TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ ALF to promote haploid cell gene expression [ChIP-seq]. Mus musculus

TRF2 is a paralogue of TATA-box binding protein (TBP) with highest expression in testis. Although Trf2 inactivation in mice leads to arrested spermatogenesis, there is no direct evidence that Trf2 is recruited to chromatin to directly regulate gene expression. We used genetically modified mice where endogenous Trf2 has been modified to carry a TAP-TAG to perform ChIP-reChIP followed by deep sequencing. We found that Trf2 is recruited to all active promoters as a subunit of TFIIA/ALF complex together with TBP. Overall design: TRF2 ChIP-reChIP was done using anti-FLAG immobilized antibody (M2, Sigma) followed by anti-TRF2 antibody (house made) on MNase I digested chromatin from testes of adult Trf2-tag/tag mice. We performed the same ChIP-reChIP procedure on Trf2-/- testes in parallel as a control. TBP ChIP-reChIP was done using anti-TBP ab28175 (Abcam) followed by anti-TBP ab51841 (Abcam) on sonicated chromatin from Trf2tag/tag and Trf2-/- testes. PolII ChIP was performed using anti-PolII antibody (sc-9001X, Santa Cruz) on sonicated chromatin from Trf2tag/tag and Trf2-/- testes. H3K4Me3 ChIP was done using anti-H3K4Me3 antibody (04-745, Millipore) on sonicated chromatin from Wt and Trf2-/- testes. Please note that the alignment bed files were used to generate heat maps with ChIP-seq data interpretation platform seqMINER (seqMINER: an integrated ChIP-seq data interpretation platform. Ye T, Krebs AR, Choukrallah MA, Keime C, Plewniak F, Davidson I, Tora L. Nucleic Acids Res. 2011 Mar;39(6):e35. doi: 10.1093/nar/gkq1287. Epub 2010 Dec 21.). No other (further) processed data as seqMINER illustrates well enough our conclusions.