A chromatin immunoprecipitation (ChlP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos

AGAMOUS-like-15 (AGL15) is a member of the MADS-domain family of DNA-binding regulatory factors that accumulates preferentially in tissue developing in an embryonic mode. To better understand how AGL15 functions, we developed a chromatin immunoprecipitation (ChlP) approach to isolate genes regulated directly by AGL15. ChlP allows purification of in vivo protein-DNA complexes. The co-purified DNA is recovered and used to isolate the putatively regulated gene. Several tests must be performed to show that the putative downstream target gene is truly regulated by the DNA-binding protein. The DNA-binding regulatory protein must interact with cis regulatory elements. The downstream gene expression pattern should respond to the level of the trans-acting regulatory factor. The cis element should be able to confer regulation in response to the trans-acting factor. We describe, in this report, our ChlP protocol, and discuss in detail, tests to confirm regulation by AGL15 for two targets identified by ChlP. These targets are referred to as Downstream Target of AGL15 (DTA1 and DTA2). Expression of DTA1, which encodes a protein with high similarity to GA-2 oxidase-like proteins, is induced by AGL15. DTA2 encodes a novel protein and expression of this target is repressed by AGL15.