The down-regulation of the αMyHC gene during heart failure seems likely to contribute to the pathogenesis of the disease (see reference 1
). We have recently shown that YY1 is increased in heart failure and represses the activity of the αMyHC promoter. In this report we have identified the Ku factors as additional repressors of the activity of the αMyHC promoter. We show that Ku70/80 protein levels and binding activity are increased sixfold in failing human heart extracts and that their overexpression decreases the activity of the αMyHC promoter and endogenous αMyHC gene expression in NRVM.
There has been controversy surrounding the function of Ku. Ku was first identified as a DNA repair protein that recognizes DNA ends without any preferences for the nature of the ends (16
). Once bound to DNA, Ku can interact with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), and together they constitute the active kinase (9
). This complex can phosphorylate several nuclear proteins in vitro, e.g., p53, c-fos
, Sp1, XRCC4, DNA-PKcs
, or Ku itself, and it is involved in nonhomologous DNA end-joining repair and V(D)J recombination (16
). Recently, Ku has been shown to be important in preventing apoptosis by interacting with Bax and preventing it from entering the mitochondria (31
At the same time, various reports have shown that Ku can bind DNA in a sequence-specific manner, as shown in the cases of several genes (6
). Recent reports showed that the Ku70/80 complex interacts with different transcription factors, e.g., heat shock factor, DNA binding domain of the progesterone receptors, and homeodomain proteins (15
), suggesting that this could be a mechanism by which Ku is recruited to specific regions of promoters and enhancers. Ku has also been shown to interact with RNA polymerase II and with TATA binding protein (42
). In this report we have shown that Ku interaction with the αMyHC promoter is sequence specific. As shown in Fig. , Ku and YY1 bind to adjacent sites in the promoter. Point mutations created in the proposed Ku binding sites abolished binding of Ku but retained YY1 binding. Point mutations in the YY1 binding site prevented binding of YY1, allowing binding of Ku. Finally, point mutations generated in both YY1 and Ku binding sites prevented binding of either protein. It has been proposed that mutations in the Ku binding site that abolish binding are not enough evidence for sequence-specific binding, due to the preference that Ku has for certain nucleotides at DNA ends (5
). Another approach to show specificity of binding is the generation of a circular DNA radiolabeled probe. As shown in Fig. , Ku bound to the circular probe even after digestion with Exo III. Ku was also capable of binding to the linear probe, but its binding was inhibited following Exo III digestion. These experiments suggested that binding of Ku to the αMyHC promoter is sequence specific.
As discussed above, Ku has been recently shown to interact with different transcription factors. At the same time, YY1 has been shown to be involved in DNA repair though the interaction with PARP-1 (26
). Ku is also known to interact with PARP-1, which led us to test the hypothesis that YY1 and Ku interact. As shown in Fig. , immunoprecipitation experiments using YY1, Ku70, or Ku80 Abs suggested that these proteins interact in cells. The consequences of this interaction are important to the understanding of Ku function as a transcription factor and of YY1 function as a protein involved in repair, and vice versa. It has been recently proposed that Ku is involved in the reinitiation phase of transcription and that there would be an equilibrium between reinitiation and repair (47
). In this model, Ku would be either sequestered in a Ku-dependent reinitiation complex, where it would not be capable of interacting with DNA ends or, in the presence of DNA damage signals, Ku and DNA-PKcs
would be released from this complex and become active for repair. This would, in turn, disrupt the transcription apparatus, preventing reinitiation from occurring. YY1 has, in turn, been shown to be important for transcription initiation, and Usheva and Shenk (43
) have shown in vitro that YY1, TFIIB, and RNA polymerase II are sufficient to initiate transcription of the adeno-associated virus P5 promoter. We propose that YY1 and Ku are part of a transcription complex that can be involved in DNA repair or transcription and that their function will vary according to the integrity of the DNA.
Ku has been shown to function either as a repressor (5
) or as an activator (36
) in transient-transfection experiments. Here, we have shown that Ku functions as a repressor of the αMyHC promoter in NRVM cells and that coexpression of YY1 and Ku increases the repressive effect. Moreover, overexpression of Ku70 and Ku80 result in repression of endogenous αMyHC and up-regulation of skeletal α actin mRNA levels, indicating that up-regulation of Ku70 and Ku80 in the failing heart plays an important role in the regulation of components of the fetal gene program.
These results are consistent with our hypothesis that Ku is a repressor of αMyHC and that the increase in Ku protein levels is at least partially responsible for the down-regulation of the αMyHC gene observed in human heart failure. It is extremely interesting that both YY1 and Ku levels are increased in heart failure. Based on our results, the increase in the levels of these two proteins has a dramatic effect in the activity of the αMyHC promoter and most likely on the levels of αMyHC gene expression.
Finally, Ku has been shown to be expressed in various organisms besides humans, including monkey, Xenopus laevis
, yeast, Drosophila
, and rodents (5
). Interestingly, Ku levels in rodents are decreased in comparison to humans (5
). At the same time, αMyHC mRNA levels are increased in rodents compared to levels in humans. One can speculate that the difference in the levels of αMyHC and Ku are related and part of an evolutionary process. Further studies will allow us to elucidate the mechanism by which YY1 and Ku repress the activity of the αMyHC promoter in cardiac cells.