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Enhancer bound proteome of intronic Ig Emu CORE enhancer with reverse DNA sequence polarity control bait

ABSTRACT: The specificity of humoral immune responses depends on the functional rearrangement and expression of only one allele of immunoglobulin (Ig) genes. Here, we analyzed the comprehensive proteome of the murine Ig Emu CORE enhancer, which governs the rearrangement and expression of the Ig mu heavy chain allele. The Ig Emu consists of a CORE enhancer (harboring a multitude of transcription factor binding sites) and two 5’ and 3’ flanking MAR (matrix attachment region) elements. By mass spectrometry of proteins bound at wild type versus mutant Emu enhancers, we identified Emu-binding proteins and associated multi-protein complexes. We found that the MSL/MOF complex, a regulator of gene dosage compensation in flies, binds Emu via transcription factor YY1 and facilitates Emu-driven chromatin looping and promoter interaction. Msl2 gene knockout in primary pre-B cells or Mof heterozygosity in mice reduced mu gene expression. In this data set we compare proteins binding to the wild-type Emu versus a DNA bait control for which the Emu CORE sequence was switched to its reverse polarity sequence (the flanking MARs sequence are wild-type). The latter conserves the DNA GC content but virtually destroys all sequence-specific transcription factor binding sites. Of note, DNA repetitive sequences that can also be bound by DNA interacting proteins are kept functional by this control bait. SILAC quantitative proteomics was employed in a label swap approach incubating wild-type and control DNA with labeled and non-labled protein extracts, respectively.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): B Cell

DISEASE(S): Lymphoma

SUBMITTER: Gerhard Mittler

LAB HEAD: Gerhard Mittler

PROVIDER: PXD045637 | Pride | 2024-10-25

REPOSITORIES: Pride

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			Action	DRS
	Rep01_EnhancerMARrev_FWD_01.raw	Raw
	Rep01_EnhancerMARrev_FWD_02.raw	Raw
	Rep01_EnhancerMARrev_FWD_03.raw	Raw
	Rep01_EnhancerMARrev_FWD_04.raw	Raw
	Rep01_EnhancerMARrev_FWD_05.raw	Raw

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Project description:Background: In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-M-NM-1-stimulated endothelial cells. Nuclear factor-kappa B (NF-M-NM-:B) reporter gene assays confirmed that MOF decrease the activity of NF-M-NM-:B. Strong inter-individual variability in the leukocytesM-bM-^@M-^Y DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. However, in individuals, significant changes in DNA methylation of genes involved in leukocyte rolling, adhesion and cytoskeleton remodelling were seen. Conclusion: Our study revealed that regular consumption of MOF modulates the expression of genes in immune cells which, however, cannot be correlated to alterations in the DNA methylome. Remarkably, at the individual level, genes related to leukocyte adhesion pathways present complex variation in DNA methylation. As such, the individual transcriptional and epigenetic modulation of genes involved in early manifestation of cardiovascular diseases constitutes important subcellular mechanisms by which MOF promote vascular health in humans. DNA methylation levels of blood samples from 10 subjects were assessed using HumanMethylation450 BeadChips (Illumina), both before and after a diet intervention with MOF.

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Enhancer bound proteome of intronic Ig Emu CORE enhancer with reverse DNA sequence polarity control bait

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