IDENTIFICATION OF PROTEINS INTERACTING WITH THE PROMOTER REGION OF KLF17 GENE IN GYMNOCYPRIS ECKLONI

The Qinghai-Xizang Plateau is a unique geographical unit with the highest elevation in the world and a natural laboratory for studying organism adaptation to extreme environment. Among the fundamental and abundant cell types in vertebrate life, erythrocytes play a vital role in sustaining vertebrates. Spatially and temporally coordinated regulatory networks and feedback mechanisms are essential for fish erythropoiesis and typical development. Fish species belonging to the subfamily Sohizothoraoinae, after a long period of adaptation to high temperature and low oxygen, have become one of the significant components of the present fish fauna of the Qinghai-Tibetan Plateau. In order to study the regulatory mechanism of erythrocyte development, we selected Gymnocypris eckloni, a representative species of the Sohizothoraoinae subfamily, due to its unique geographical distribution and life history responses. Based on previous research in the laboratory, the whole genome data of G. eckloni was used to identify the gene involved in erythroid development, namely KLF17, and to obtain the promoter sequence of KLF17. The KLF17 promoter DNA was used as bait to screen the total proteins from the kidney tissues of G. eckloni and identify the candidate binding proteins by LC-MS/MS. The identified candidate binding proteins of the KLF17 promoter were subjected to GO function enrichment, and KEGG pathway analysis by bioinformatics methods. The results showed that a total of 576 proteins were screened for specific binding to the KLF17 promoter region in G. eckloni kidney tissue. After removing proteins that could not be identified, there were 306 candidate binding proteins. These include eukaryotic translation initiation factor 2, cytochrome P450 enzymes, transferrin, protein-like metalloproteinases with type I platelet binding protein motifs, pyruvate dehydrogenase, erythrocyte membrane protein band 4.1 proteins, and other hematopoiesis-related proteins. Furthermore, GO functional enrichment analysis indicated that these proteins were involved in various biological functions, including cell growth, cell cycle, immune response, signaling and nucleic acid binding to transcription factors. KEGG pathway analysis showed that these proteins participated in several signaling pathways, such as amino acid metabolism pathway, apoptosis, PPAR signaling, Cytochrome P450 metabolism, HIF-1signaling, Hippo signaling and the neurotrophic factor pathway. The candidate binding proteins of the KLF17 promoter screened in this study mainly include eukaryotic translation initiation factor 2, cytochrome P450 enzymes and transferrin. Functional analyses revealed that they are involved in the HIF-1signaling pathway, which stimulates erythropoiesis and erythrocyte development through the regulation of hemoglobin and cadherin gene expression, iron metabolism, angiogenesis, glycolysis and other pathways. KLF17 is involved in regulating the interactions between haem, iron and globin proteins in erythrocytes at the transcriptional level, providing important information for studying the mechanism of erythrocyte differentiation. Meanwhile, transferrin, as a cross-regulator involved in the regulation of iron and oxygen, can promote the transport of iron in the blood and enhance the binding capacity of blood oxygen, which enables the organism to survive for a long time in a low-oxygen environment, which also shows that the mechanism of hypoxic adaptation in G. eckloni is related to transferrin downstream of HIF-1. In summary, the present study provide a solid foundation for future investigations into the interaction mechanism between this protein and KLF17. It also provide scientific data for dissecting the molecular mechanism underlying hematopoietic system development and adaptation to low-oxygen environment in native plateau fishes.