Investigating Th9 Cell Transcriptional Regulation

CD4⁺ T cells were purified by MACS sorting and were polarized to Th9 phenotype. ChIP was performed according to the protocol described in (25 (link)) with the following modifications: Chromatin was sheared using Micrococcal Nuclease (New England Biolabs) and Protein A Magnetic Beads (New England Biolabs) were used. Cell lysates were used for immunoprecipitation with anti-STAT5, anti-BCL6, anti-mono-methyl-Histone H3 lysine 4 (H3K4me1), and anti-pan-methyl-H3K9 (all from Cell Signaling Technology) and were compared to control IgG. One region of the Il9 promoter containing putative STAT5 and BCL6 binding sites as well as a region of the Bcl6 promoter containing putative STAT5 binding sites was amplified by SYBR Green qPCR (Applied Biosystems) and quantified in duplicate with the percentage of input method. The following primers were used: Il9 Promoter: Site 1 Fwd: ACTGAGTTCCAGACTCCCGT, Rev: GCCCAGCACAGAACTGAAGA; Site 2 Fwd: GGATCCTCAAGGCCAATGCT, Rev: ACACCTCTGAGAAGTCGCTC; Site 3 Fwd: ACAGAAGTGTGCTGTCTGGT; Rev: CCCCTTGAGCCACTGGATAC. Bcl6 Promoter: Site 1 Fwd: CTGCGGAGCAATGGTAAAGC, Rev: ATAATCACCTGGTGTCCGGC; Site 2 Fwd: CGAGGAGCCGAGTTTATGGG, Rev: GAGAGTGCGCTTTGCTTTCC; Site 3 Fwd: CGAATGACAGTCCCGACGAT, Rev: GCTTGGGATGCTCCTGTTGT.

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Bassil R., Orent W., Olah M., Kurdi A.T., Frangieh M., Buttrick T., Khoury S.J, & Elyaman W. (2014). BCL6 Controls Th9 Cell Development by Repressing Il9 Transcription. Journal of immunology (Baltimore, Md. : 1950), 193(1), 198-207.

Publication 2014

Micrococcal Nuclease Protein A Magnetic Beads

Bcl6 Binding sites Cd4 t cells Cell Chip Chromatin Histone h3 Immunoprecipitation Lysine Micrococcal nuclease Phenotype Primers Protein a Stat5 Sybr green

Corresponding Organization : American University of Beirut

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Variable analysis

independent variables

Polarization of CD4+ T cells to Th9 phenotype

dependent variables

Binding of STAT5, BCL6, mono-methyl-Histone H3 lysine 4 (H3K4me1), and pan-methyl-H3K9 to the Il9 promoter and Bcl6 promoter regions

control variables

Chromatin shearing using Micrococcal Nuclease
Protein A Magnetic Beads for immunoprecipitation
Control IgG for immunoprecipitation

controls

Positive control: Immunoprecipitation with anti-STAT5, anti-BCL6, anti-mono-methyl-Histone H3 lysine 4 (H3K4me1), and anti-pan-methyl-H3K9
Negative control: Immunoprecipitation with control IgG

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