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Transcriptionpath Applications Note APPLICATION NOTE TranscriptionPath™: A Method for RNA-free Transcription Profiling Brian Egan Active Motif’s TranscriptionPath™ Assay is an alternate approach to RNA-based gene expression methods. The TranscriptionPath method utilizes Chromatin Immunoprecipitation (ChIP) to measure transcription rates as a function of RNA Polymerase II (RNAPII) occupancy across the genome. The assay can be combined with Next-Generation sequencing (TranscriptionPath-Seq) for a genome-wide readout, or it can be used with qPCR for a gene-specific readout. TranscriptionPath offers several advantages when compared to RNA-based approaches including the ability to look at early time points that are not accurately measured with mRNA approaches and the ability to measure gene expression in the same sample as was used to measure transcription factor (TF) binding by ChIP. In this study, TranscriptionPath data was compared to mRNA data to provide evidence that RNAPII occupancy is a reliable measure of transcription rates, and to show that TranscriptionPath data is rich in additional information such as locations of alternative transcription start sites (TSS) and identification of unannotated genes. Introduction Gene expression is a complex and tightly regulated process complex (PIC) formation or pausing and transcription that is influenced by both transcription rates and mRNA rates. Described below are the methodology and data degradation rates. For many genes the main regulatory interpretation, as well as the benefits of using this mechanism of gene expression is at the level of transcrip- approach rather than RNA-based approaches. tion. This process can be described by a cycle of successive steps that 7$FWki[ZWdZJhWdiYh_X[Z 8$DejFWki[ZWdZJhWdiYh_X[Z includes: 1) the recruitment of an F[WaWjJII RNAPII containing complex; I_]dWbWYheiij^[][d[ ;l[di_]dWbWYheiij^[][d[ 2) initiation; 3) clearance of RNAPII from the site of initiation; Def[WaWjJII 4) pausing; 5) escape from pause; 6) elongation, and 7) termination. Even though so many different Huwe1 events influence gene transcription, Trps1 most approaches still measure only mRNA steady state levels, which gives no information regarding the mechanistic regulation of 9$FWki[ZWdZDejJhWdiYh_X[Z :$DejFWki[ZWdZDejJhWdiYh_X[Z Dei_]dWbWYheiij^[][d[ Dei_]dWbWYheiij^[][d[ gene expression. Kdjh[Wj[Z A more thorough and mechanistic F[WaWjJII Def[WaWjJII understanding of gene regulation Jh[Wj[ Z Aff2 can be obtained by focusing on Dnajb9 the actual process of transcription. This can be achieved by measur- ing RNAPII occupancy across the Figure 1: TranscriptionPath data reveals four states of RNAPII occupancy. genome using chromatin immuno- TranscriptionPath-Seq was performed using chromatin from mouse neuroblastoma cells. Four patterns of RNAPII occupancy precipitation. Using this approach, were detected; examples are shown above. (A) Promoter Paused and Transcribed – Transcription is rate-limited post-initiation. (B) Not Paused and Transcribed – Transcription is rate-limited by RNAPII recruitment. (C) Promoter Paused and Not Transcribed information can be collected on – These genes are poised for activation, but are not transcribed. In this example, the poised pattern shown is inducible. (D) Not start site selection, pre-initiation Paused and Not Transcribed – These genes are inactive. Active Motif Application Note Page 1 Results often extends beyond where the 3´ end of the mature mRNA will be. This extended transcription is a known The Method and Data Readout phenomenon and “extra” 3´ sequence will be cleaved during TranscriptionPath is based on the ChIP method utilizing mRNA processing to yield the mature transcript1. Addi- an antibody that targets RNAPII. Four distinct patterns tionally, signals will often be elevated at the 3´ end of the are observed when using ChIP-Seq to analyze the immu- transcribed region relative to the gene body. This pattern noprecipitated DNA (Figure 1, previous page). These are: indicates slowed extension or polymerase pausing as A. Paused and Transcribed – This pattern is defined by a transcription is terminated (Figure 2). significant peak at the TSS followed by a lower, more even signal across the gene body. The peak at the TSS is indica- Accessing Early Time Points tive of either formation of the PIC or pausing following Many gene expression studies are initiated in an effort to RNAPII release from the PIC. The elevated signal across understand how gene regulation is being altered in response the gene body is indicative of transcription of the gene. B. to a stimulus such as a drug. Gene expression in these stud- Not Paused and Transcribed – This pattern is defined by ies is often measured at time points beyond 24 hours, with even signal across the gene body with no peak at the TSS. six hours being considered an early measurement. However, C. Paused and Not Transcribed – This pattern is defined gene expression changes at 24 hours are not primary events. by a peak at the TSS and no signal in the gene body. These The primary events occur within minutes of treatment, genes have assembled the PIC and are poised for transcrip- often with the induction of transcription factors that are tion, however the genes are not being transcribed. D. Not transcribed, translated and imported back into the nucleus Paused and Not Transcribed – This pattern is defined by resulting in a second wave of transcriptional changes. In no recruitment of RNAPII to the gene body or TSS. In order to truly understand a cellular response it is important other words, these genes are inactive. The different patterns to understand the primary response, that is, the changes provide information on the mechanisms of transcription of that are happening within minutes of treatment2. Measuring each gene. Patterns A and C, which have RNAPII peaks at mRNA at these early time points has limitations, especially the TSS, have rate-limiting steps that occur after RNAPII when studying transcripts derived from longer genes recruitment. Pattern B, which does not display a RNAPII (i.e. genes over 100 Kb)3. Transcripts over 100 Kb will peak at the promoter, is rate-limited by the recruitment take over one hour to be fully transcribed, processed and of RNAPII. exported to the cytoplasm where they can be detected. As a result, many changes in gene expression will be missed Some additional features of the TranscriptionPath signal when using mRNA-based methods at early time points. that are worth noting concern RNAPII occupancy at the The TranscriptionPath method can detect gene expres- 3´ end of genes. RNAPII occupancy is strongly correlated sion changes that are missed by mRNA detection methods with gene annotations, except at the 3´ end where signal because it measures RNAPII occupancy at DNA in real ;b[lWj[Zi_]dWbWj) [dZ F[WaWjJII time. The occupancy of the RNAPII on the DNA is a sur- rogate readout for transcription rates and the Transcrip- tionPath data presented in this paper support this assertion. Additionally, studies by others support these findings and provide evidence that RNAPII occupancy is a reliable read- out for gene transcription4. In order to demonstrate the ability of TranscriptionPath to assess gene expression at early time points and to compare it to mRNA-based methods, studies were initiated in a mouse pancreatic islet cell line treated with forskolin to stimulate changes in gene expression. TranscriptionPath DNA and mRNA from these cells were used to analyze specific genes Figure 2: A representative TranscriptionPath gene profile. of varying lengths. For TranscriptionPath, RNAPII ChIP TranscriptionPath-Seq was performed using chromatin from mouse neuroblastoma was performed followed by qPCR using primers that were cells. Most genes will display an RNAPII peak at the promoter indicative of the paused state. RNAPII occupancy in the gene body tends to be fairly even across the gene body. designed approximately 1500 bp 3´ of the TSS of each gene. Occupancy then increases toward the 3´ end of the gene and often extends beyond the Because the polymerase extends at a rate of approximately 3´ gene boundary. The increased occupancy at the 3´ end is associated with pausing, as 5 transcription is terminated. Red arrows mark the start and end of the annotation and 1500 bp/min in vivo , this method enables us to detect serve to highlight these features in relation to the TranscriptionPath data. changes in RNAPII occupancy within these genes at one minute after treatment. Figure 3 (next page) shows that for Page 2 TranscriptionPath™: A Method for RNA-free Transcription Profiling 7$ ?]\'h"=[d[B[d]j^3(.'"*''Xf 8$ ?hi("=[d[B[d]j^3('"*,-Xf , + JhWdiYh_fj_edFWj^ JhWdiYh_fj_edFWj^ HD7 HD7 + * * ) ) ( ( <ebZ?dZkYj_ed ' ' & & & (& *& ,& .& '&& '(& '*& ',& '.& (&& & (& *& ,& .& '&& '(& '*& ',& '.& (&& 9$ ;]h'"=[d[B[d]j^3)"-+&Xf :$ IbY(W("=[d[B[d]j^3)&")+)Xf (+ '$( JhWdiYh_fj_edFWj^ JhWdiYh_fj_edFWj^ HD7 HD7 (& '$& &$. '+ &$, '& &$* <ebZ?dZkYj_ed + &$( & & & (& *& ,& .& '&& '(& '*& ',& '.& (&& & (& *& ,& .& '&& '(& '*& ',& '.& (&& J_c[c_dkj[i J_c[c_dkj[i Figure 3: TranscriptionPath is better than mRNA-based methods at detecting changes in gene expression at early time points. Mouse Min6 cells were treated with forskolin to induce changes in gene expression. Cells were fixed, chromatin was prepared and TranscriptionPath analysis was performed using qPCR primers specific to sequences approximately 1500 bp 3´ of the transcription start site (TSS). mRNA was measured by preparing cDNA and designing primers that span splice junctions. (A)
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