#METABOLOMICS WORKBENCH Ashlley_20240331_013956 DATATRACK_ID:4751 STUDY_ID:ST003153 ANALYSIS_ID:AN005173 PROJECT_ID:PR001959 VERSION 1 CREATED_ON April 1, 2024, 1:47 am #PROJECT PR:PROJECT_TITLE YEATS4 Links Histone Crotonylation to Fatty-acid Uptake/Metabolism and Breast PR:PROJECT_TITLE Cancer Stemness PR:PROJECT_SUMMARY The integration between epigenetic regulation and metabolism is critical to PR:PROJECT_SUMMARY maintaining cellular homeostasis. As an epigenetic mark mainly linked to gene PR:PROJECT_SUMMARY activation, histone crotonylation (Kcr) uses the donor of crotonyl-CoA, a PR:PROJECT_SUMMARY metabolite generated primarily from fatty acid oxidation. Whether there is an PR:PROJECT_SUMMARY intrinsic crosstalk between histone Kcr and fatty acid metabolism remains to be PR:PROJECT_SUMMARY explored. We report here that the YEATS family protein YEATS4 is a reader of PR:PROJECT_SUMMARY histone Kcr preferentially towards H3K14cr. YEATS4 is amplified and PR:PROJECT_SUMMARY overexpressed in breast cancer cells, mainly in the ER+ subtype. Integrative PR:PROJECT_SUMMARY epigenomic and transcriptomic analyses reveal extensively overlapped chromatin PR:PROJECT_SUMMARY distribution of YEATS4 with H3K14cr, leading to activation of multiple genes PR:PROJECT_SUMMARY involved in fatty acid trafficking and metabolism, such as CD36, CPT1/2, and PR:PROJECT_SUMMARY ACOX1. Depletion of YEATS4 in breast cancer cells led to compromised fatty acid PR:PROJECT_SUMMARY uptake and β-oxidation. Interestingly, YEATS4 is upregulated in ALDH+ breast PR:PROJECT_SUMMARY cancer stem cells, leading to boosted fatty acid metabolism, enhanced PR:PROJECT_SUMMARY self-renewal, and accelerated tumor growth. Clinical pathological evidence PR:PROJECT_SUMMARY indicates that elevated YEATS4 expression is correlated with a poor prognosis PR:PROJECT_SUMMARY and worse overall survival in ER+ breast cancer patients. Together, our study PR:PROJECT_SUMMARY uncovers a feedforward epigenetic-metabolic loop implicated in breast PR:PROJECT_SUMMARY carcinogenesis, supporting the pursuit of YEATS4 as a potential therapeutic PR:PROJECT_SUMMARY target for breast cancer intervention. PR:INSTITUTE Peking Universty PR:LAST_NAME Peng PR:FIRST_NAME Zijun PR:ADDRESS Xueyuan Road No.38, Beijing, Bejing, 100191, China PR:EMAIL zj_peng@126.com PR:PHONE +8617317790671 #STUDY ST:STUDY_TITLE Untargeted metabolomic profile in control and YEATS4 knockdown MCF-7 cells ST:STUDY_SUMMARY The integration between epigenetic regulation and metabolism is critical to ST:STUDY_SUMMARY maintain cellular homeostasis. As an epigenetic mark mainly linked to gene ST:STUDY_SUMMARY activation, histone crotonylation (Kcr) uses the donor of crotonyl-CoA, a ST:STUDY_SUMMARY metabolite generated primarily from fatty acid oxidation. Whether there is an ST:STUDY_SUMMARY intrinsic crosstalk between histone Kcr and fatty acid metabolism remains to be ST:STUDY_SUMMARY explored. We report here that YEATS family protein YEATS4 is a reader of histone ST:STUDY_SUMMARY Kcr preferentially towards H3K14cr. YEATS4 is amplified and overexpressed in ST:STUDY_SUMMARY breast cancer cells, mainly in the ER+ subtype. Integrative epigenomic and ST:STUDY_SUMMARY transcriptomic analyses reveals extensively overlapped chromatin distribution of ST:STUDY_SUMMARY YEATS4 with H3K14cr, leading to activation of multiple genes involved in ST:STUDY_SUMMARY fatty-acid trafficking and metabolism, such as CD36, CPT1/2, and ACOX1. ST:STUDY_SUMMARY Depletion of YEATS4 in breast cancer cells led to compromised fatty acid uptake ST:STUDY_SUMMARY and -oxidation. Interestingly, YEATS4 is upregulated in ALDH+ breast cancer ST:STUDY_SUMMARY stem cells, leading to boosted fatty acid metabolism, enhanced self-renewal, and ST:STUDY_SUMMARY accelerated tumor growth. Clinicalpathological evidence indicates that elevated ST:STUDY_SUMMARY YEATS4 expression is correlated with poor prognosis and worse overall survival ST:STUDY_SUMMARY of ER+ breast cancer patients. Together, our study uncovers a feedforward ST:STUDY_SUMMARY epigenetic-metabolic loop implicated in breast carcinogenesis, supporting the ST:STUDY_SUMMARY pursuit of YEATS4 as a potential therapeutic target for breast cancer ST:STUDY_SUMMARY intervention. ST:INSTITUTE Peking Universty ST:LAST_NAME Peng ST:FIRST_NAME Zijun ST:ADDRESS Xueyuan Road No.38, Beijing, Bejing, 100191, China ST:EMAIL zj_peng@126.com ST:PHONE +86 17317790671 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - shNC_1 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC1.mzXML SUBJECT_SAMPLE_FACTORS - shNC_2 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC2.mzXML SUBJECT_SAMPLE_FACTORS - shNC_3 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC3.mzXML SUBJECT_SAMPLE_FACTORS - shNC_4 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC4.mzXML SUBJECT_SAMPLE_FACTORS - shNC_5 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC5.mzXML SUBJECT_SAMPLE_FACTORS - shNC_6 Sample source:MCF-7 cell | Treatment:Wild-type RAW_FILE_NAME(Raw file name)=NC1_pos-NC6.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_1 Sample source:MCF-7 cell | Treatment:YEATS4 knockdown RAW_FILE_NAME(Raw file name)=SHY1_pos-SHY1.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_2 Sample source:MCF-7 cell | Treatment:YEATS5 knockdown RAW_FILE_NAME(Raw file name)=SHY2_pos-SHY2.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_3 Sample source:MCF-7 cell | Treatment:YEATS6 knockdown RAW_FILE_NAME(Raw file name)=SHY3_pos-SHY3.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_4 Sample source:MCF-7 cell | Treatment:YEATS7 knockdown RAW_FILE_NAME(Raw file name)=SHY4_pos-SHY4.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_5 Sample source:MCF-7 cell | Treatment:YEATS8 knockdown RAW_FILE_NAME(Raw file name)=SHY5_pos-SHY5.mzXML SUBJECT_SAMPLE_FACTORS - shYEATS4_6 Sample source:MCF-7 cell | Treatment:YEATS9 knockdown RAW_FILE_NAME(Raw file name)=SHY6_pos-SHY6.mzXML SUBJECT_SAMPLE_FACTORS - QC_1 Sample source:MCF-7 cell | Treatment:quality control RAW_FILE_NAME(Raw file name)=QC01_pos-QC01.mzXML SUBJECT_SAMPLE_FACTORS - QC_2 Sample source:MCF-7 cell | Treatment:quality control RAW_FILE_NAME(Raw file name)=QC02_pos-QC02.mzXML SUBJECT_SAMPLE_FACTORS - QC_3 Sample source:MCF-7 cell | Treatment:quality control RAW_FILE_NAME(Raw file name)=QC03_pos-QC03.mzXML #COLLECTION CO:COLLECTION_SUMMARY Cells were counted, washed with cold PBS and then flash-frozen in liguid N2. CO:COLLECTION_SUMMARY Then for each sample, 1000 μL extract solution (acetonitrile: methanol: water = CO:COLLECTION_SUMMARY 2: 2: 1) containing the isotopically-labeled internal standard mixture was CO:COLLECTION_SUMMARY added. After 30 seconds of vortex, the samples were homogenized at 35 Hz for 4 CO:COLLECTION_SUMMARY min and sonicated for 5 min in an ice-water bath. The homogenization and CO:COLLECTION_SUMMARY sonication cycle was repeated 2 times. The samples were sonicated for 5 min in CO:COLLECTION_SUMMARY an ice-water bath incubated at -40 ℃ for 1 h and centrifuged at 12000 rpm for CO:COLLECTION_SUMMARY 15 min at 4°C. 800 μL of supernatant was transferred to a fresh tube and dried CO:COLLECTION_SUMMARY in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted CO:COLLECTION_SUMMARY in 200 μL of 50% acetonitrile by sonication on ice for 10 min. The constitution CO:COLLECTION_SUMMARY was then centrifuged at 13000 rpm for 15 min at 4 ℃, and 75 μL of supernatant CO:COLLECTION_SUMMARY was transferred to a fresh glass vial for LC/MS analysis. The quality control CO:COLLECTION_SUMMARY (QC) sample was prepared by mixing an equal aliquot of the supernatants from all CO:COLLECTION_SUMMARY of the samples. CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY no treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For each sample, 1000 μL extract solution (acetonitrile: methanol: water = 2: SP:SAMPLEPREP_SUMMARY 2: 1) containing the isotopically-labeled internal standard mixture was added. SP:SAMPLEPREP_SUMMARY After 30 seconds of vortex, the samples were homogenized at 35 Hz for 4 min and SP:SAMPLEPREP_SUMMARY sonicated for 5 min in an ice-water bath. The homogenization and sonication SP:SAMPLEPREP_SUMMARY cycle was repeated 2 times. The samples were sonicated for 5 min in an ice-water SP:SAMPLEPREP_SUMMARY bath incubated at -40 ℃ for 1 h and centrifuged at 12000 rpm for 15 min at 4 SP:SAMPLEPREP_SUMMARY ℃. 800 μL of supernatant was transferred to a fresh tube and dried in a SP:SAMPLEPREP_SUMMARY vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted in 200 SP:SAMPLEPREP_SUMMARY μL of 50% acetonitrile by sonication on ice for 10 min. The constitution was SP:SAMPLEPREP_SUMMARY then centrifuged at 13000 rpm for 15 min at 4 ℃, and 75 μL of supernatant was SP:SAMPLEPREP_SUMMARY transferred to a fresh glass vial for LC/MS analysis. The quality control (QC) SP:SAMPLEPREP_SUMMARY sample was prepared by mixing an equal aliquot of the supernatants from all of SP:SAMPLEPREP_SUMMARY the samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 25 mmol/L ammonium acetate; 25 mmol/L ammonium hydroxide (pH = CH:SOLVENT_A 9.75） CH:SOLVENT_B 100% acetonitrile;25 mmol/L ammonium acetate; 25 mmol/L ammonia hydroxide CH:FLOW_GRADIENT 0-0.5 min, 95%B; 0.5-7.0 min, 95%-65% B; 7.0-8.0 min, 65%-40% B; 8.0-9.0 min, CH:FLOW_GRADIENT 40% B; 9.0-9.1 min, 40%-95% B; 9.1-12.0 min, 95% B CH:FLOW_RATE 2 μl/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The TripleTOF 6600 mass spectrometry (AB Sciex) was used for its ability to MS:MS_COMMENTS acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS MS:MS_COMMENTS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) MS:MS_COMMENTS continuously evaluates the full scan survey MS data as it collects and triggers MS:MS_COMMENTS the acquisition of MS/MS spectra depending on preselected criteria. In each MS:MS_COMMENTS cycle, the most intensive 12 precursor ions with intensity above 100 were chosen MS:MS_COMMENTS for MS/MS at collision energy (CE) of 30 eV. The cycle time was 0.56 s. ESI MS:MS_COMMENTS source conditions were set as follows: Gas 1 as 60 psi, Gas 2 as 60 psi, Curtain MS:MS_COMMENTS Gas as 35 psi, Source Temperature as 600 ℃, Declustering potential as 60 V, MS:MS_COMMENTS Ion Spray Voltage Floating (ISVF) as 5000 V or -4000 V in positive or negative MS:MS_COMMENTS modes, respectively. MS raw data (.wiff) files were converted to the mzXML MS:MS_COMMENTS format by ProteoWizard, and processed by R package XCMS (version 3.2). The MS:MS_COMMENTS process includes peak deconvolution, alignment, and integration. Minfrac and MS:MS_COMMENTS cut-off are set as 0.5 and 0.3 respectively. An In-house MS2 database was MS:MS_COMMENTS applied for metabolite identification. MS:MS_RESULTS_FILE ST003153_AN005173_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END