) ChIP-qPCR analysis showing that the Pol II occupancy change at FOS gene in control and AFF4 KO cells. (D) Genome browser track examples of Pol II occupancy in control, AFF1 KO and AFF4 KO cells. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift towards 5’ ends.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Western Blot, Knock-Out

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A-B) Metagene analysis comparing Pol II occupancy on serum induction genes between wildtype, AFF1 KO and AFF4 KO cell lines. Pol II density is mapping to a 6 kb window around the genes. Grey vertical dotted lines represent Pol II pause sites in termination zone. (C) Pol II ChIP-seq for FOS and DUSP2 two individual genes are shown in wildtype, AFF1 KO and AFF4 KO cell lines. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift towards 5’ ends. (D) Images of FOS RNA FISH showing differences of nascent transcript in wildtype, AFF1 KO and AFF4 KO upon serum induction at three periods. DNA was stained with DAPI in blue. (E) Mathematical statistics analysis of mean transcription sites and transcription site intensity at FOS per cell after serum induction. (F) Real-time qPCR detection of FOS and DUSP2 levels in WT, AFF1 KO and AFF4 KO cells.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: ChIP-sequencing, Staining

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Schematic diagrams indicate AFF4 interaction regions and the deletion region of AFF4 mut cell. (B) Western blot analysis of AFF4, ENL and CDK9 in the wild type and AFF4 depletion as well as mutation HCT116 cells through AFF4 immunoprecipitation. (C) Metagene analysis comparing Pol II ChIP-seq signals at the serum-induced genes between wildtype and AFF4 MT cell lines. Pol II density is mapping to a 6 kb window around the genes. Grey vertical dotted lines represent Pol II pause sites in termination zone. (D) Pol II ChIP-seq for FOS and DUSP2 two individual genes are shown in wildtype and AFF4 MT cell lines. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift towards 5’ ends. (E) Images of FOS RNA FISH showing differences of nascent transcript in wildtype and AFF4 MT cells upon serum induction at three periods. DNA was stained with DAPI in blue. (F) Mathematical statistics analysis of mean transcription sites and transcription site intensity at FOS per cell after serum induction. (G) Heatmap of RNA sequencing datagenerating by log 2 fold change shows differential expression levels of serum induction genes in wildtype and AFF4 MT cells.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, ChIP-sequencing, Staining, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Western blot analyses showing the protein levels of AFF4 and Pol II in AFF4 mutation HCT116 cells. α-Tubulin was used as a loading control. (B) ChIP-qPCR analysis showing that the Pol II occupancy change at FOS gene in control and AFF4 MT cells. (C) Genome browser track examples of Pol II occupancy in control and AFF4 MT cells. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift towards 5’ ends. (D) Two individual gene tracks of RNA-seq in WT and AFF4 mut are shown.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Western Blot, Mutagenesis, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A-C) ChIP-qPCR analysis showed the occupancies of elongation factors (AFF1, CDK9, and SPT5) at the FOS gene in AFF4 mutation serum induced HCT116 cells. (D-E) ChIP-qPCR analysis showing that the elongation factors PAF1 and SPT6 occupancy change at the FOS gene in control and AFF4 mutant cells. (F) The protein levels of Ser2P and Ser5P in AFF4 mutation HCT116 cells. α-Tubulin was used as a loading control. (G-H) Metaplot analysis of Ser2P and Ser5P ChIP-seq for the serum induced genes in AFF4 MT cell line. Ser2P and Ser5P density are plotted in a −3 kb and +5 kb window around the genes. (I) Genome browser tracks of Ser2P and Ser5P ChIP-seq at induction genes FOS and EGR1 in control and AFF4 mutation cells in HCT116.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Mutagenesis, ChIP-sequencing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) The protein levels of Ser2P and Ser5P Pol II in AFF4 mutation HCT116 cells under serum induction. α-Tubulin was used as a loading control. (B) ChIP-qPCR analysis showing that the Ser2P Pol II occupancy change at the FOS gene in control and AFF4 mut serum induced HCT116 cells. (C) ChIP-qPCR analysis showing that the Ser5P occupancy change at the FOS gene in wildtype and AFF4 MT serum induced HCT116 cells.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) The protein levels of CPF components CSTF2, CPSF3, SYMPK, Xrn2, PPP1CB and PPP1CC in AFF4 depletion and AFF4 mutation HCT116 cells. α-Tubulin was used as a loading control. (B) Schematic diagram shows the primers sites around FOS gene. ChIP-qPCR analysis showing the occupancy of CSTF2 at FOS gene in AFF4 depletion and AFF4 mutation serum induced HCT116 cells. The HEMO gene acts as a negative control for ChIP-qPCR. (C) RT-qPCR analysis of CSTF2 gene expression after CSTF2 knockdown in wildtype and AFF4 mutation HCT116 cell lines. (D) RT-qPCR analysis of FOS gene expression after CSTF2 knockdown in wildtype and AFF4 mutation HCT116 cell lines. (E) ChIP-qPCR showing the occupancy of Pol II after CSTF2 knockdown in AFF4 mut. The HEMO gene acts as a negative control for ChIP-qPCR.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Mutagenesis, Negative Control, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) The protein levels of CPF components CSTF2, CPSF3, SYMPK, Xrn2, PPP1CB and PPP1CC in AFF4 depletion and AFF4 mutation serum induced HCT116 cells. α-Tubulin was used as a loading control. (B) RT-qPCR showing the RNA expression level of CPSF3 after CPSF3 knockdown in wildtype and AFF4 MT cells. (C) RT-qPCR showing the RNA expression level of FOS after CPSF3 knockdown in wildtype and AFF4 MT cells. (D) The change of Pol II enrichment around the FOS gene after knockdown CPSF3 in WT and AFF4 MT cells analyzed by ChIP-qPCR. The HEMO gene acts as a negative control for ChIP-qPCR.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Mutagenesis, Quantitative RT-PCR, RNA Expression, Negative Control

Journal: bioRxiv

Article Title: Distinct roles of the two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA Pol II transcription elongation

doi: 10.1101/2022.08.09.503397

Figure Lengend Snippet: (A) Western blot analyses showing the protein level change of AFF4 in AFF1 knockdown and AFF1 in AFF4 knockdown A549 cells. α-Tubulin was used as a loading control. (B) Metaplots of Pol II ChIP-seq read densities within −3 kb to TSS and +3 kb of TES regions in the control and AFF1 knockdown A549 cells in the subset of active genes with strong AFF4 binding peaks. Vertical dotted lines represent Pol II pause site in the termination region. Black arrows indicate the direction of Pol II peak shift. (C) Genome browser track examples of the Pol II ChIP-seq signals and pro-seq signals in wildtype and AFF1 KD A549 cells. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift towards 3’ ends. (D) qRT-PCR analysis showing pre-mRNA production in different positions of PLK2 and UGDH at different DRB release times in wildtype and AFF1 knockdown A549 cells. (E) Metaplots showing that the increased AFF4 occupancy in the subset of active genes with strong AFF4 binding peaks. The analysis area is −3 kb of TSS and +3 kb of TES. (F) Genome browser track examples of the AFF1 ChIP-seq, and AFF4 occupancy profiles in control and AFF1 KD cells.

Article Snippet: AFF1, AFF4 and Pol II ChIP-seq data in A549 cells, Pol II, Ser2P Pol II and Ser5P Pol II ChIP-seq data in serum-treated HCT116 cells were generated in this study, and acquired through the default Illumina pipeline using Casava v1.8.

Techniques: Western Blot, ChIP-sequencing, Binding Assay, Quantitative RT-PCR