T-bet Binding to Newly Identified Target Gene Promoters Is Cell Type-independent but Results in Variable Context-dependent Functional Effects

Kristin M Beima,Michael M Miazgowicz,Megan D Lewis,Pearlly S Yan,Tim H-M Huang,Amy S Weinmann

doi:10.1074/jbc.m513613200

Kristin M Beima, Michael M Miazgowicz + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m513613200

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Abstract

Recently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rbeta, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters.

Highlights

T-box expressed in T cells (T-bet) is a member of the T-box transcription factor family and plays a critical role in the generation of CD4ϩ T helper 1 (Th1) cells [1]
T-bet Associates with the IFN␥ Promoter in B Cells—T-bet is expressed in B cells in response to several stimuli [1, 10], but very little is known about the genes that are directly regulated by T-bet in this cellular setting
We examined if T-bet can interact with the IFN␥ promoter in B cells

Summary

The abbreviations used are

ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP assay coupled to genomic microarray technology; IFN, interferon; IL, interleukin; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P/I, phorbol 12-myristate 13-acetate and ionomycin; Eomes, eomesodermin. Variable Consequences for T-bet Association with Target Genes factor family members, such as the T-box family, often have highly homologous DNA binding domains but diverge in the domains required for functional activity This presents the real possibility that divergent family members may have the ability to associate with similar genomic loci, but they differ in their ability to regulate gene expression. A rigorous analysis examining both the ability of a transcription factor to bind to a locus and cause a functional consequence on gene expression in diverse cellular settings is important. The data demonstrate that the association of T-bet with a target promoter can result in several different outcomes, including obligatory, modest context-dependent, or no detectable regulatory role The mechanism for these variable outcomes may involve the differential requirement for T-bet at distinct steps in transcriptional activity at each promoter. In support of this possibility, histone H3K9 acetylation is dependent upon T-bet expression only at the promoters that require T-bet for gene expression

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION