Histone Acetylation Mediated by Brd1 Is Crucial for Cd8 Gene Activation during Early Thymocyte Development

During T cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficient enhancer activity during initial Cd8 activation is known to cause a variegated CD8 expression, thereby generating a CD4⁺CD8^-TCRb^-/low population that should appear as CD4⁺CD8⁺ double-positive (DP) thymocytes. However, epigenetic and molecular mechanisms involved in initial Cd8 gene activation remain elusive. We previously reported that Brd1, also known as Brpf2, is responsible for the global acetylation of H3K14 as a subunit of the Hbo1 histone acetyltransferase (HAT) complex.

In this study, we generated conditional Brd1 knockout mice (Tie2-Cre;Brd1^fl/fl mice), in which Brd1 is inactivated in all hematopoietic cells. Although Tie2-Cre;Brd1^fl/fl mice were born and grew normally, detailed analysis revealed an abnormal thymocyte differentiation. Deletion of Brd1 resulted in the appearance of CD4⁺CD8^-TCRβ^-/low thymocytes that was indistinguishable from DP thymocytes in their properties. Hierarchical clustering of gene expression profile showed that Brd1^Δ/Δ CD4⁺CD8^-TCRβ^-/low thymocytes retain an expression profile nearly identical to one observed in DP thymocytes. These results indicate that Brd1^Δ/Δ CD4⁺CD8^-TCRβ^-/low thymocytes correspond to immature thymocytes that would normally appear as CD4⁺CD8⁺ DP thymocytes, and was generated because of a variegated activation of Cd8 gene. Importantly, in the gut intraepithelial lymphocyte (IEL) compartment, the population of CD4⁺CD8aa⁺ aβT cells, whose generation requires Cd8 gene reactivation in CD4⁺CD8⁻ helper T cells, were significantly decreased in Tie2-Cre;Brd1^fl/fl mice compared to the Tie2-Cre control mice. In addition, the percentage of CD8aa expressing gdT cells was significantly reduced. These findings suggest that Brd1 is also required to initiate Cd8a activation in gdT cells as well as Cd8 reactivation in CD4⁺T cells.

We further conducted retroviral transduction of Brd1 into Brd1^Δ/Δ DN3 cells that were flowed by culture on TSt-4/DLL stromal cells. Upon exogenous Brd1 expression, CD8 expression was completely restored and thereby Brd1^Δ/Δ DN3 cells differentiated into DP cells.

ChIP analysis demonstrated that Brd1 and Hbo1 co-localize at the known enhancers in the Cd8 locus and that their bindings are responsible for acetylation at H3K14. Biochemical results confirmed a presence of Brd1 in Hbo1 HAT complexes. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which would serve as an epigenetic mark that promotes the recruitment of transcription machineries to the Cd8 enhancers.