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LC-MSMS validation of LtpM-mediated O-Glc modification of purified LtpM, CUGBP1, PCBP1 and PCBP2

ABSTRACT: Validation of LtpM autoglucosylation; validation of the identified O-Glc-modified proteins (CUGBP1, PCBP1 and PCBP2)

INSTRUMENT(S):

ORGANISM(S): Legionella Pneumophila Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Peng Dai

LAB HEAD: Professor Peng

PROVIDER: PXD058433 | Pride | 2025-07-14

REPOSITORIES: Pride

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Project description:5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicated 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. We describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occur in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. We developed a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. In this method, we took advantage of the differential enzymatic sensitivities of the isoschizomers MspI and HpaII. HpaII cleaves only a completely unmodified site, any modification at either cytosine blocks the cleavage, while MspI recognizes and cleaves both 5-mC and 5-hmC, but not the newly discovered 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Furthermore, beta-glucosyltransferase (beta-GT) can transfer a glucose to the hydroxyl group of 5-hmC and generate beta-glucosyl-5-hydroxymethylcytosine (5-ghmC) that blocks MspI digestion. Thus, either by combining beta-GT treatment with MspI digestion or simply applying MspI/HpaII digestion, short sequence tags generated can be used for inferring hydroxymethylation or methylation status in around 1.8 million cytosine sites in the human genome. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells.

			Action	DRS
	CUGBP1_OGlc.raw	Raw
	LtpM_OGlc.raw	Raw
	PCBP1_OGlc.raw	Raw
	PCBP2_OGlc.raw	Raw
	Supplementary_Dataset_1-2_20241204.xlsx	Xlsx

Dataset Information

LC-MSMS validation of LtpM-mediated O-Glc modification of purified LtpM, CUGBP1, PCBP1 and PCBP2

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