Here we show a novel pathway of transcriptional regulation of a DNA-binding transcription factor by coupled interaction and modification (e.g., acetylation) through the DNA-binding domain (DBD). The oncogenic regulator SET was isolated by affinity purification of factors interacting with the DBD of the cardiovascular transcription factor KLF5. SET negatively regulated KLF5 DNA binding, transactivation, and cellproliferative activities. Down-regulation of the negative regulator SET was seen in response to KLF5-mediated gene activation. The coactivator/acetylase p300, on the other hand, interacted with and acetylated KLF5 DBD, and activated its transcription. Interestingly, SET inhibited KLF5 acetylation, and a nonacetylated mutant of KLF5 showed reduced transcriptional activation and cell growth complementary to the actions of SET. These findings suggest a new pathway for regulation of a DNA-binding transcription factor on the DBD through interaction and coupled acetylation by two opposing regulatory factors of a coactivator/acetylase and a negative cofactor harboring activity to inhibit acetylation.The Sp/KLF (for Sp1-and Krüppel-like factor) family of zinc finger transcription factors has received recent attention due to important roles in developmental, differentiation, and oncogenic processes, among others (2, 3,35). It is comprised of over 15 mammalian family members which have in common three similar C 2 H 2 -type zinc fingers at the carboxyl terminus which comprises the DNA-binding domain (DBD). Sp/KLF family members include the founding ubiquitous factor Sp1 (9), the erythroid differentiation factor EKLF/KLF1 (27), and the tumor suppressor gene KLF6/GBF/Zf9/COPEB, which we and others identified as a cellular factor possibly involved in human immunodeficiency virus type 1 transcription (18,32,44). It was recently shown by gene knockout studies that the proto-oncogene KLF5/BTEB2/IKLF (40, 42) is important for cardiovascular remodeling in response to stress (41). Contrary to initial expectations that this family of factors would likely have redundant functions, they in fact have important individual biological functions. However, the underlying mechanisms governing their specific functions and regulation are poorly understood.We have studied the regulatory mechanisms of action of Sp/KLF family members in the past and have shown differential regulation through interaction and acetylation on the DBD by the coactivator/acetylase p300 (45). Acetylation is an important nuclear regulatory signal which regulates transcriptional processes with biological implications, including regulation of development, differentiation, and oncogenesis (5, 10, 31), which closely resembles the roles of Sp/KLF family members. We therefore thought that the Sp/KLF factors may be differently regulated by acetylation and showed that the coactivator/acetylase p300, but not the MYST-type acetylase Tip60, specifically interacts and acetylates Sp1 but not KLF6 through the zinc finger DBD and that DNA binding inhibits this interaction and ace...

The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10–C25) terpenes that are formed from polyisoprenyl diphosphates2–4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.