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EnChIP Mediated proteome profiling reveals the role of SMAD-p300 regulatory axis in GM2 synthase transcriptional regulation.

ABSTRACT: Aberrant expression of sialic acid containing glycolipids, or gangliosides and their corresponding synthases have been associated with a variety of cancers. GM2-synthase,the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be overexpressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (EMT) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic mechanism underlying the transcriptional regulation of GM2-synthase gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a modified CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn’t clear. This study demonstrated that the histone acetyltransferase (HAT), p300 acts a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand modulates SMAD2/4 to have a direct positive impact on GM2 synthase gene transcription. We identified p300 to have an activator role in GM2 synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns shows the important role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2--synthase transcriptional regulation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Kaushik Biswas

LAB HEAD: Kaushik Biswas

PROVIDER: PXD065551 | Pride | 2026-03-27

REPOSITORIES: Pride

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Source:

			Action	DRS
	NonTarget_Flag_Fr1.d.zip	Other
	NonTarget_Flag_Fr1.mzML	Mzml
	NonTarget_Flag_Fr10.d.zip	Other
	NonTarget_Flag_Fr10.mzML	Mzml
	NonTarget_Flag_Fr11.d.zip	Other

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Project description:TGF-b is an important pleiotropic cytokine with potent immunoregulatory properties. Although many previous reports have been proposed for the immunoregulatory functions of TGF-b on T cells, such as the suppression of cell proliferation, cytokine production and cytokine signaling, as well as the induction of apoptosis, it is not well elucidated whether the each effect of TGF-b on T cells is dependent on Smad signaling or Smad-independent other signaling pathways. The aim of the study was to clarify the involvement of Smad signaling and to investigate the redundancy of Smad2 and Smad3 on various TGF-b-mediated regulation of gene expression in CD4+ T cells. We used microarrays to detail the global program of gene expression regulated by TGF-b in CD4+ T cells, and identified distinct classes of up/down-regulated genes which are dependent on or independent of TGF-b-Smad signaling. Most of genes regulated by TGF-b were redundantly dependent on Smad2 and Smad3, including Foxp3 and IL-2. In addition, some genes were sufficiently regulated via Smad2 or Smad3 signaling alone. In contrast, TGF-b-mediated RORgt induction was independent of Smad signaling. CD4+CD25-CD44loCD62Lhi T cells (naive) were isolated from the spleens in wild-type (WT), T cell-specific Smad2 conditional knockout (Smad2KO or Smad2del/del), Smad3 knockout (Smad3KO or Smad3-/-) or Smad2del/delSmad3+/- mice by using a BD FACS ariaTM cell sorter (BD Bioscience) (purity: >98%). Freshly purified cells were then stimulated with anti-TCR stimuli in the absence or presence of TGF-b for 24 hr, respectively. A complete and precise experimental procedure is given in the "treatment protocol". It was very difficult to obtain the enough number of CD4+CD25-CD44loCD62Lhi naive T cells from Smad2del/delSmad3-/- mice because alomost all of CD4+ T cells were activated in Smad2del/delSmad3-/- mice. We confirmed that the several known Smad-regulated genes were almost out of control in Smad2del/delSmad3+/- CD4+ T cells by using quantitative RT-PCR. Furthermore, previous studies have reported the similar results in other cell types deficit in two alleles of Smad2 and one allele of Smad3. For these reasons, we substituted Smad2del/delSmad3+/- naive T cells for Smad2/3-deficient naive T cells. Cells were quickly collected 24 hr after culture for RNA extraction and hybridization on Affymetrix microarrays.

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EnChIP Mediated proteome profiling reveals the role of SMAD-p300 regulatory axis in GM2 synthase transcriptional regulation.

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