Summary of Study ST002323

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001489. The data can be accessed directly via it's Project DOI: 10.21228/M88414 This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST002323
Study Title	SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia
Study Summary	Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAP2) at TSS, and induces the promoter-proximal pausing of RNAP2 in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAP2 pausing and its function in cancer.
Institute	Chiba University
Last Name	Hoshii
First Name	Takayuki
Address	1-8-1 Inohana Chuo-ku, Chiba, Chiba, 2608670, Japan
Email	hoshiit@chiba-u.jp
Phone	81432262039
Submit Date	2022-10-12
Analysis Type Detail	LC-MS
Release Date	2022-11-18
Release Version	1

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Project:

Project ID:	PR001489
Project DOI:	doi: 10.21228/M88414
Project Title:	SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia
Project Summary:	Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAP2) at TSS, and induces the promoter-proximal pausing of RNAP2 in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAP2 pausing and its function in cancer.
Institute:	Chiba University
Last Name:	Hoshii
First Name:	Takayuki
Address:	1-8-1 Inohana Chuo-ku, Chiba, Chiba, 2608670, Japan
Email:	hoshiit@chiba-u.jp
Phone:	81432262039

Subject:

Subject ID:	SU002410
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	MOLM-13 cells expressing FKBPF36V-tagged SETD1A

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Condition
SA229079	1	DMSO_24h
SA229080	3	DMSO_24h
SA229081	2	DMSO_24h
SA229082	8	DMSO_48h
SA229083	9	DMSO_48h
SA229084	7	DMSO_48h
SA229085	4	dTAG-13_24h
SA229086	6	dTAG-13_24h
SA229087	5	dTAG-13_24h
SA229088	12	dTAG-13_48h
SA229089	10	dTAG-13_48h
SA229090	11	dTAG-13_48h

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002403
Collection Summary:	Metabolomic profiling was conducted for triplicated DMSO/dTAG-13 treated leukemia cell extracts via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) by using Agilent 7100 CE capillary electrophoresis (Agilent Technologies), the Agilent 6230 LC/MSD TOF system (Agilent Technologies), an Agilent1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter- and G1607A Agilent CE-ESI-MS sprayer kit.
Sample Type:	Leukemia cells

Treatment:

Treatment ID:	TR002422
Treatment Summary:	Cells were treated with dTAG-13 for 24 h or 48 h.
Treatment Compound:	dTAG-13

Sample Preparation:

Sampleprep ID:	SP002416
Sampleprep Summary:	For anionic metabolite analysis, the original Agilent stainless ESI needle was replaced with the Agilent G7100-60041 platinum ESI needle. System control and data acquisition were performed by Agilent MassHunter Workstation, and data analysis was done by Keio MasterHands software. For cationic metabolite analysis, separations were carried out in a fused silica capillary (50 µm i.d. x 100 cm total length) filled with 1 M formic acid as the electrolyte. Approximately 5 nL of sample solution were injected at 50 mbar for 5 sec and 30 kV of voltage was applied. The capillary temperature was maintained at 20 ºC and the sample tray was cooled below 5 ºC. Methanol-water (50 % v/v) containing 0.01 µM Hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µL/min.

Sampleprep ID:

SP002416

Sampleprep Summary:

For anionic metabolite analysis, the original Agilent stainless ESI needle was replaced with the Agilent G7100-60041 platinum ESI needle. System control and data acquisition were performed by Agilent MassHunter Workstation, and data analysis was done by Keio MasterHands software. For cationic metabolite analysis, separations were carried out in a fused silica capillary (50 µm i.d. x 100 cm total length) filled with 1 M formic acid as the electrolyte. Approximately 5 nL of sample solution were injected at 50 mbar for 5 sec and 30 kV of voltage was applied. The capillary temperature was maintained at 20 ºC and the sample tray was cooled below 5 ºC. Methanol-water (50 % v/v) containing 0.01 µM Hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µL/min.

Combined analysis:

Analysis ID	AN003791	AN003792
Analysis type	MS	MS
Chromatography type	CE	CE
Chromatography system	Agilent 7100 CE	Agilent 7100 CE
Column	COSMO(+) (chemically coated with cationic polymer) capillary (50um x 105cm total length) (Nacalai Tesque,Kyoto,Japan)	COSMO(+) (chemically coated with cationic polymer) capillary (50um x 105 cm total length) (Nacalai Tesque,Kyoto,Japan)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6230 TOF	Agilent 6230 TOF
Ion Mode	POSITIVE	NEGATIVE
Units	fmol/cell	fmol/cell

Chromatography:

Chromatography ID:	CH002804
Chromatography Summary:	For cationic metabolite analysis, separations were carried out in a fused silica capillary (50 µm i.d. x 100 cm total length) filled with 1 M formic acid as the electrolyte. Approximately 5 nL of sample solution were injected at 50 mbar for 5 sec and 30 kV of voltage was applied. The capillary temperature was maintained at 20 ºC and the sample tray was cooled below 5 ºC. Methanol-water (50 % v/v) containing 0.01 µM Hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µL/min.
Instrument Name:	Agilent 7100 CE
Column Name:	COSMO(+) (chemically coated with cationic polymer) capillary (50um x 105cm total length) (Nacalai Tesque,Kyoto,Japan)
Chromatography Type:	CE

Chromatography ID:	CH002805
Chromatography Summary:	For anionic metabolite analysis, a commercially available COSMO(+) (chemically coated with cationic polymer) capillary (50 µm i.d. x 105 cm total length) (Nacalai Tesque, Kyoto, Japan) was used with a 50 mM ammonium acetate solution (pH 8.5) as the electrolyte. Sample solution (30 nL) was injected at 50 mbar for 30 sec and -30 kV of voltage was applied. Ammonium acetate (5 mM) in 50 % methanol-water (v/v) containing 0.01 µM Hexakis(2,2-difluoroethoxy)phosphazene was delivered as the sheath liquid at 10 µl/min.
Instrument Name:	Agilent 7100 CE
Column Name:	COSMO(+) (chemically coated with cationic polymer) capillary (50um x 105 cm total length) (Nacalai Tesque,Kyoto,Japan)
Chromatography Type:	CE

MS:

MS ID:	MS003533
Analysis ID:	AN003791
Instrument Name:	Agilent 6230 TOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Electrospray ionization (ESI)-time-of-flight mass spectrometry (TOFMS) was conducted in the positive ion mode and the capillary voltage was set at 4,000V. A flow rate of heated dry nitrogen gas (heater temperature 300 ºC) was maintained at 7 psig. In TOFMS, the fragmentor-, skimmer-, and Oct RFV voltage was set at 75 V, 50 V, and 500V, respectively. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The 13C isotopic ion of a protonated methanol dimer ([2MeOH+H]+, m/z 66.0631) and Hexakis(2,2-difluoroethoxy)phosphazene ([M+H]+, m/z 622.0290) provided the lock mass for exact mass measurements.
Ion Mode:	POSITIVE

MS ID:	MS003534
Analysis ID:	AN003792
Instrument Name:	Agilent 6230 TOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	ESI-TOFMS was conducted in the negative ion mode; the capillary voltage was set at 3,500 V. For TOFMS, the fragmentor-, skimmer-, and Oct RFV voltage was set at 100 V, 50 V, and 500 V, respectively. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards, i.e., 13C isotopic ion of deprotonated acetic acid dimer ([2CH3COOH-H]-, m/z 120.0384), and Hexakis + deprotonated acetic acid (m/z 680.03554) provided the lock mass for exact mass measurements.
Ion Mode:	NEGATIVE