B cell activating factor (BAFF) gene promoter activity depends upon co-activator, p300

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• B-cell activating factor (BAFF) is crucial for mature B cell development and survival.
• Previous studies showed lipopolysaccharide (LPS) increases mouse BAFF (mBAFF) via toll-like receptor 4 (TLR4), reactive oxygen species (ROS), and NF-kappaB activation.

Purpose of the Study:

• To investigate the regulation of mBAFF expression through promoter activation.
• To explore the roles of NF-kappaB and the co-activator p300 in mBAFF promoter activity.

Main Methods:

• Cloning of the mBAFF promoter into a luciferase reporter vector (pGL3-basic).
• Analysis of NF-kappaB binding motifs within the mBAFF promoter.
• Stimulation of Raw 264.7 murine macrophages with LPS, serum deprivation, and hydrogen peroxide (H2O2).
• Treatment with N-acetyl-l-cysteine (NAC) as a ROS scavenger.
• Co-transfection experiments with p65 and p300 (including dominant-negative p300).
• Chromatin immunoprecipitation (ChIP) assay to detect p300 binding to the BAFF promoter.

Main Results:

• A 2.0 kb mBAFF promoter exhibited higher activity than shorter constructs, correlating with NF-kappaB binding sites.
• LPS stimulation, serum deprivation, and H2O2 treatment increased mBAFF promoter activity in a time-dependent manner.
• ROS production was linked to mBAFF promoter activity, as evidenced by NAC's inhibitory effect.
• LPS and serum starvation enhanced NF-kappaB activation, correlating with increased mBAFF promoter activity.
• Co-expression of p65 and p300 augmented mBAFF promoter activity, while dominant-negative p300 inhibited it.
• ChIP assays confirmed p300 binding to the BAFF promoter.

Conclusions:

• mBAFF expression is regulated by promoter activation involving NF-kappaB.
• The co-activator p300 plays a critical role in this process, cooperating with NF-kappaB.
• ROS signaling pathways are involved in the regulation of mBAFF promoter activity.