Long Vo ngoc, Cassidy Yunjing Huang, California Jack Cassidy, Claudia Medrano, James T. Kadonaga
The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription, but the downstream core promoter in humans has been difficult to understand. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.
Long Vo ngoc, Cassidy Yunjing Huang, California Jack Cassidy, Claudia Medrano, James T. Kadonaga. Identification of the human DPR core promoter element using machine learning. 2020-09-09. https://doi.org/10.1038/s41586-020-2689-7
A HARPE plasmid library with a 12-nucleotide randomized DPE-region segment (+23 to +34 relative to the initiator +1 TSS) was transcribed in vitro with HeLa nuclear extract in an SCP1m core-promoter background. Reporter RNA/cDNA and plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transiently transfected into HeLa cells using the human IRF1 core-promoter cassette. Reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A modified HARPE plasmid library randomized the long TATA region from -32 to -21 relative to the initiator +1 TSS and linked each TATA variant to a downstream barcode. The library was transiently transfected into HeLa cells; reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify TATA-dependent transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transcribed in vitro with HeLa nuclear extract in a TATA-less SCP1m core-promoter background. Reporter RNA/cDNA and plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transcribed in vitro with HeLa nuclear extract. Sarkosyl was added to 0.2% (w/v) 20 seconds after rNTP initiation to restrict transcriptional progression, and RNA/cDNA and plasmid DNA were sequenced for each variant.
A HARPE plasmid library with the 12-nucleotide MTE-region segment randomized from +18 to +29 relative to the initiator +1 TSS was transcribed in vitro with HeLa nuclear extract in an SCP1m core-promoter background. Reporter RNA/cDNA and plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transcribed in vitro with HeLa nuclear extract in the TATA-containing SCP1 core-promoter background. Reporter RNA/cDNA and plasmid DNA were sequenced to quantify DPR-dependent transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transiently transfected into HeLa cells in a TATA-less SCP1m core-promoter background. Reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify DPR-dependent transcription strength.
A HARPE plasmid library with the 12-nucleotide MTE-region segment randomized from +18 to +29 relative to the initiator +1 TSS was transiently transfected into HeLa cells in an SCP1m core-promoter background. Reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transiently transfected into HeLa cells in the TATA-containing SCP1 core-promoter background. Reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify DPR-dependent transcription strength.
A HARPE plasmid library with the 19-nucleotide DPR segment randomized from +17 to +35 relative to the initiator +1 TSS was transcribed in vitro with HeLa nuclear extract using the human IRF1 core-promoter cassette. Reporter RNA/cDNA and plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A low-complexity HARPE library tested a 13-nucleotide DPR sequence variant together with randomized 3-nucleotide tags after transient transfection into HeLa cells. Reporter RNA/cDNA and recovered plasmid DNA were sequenced, and tag-level observations were averaged to obtain one transcription-strength measurement per DPR sequence.
A HARPE plasmid library with a 12-nucleotide randomized DPE-region segment (+23 to +34 relative to the initiator +1 TSS) was transiently transfected into HeLa cells in an SCP1m core-promoter background. Reporter RNA/cDNA and recovered plasmid DNA were sequenced to quantify sequence-specific transcription strength.
A modified HARPE plasmid library randomized the short TATA region from -30 to -23 relative to the initiator +1 TSS, with a fixed TA dinucleotide at -32 and -31 to set TATA phasing, and linked each TATA variant to a downstream barcode. The library was transcribed in vitro with HeLa nuclear extract and assayed by paired RNA/cDNA and plasmid-DNA sequencing.
A low-complexity HARPE library tested a 13-nucleotide DPR sequence variant together with randomized 3-nucleotide tags during in vitro transcription with HeLa nuclear extract. Reporter RNA/cDNA and plasmid DNA were sequenced, and tag-level observations were averaged to obtain one transcription-strength measurement per DPR sequence.
A modified HARPE plasmid library randomized the long TATA region from -32 to -21 relative to the initiator +1 TSS and linked each TATA variant to a downstream barcode. The library was transcribed in vitro with HeLa nuclear extract; reporter RNA/cDNA and plasmid DNA were sequenced to quantify TATA-dependent transcription strength.
A modified HARPE plasmid library randomized the short TATA region from -30 to -23 relative to the initiator +1 TSS, with a fixed TA dinucleotide at -32 and -31 to set TATA phasing, and linked each TATA variant to a downstream barcode. The library was transiently transfected into HeLa cells and assayed by paired RNA/cDNA and plasmid-DNA sequencing.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 46 files (ZIP)filelist.txtGSE139635_family.soft.gzGSE139635_GM12878_SVRb_SVRc_SVRtata.bed.gzGSE139635_HeLa_SVRb_SVRc_SVRtata.bed.gzGSE139635_MCF7_SVRb_SVRc_SVRtata.bed.gzGSE139635_series_matrix.txt.gzGSE139635_SVRb_DPR.model.gzGSE139635_SVRc_DPR.model.gzGSE139635_SVRpredict.R.gzGSE139635_SVRtata_TATAbox.model.gzGSE139635_TATAlong_ConversionTable.txt.gzGSE139635_TATAshort_ConversionTable.txt.gzGSM4144990_DPE_cell_rep1.txt.gzGSM4144991_DPE_cell_rep2.txt.gzGSM4144992_DPE_inVitro_rep1.txt.gzGSM4144993_DPE_inVitro_rep2.txt.gzGSM4144994_DPR_cell_rep1.txt.gzGSM4144995_DPR_cell_rep2.txt.gzGSM4144996_DPR_inVitro_Sarkosylrep1.txt.gzGSM4144997_DPR_inVitro_Sarkosylrep2.txt.gzGSM4144998_DPRinVitro_rep1.txt.gzGSM4144999_DPRinVitro_rep2.txt.gzGSM4145000_IRF_cell_rep1.txt.gzGSM4145001_IRF_cell_rep2.txt.gzGSM4145002_IRF_inVitro_rep1.txt.gzGSM4145003_IRF_inVitro_rep2.txt.gzGSM4145004_LowComp_cell_rep1.txt.gzGSM4145005_LowComp_cell_rep2.txt.gzGSM4145006_LowComp_inVitro_rep1.txt.gzGSM4145007_LowComp_inVitro_rep2.txt.gzGSM4145008_MTE_cell_rep1.txt.gzGSM4145009_MTE_cell_rep2.txt.gzGSM4145010_MTE_inVitro_rep1.txt.gzGSM4145011_MTE_inVitro_rep2.txt.gzGSM4145012_TATAlong_cell_rep1.txt.gzGSM4145013_TATAlong_cell_rep2.txt.gzGSM4145014_TATAlong_inVitro_rep1.txt.gzGSM4145015_TATAlong_inVitro_rep2.txt.gzGSM4145016_TATAshort_cell_rep1.txt.gzGSM4145017_TATAshort_cell_rep2.txt.gzGSM4145018_TATAshort_inVitro_rep1.txt.gzGSM4145019_TATAshort_inVitro_rep2.txt.gzGSM4411945_SCP1withTATA_cell_rep1.txt.gzGSM4411946_SCP1withTATA_cell_rep2.txt.gzGSM4411947_SCP1withTATA_inVitro_rep1.txt.gzGSM4411948_SCP1withTATA_inVitro_rep2.txt.gz