Characterization Of The Phenotypes Of KRAB-type Zinc Finger Proteins PITA And PISA Transgenic Mice & Investigation Of SUMO Ylation Of Apak For The Regulation Of Ribosome RNA Synthesis

It was discovered that the p53 protein is also well known for its role as a transcription factor. p53 is activated by a variety of cellular intrinsic and extrinsic stress signals, including oxygen deprivation, DNA damage and oncogene activation and regulate different downstream target genes to maintain normal cellular homeostasis.Previous studies mainly focused on p53 functions as a transcription factor to induce cell cycle arrest or apoptosis. In addition to these functions for pro-oncogenic cells, in recent years new activities of p53 such as regulation of cell metabolism have been described and p53-regulated genes responsible for cell metabolism have constantly been identified.Research have shown that p53 can promote the expression of downstream target gene TIGAR to reduce PFK1 activity thereby inhibiting glycolysis. p53 transcriptionally activated SCO2 to promote the oxidative phosphorylation. To our knowledge, however, few p53 regulators have been shown to take part in selectively regulating p53-mediated cell metabolism. how p53-mediated cell metabolism is negatively regulated has become a hot issue.In mammals, KRAB zinc finger protein(KZNF protein) is the largest single family of transcriptional regulators. The proteins of this family exhibit potent transcriptional repressor activity. In the human genome, numerous studies have found such repression of KZNF protein involved in regulating proliferation, apoptosis, cell metabolism many other processes. We previously identified the KZNF protein Apak(ATM and p53associated KZNF protein, also known as ZNF420) specifically inhibits the expression of p53 target genes associated with apoptosis but has no significant effect on the transcription of cell cycle arrest-related genes. So whether or not KZNF protein family to participate in the regulation of p53 mediated metabolism remains unknown.Previously,we identify PITA(p53 inhibitor on TIGAR activation) and PISA(p53 inhibitor on SCO2 activation), the other two KRAB zinc finger protein family members, can interact with p53.When PITA or PISA binds to p53, they inhibit the expression of p53 downstream target genes TIGAR or SCO2, respectively. PITA promoted PFK1 activity through inhibiting TIGAR and increase glucose consumption and lower lactate production in cells. PISA had significant inhibit on the SCO2, thus inhibiting cytochrome c oxidase activity and promoting cell glycolysis. The different outcome of the p53-mediated stress response depends on degrees of stress.On one hand, under mild stress, PITA dissociated from p53 and resulted in the expression of TIGAR. Overexpression of TIGAR was associated with increased NADPH levels and resulted in cell survival.On the other hand, under severe stress, PISA dissociated from p53, resulting in activation of transcription of SCO2 and inducing cell death.In this study, In order to further validate the physiological functions of PITA and PISA, we attempted to generate transgenic mice. we found that the tissues from PITA transgenic mice exhibited elevated PFK1 activity, increased glucose consumption and lactate production and lowed energy expenditure, compared with those in the corresponding tissues from WT mice. PISA transgenic mice exhibited substantially lowered cytochrome C oxidase activity, increased glycolysis and PISA transgenic mice presented lower energy expenditure than wild-type mice. When the p53 deletion, shifting ATP generation from the oxidative phosphorylation pathway to glycolysis, a phenomenon widely observed in cancer cells and known as the Warburg effect. p53plays a important role in inhibiting cancer development by regulating cellular growth, cell cycle arrest, apoptosis, aging processes and DNA repair. Functional regulation of p53 in metabolism are becoming as important components to the control of tumor development. By multi-organ cancer screening,we found the expression of PITA and PISA in colorectal cancer tissues was significantly higher than the adjacent tissues.In summary, we generated PITA and PISA transgenic mouse model to obtain evidence about the PITA and PISA physiological functions and found that PITA and PISA play significant roles in regulating glycolytic and oxidative phosphorylation steps via transcriptional regulation of TIGAR and SCO2.We also explored the relevance of PITA and PISA with colorectal cancer.Ribosome is a complex consisted by ribosomal RNA and ribosomal proteins, which is required for protein synthesis. SUMOylation of substrate proteins have an important role in ribosome biogenesis. Previous studies found that the KRAB-type zinc-finger protein Apak specifically suppresses p53-mediated apoptosis. Upon ribosomal stress, the tumor suppressor protein ARF promotes Apak SUMOylation and translocation to the nucleolus. To evaluate the regulation of ribosome RNA synthesis by SUMOylation of Apak, in this study, Northern Blot assay was used to detect the level of ribosome RNA to evaluate the regulation of ribosome RNA synthesis by SUMOylation of Apak. Real-time PCR detect the level of ribosome RNA transcripts. RNA-Ch IP examined the interaction between Apak and ribosomal RNA. Here we showed that SUMOylation of Apak inhibits 47 S ribosomal RNA precursor synthesis and specifically reduced 18 S r RNA and 5.8S r RNA transcribed by RNA polymerase I. Moreover, our data demonstrated the specific interaction between endogenous Apak and 18 S r RNA or 5.8S r RNA in human cells following Act D treatment and oncogenic stress. These finding provide new evidences for understanding functions of Apak in cellular process and new insights into the regulatory mechanism of KRAB type zinc finger proteins in ribosomal RNA synthesis.