Gene Regulation Of Angiogenin And Ribonuclease-4and Their Biological Functions

Ribonuclease A superfamily is a class of endonucleases which can hydrolyze the RNA nuclear acid. Their homologous genes are clustered on the chromatin. Ribonuclease-4(RNase-4) and angiogenin (ANG) are the fourth and fifth member of superfamily. Their genes locus has a unique gene arrangement, which the two genes share the same promoters and partial5’-untranslated region followed by two distinct exons encoding the two proteins, respectively. This gene arrangement is unique in the ribonuclease A superfamily, and is very rare in the whole human genome. However, gene regulation of this locus is still poorly understood. Thanks to the Encyclopedia of DNA Elements (ENCODE) project data sets, we located the ANG and RNase-4regulation region and identified the transcription site as the "broad class" that can initiate transcription over a～200bp region. Meanwhile, we verified the gene promoter region by dual luciferase reporter assay. Combined with ENCODE data sets, we identified two RNA polymerase Ⅱ promoter active regions (P1and P2) on the ANG and RNase-4gene promoter region. The distal RNA polymerase Ⅱ promoter active region PI followed by a tRNA cis-element (T1) and its activity was inhibited, while the proximal RNA polymerase Ⅱ promoter active region P2was preceded by the second tRNA cis-element (T2) and benefited by the elements. We have further shown that CCCTC-binding factor (CTCF) formed a chromatin loop between the two introns flanking the ANG coding exon and thereby regulating the transcription levels of ANG and RNase-4genes.Our data represents a new layer of transcriptional regulation of ANG and RNase-4genes that share the same promoters and are supposed to be co-expressed.The human ribonuclease A superfamily contains13members, each member has its own unique biological functions, such as host defense, angiogenesis, enhance\inhibition of tumor growth and activation of the immune system. ANG was originally isolated and purified from the condition medium of tumor cells based on its angiogenic activity. With nearly30years of researches, we have a certain understanding of the mechanism of action of ANG under physiological and pathological conditions. ANG undergoes nuclear translocation and enhances ribosomal RNA (rRNA) transcription by binding the ABE (Angiogenin binding element, ABE) sequence region on rDNA in a variety of cells. However, the underlying mechanism remains elusive. Here, we demonstrated that ANG not only binds ABE sites in rDNA, but also can bind on the UCE (upstream control element, UCE) site. Down-regulation of ANG gene depleted the occupancy of RNA polymerase Ⅰ, TAF1110, TAF148, but not upstream binding factor (UBF), to the promoter of rDNA. Notably, our data showed that ANG enhanced the demethylation of rDNA promoter region and regulated the levels of histone modification at this region. Besides ANG binds on the rDNA region, whether ANG has other binding sites through the whole genome needs to be validated. We mapped the ANG genome-wide binding sites by chromatin immunoprecipitation combined with the promoter gene array technology. We first discloses that ANG enhances rRNA transcription through an epigenetic activation on rDNA promoter region, furthermore, we have screened a number of potential genes directly regulation by ANG.RNase-4and ANG were isolated at the same time, however, RNase-4biological functions is still poorly understood. We found that RNase-4and ANG have the same gene expression profiles by bioinformatic analysis, and RNase-4may be highly expressed in the tumor tissue. At the same time, our previous work also proved RNase-4protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress. We studied the role of RNase-4in tumorigenesis, and the preliminary results show that RNase-4is highly expressed in tumor tissues and has the dual functions of angiogenesis and enhance tumor growth. Therefore, RNase-4may be a new tumorogenesis factor, and as a potential target for cancer treatment.In summary, our thesis found that:1. ANG and RNase-4genes are regulated by tRNA cis-elements and CTCF;2. ANG enhances rRNA transcription an epigenetic activation on rDNA promoter region;3. genome-wide binding sites of ANG;4RNase-4is a novel tumorigenesis factor.