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...

We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.

Dysferlinopathy is a progressive muscle disorder that includes limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG‐132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient‐derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017–1029

Krüppel-like factor 5 (KLF5) is a transcription factor important in regulation of the cardiovascular response to external stress. KLF5 regulates pathological cell growth, and its acetylation is important for this effect. Its mechanisms of action, however, are still unclear. Analysis in KLF5-deficient mice showed that KLF5 confers apoptotic resistance in vascular lesions. Mechanistic analysis further showed that it specifically interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme important in DNA repair and apoptosis. KLF5 interacted with a proteolytic fragment of PARP-1, and acetylation of KLF5 under apoptotic conditions increased their affinity. Moreover, KLF5 wild-type (but not a non-acetylatable point mutant) inhibited apoptosis as induced by the PARP-1 fragment. Collectively, we have found that KLF5 regulates apoptosis and targets PARP-1, and further, for acetylation to regulate these effects. Our findings thus implicate functional interaction between the transcription factor KLF5 and PARP-1 in cardiovascular apoptosis.The cardiovasculature adapts dynamically to metabolic and/or mechanical stresses (i.e. blood vessel remodeling in response to oxidative and hypertensive stress). Although this response initially compensates for the pathological stimulus, chronic and excessive load ultimately leads to decompensatory maladaptation, which is the underlying pathology of heart failure and atherosclerosis (1, 2).The cellular mechanisms underlying cardiovascular adaptation processes are characterized by cellular hyperplasia, hypertrophy and death. Previous studies have begun to clarify the molecular basis of the process centered on signaling pathways linking extracellular stimuli to intracellular processes characterized by the intracellular signaling cascade and downstream gene expression events, which include roles of transcription factors, such as NFAT (nuclear factor of activated T cells) through the calcineurin pathway and histone deacetylases in cardiac hypertrophy (3-6). We have recently shown that the transcription factor, Krüppel-like factor 5 (KLF5), 4 regulates the cardiovascular response to pathological stress (e.g. angiotensin II) by modulating atherosclerosis, angiogenesis, and cardiac hypertrophy (7-10).Although the transcriptional and signaling networks regulating the cardiac adaptation response have begun to be unraveled, further investigation is needed to better understand the pathogenic roles of the involved factors and pathways. Regulation of cell death/survival, in particular, remains poorly understood. Here, we have shown that KLF5 inhibits cell death/apoptosis and that it functionally interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in the response to DNA damage (11). MATERIALS AND METHODSCell Culture and Apoptotic Assays-3T3 and HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum. Human umbilical vein-derived endothelial cells were cultured in EBM-2 medium with EGM-2 supplement (Clonetics). Stable transforman...

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