We previously showed that ZNF76 is a general transcription repressor targeting TATA-binding protein (TBP), through a process regulated by sumoylation [G. Zheng, Y.C. Yang, ZNF76, a novel transcriptional repressor targeting TATA-binding protein, is modulated by sumoylation, J. Biol. Chem. 279 (2004) 42410-42421]. In this study, two additional regulatory mechanisms for ZNF76 were identified. ZNF76 is acetylated by p300 and deacetylated by HDAC1, and acetylation of ZNF76 leads to its loss of sumoylation and attenuation of TBP interaction. Consistent with their physical antagonism, acetylation, and sumoylation play opposite roles in regulating the transactivation of ZNF76. Besides acetylation and sumoylation, ZNF76 is also regulated through mRNA splicing: two isoforms of ZNF76 have different abilities of interacting with TBP. Our study shows that ZNF76, a TBP-interacting transcriptional modulator, is regulated by both lysine modifications and alternative splicing.