Research About The Nuclear Import Of KRAB Domain And The Function Of ZNF300during The Induced Differentiation Of K562Cells

As the major transcriptional factors, the zinc finger proteins play very important role during the regulation of RNA transcription. The zinc finger proteins can be divided into a number of different types based on different classification methods. There is a kind of zinc finger protein called KRAB zinc finger proteins which contain a very conservative KRAB domain in its N-terminus that account for roughly40%of the total zinc finger transcriptional factors. Thus the KRAB zinc finger proteins play very important role in all the life activities, but little is known about its exact function.Previously our lab cloned two novel zinc finger protein genes ZNF268and ZNF300from the cDNA library of the early human embryo. And we found that these two genes can be translated into the zinc finger proteins which contain the KRAB domain. The existing research data shows that the gene product of these two genes can be served as a transcription factor existing in the cells and have the function of transcriptional repression. Our earlier results suggested that these two genes may play an important role in the process of embryonic development, hemotopoiesis, and our previous immunohistochemical results also suggested that these two genes may be associated with the occurrence of leukemia. Despite such clues exist, the exact function and mechanism of these two genes during those processes is still unknown.The ZNF268gene encodes different protein products including ZNF268a and ZNF268b2. During our research, we found that there are great differences between ZNF268a and ZNF268b2in the subcellular localization. The ZNF268a which contains the KRAB domain and the zinc finger domain only localized in nuclear, while the ZNF268b2which consists of the zinc finger protein without the KRAB domain was found existed both in the nuclear and the cytoplasm. These results suggested that the KRAB domain might have the function of nuclear entrance. We also found that the cell distribution of ZNF268b2is result from its lack of the KRAB domain but not because of a nuclear export sequence existed in the ZNF268b2. We expressed the KRAB domain of ZNF268a, ZNF300and other zinc finger proteins together with GFP and tested the cellular distribution of these fusion proteins. The result showed that the phenomenon of KRAB domain imported into the nuclear is universal. Further study found that the ability of KRAB domain imported into the nuclear is closely related with the conservative sites of its acid amino sequence and depends on the interaction with its co-transcription factor KAP1. Due to the RBCC domain through which KAP1interacts with KRAB domain could not enter into the nuclear, so the mutation of RBCC domain resulted in a fine improvement of the ability of KRAB domain imported into the nuclear, while the overexpression of RBCC domain significantly inhibited the ability of the KRAB domain imported into the nuclear. We also found that the KRAB domain could not interact with the nuclear transporter Importin α/β directly, while they could form nuclear transport complex indirectly under the participation of KAP1. Based on these results and literature, we proposed the following model for explaining the ability of the KRAB domain imported into the nuclear, which is that KRAB directly interact with the RBCC domain of KAP1, whereas KAP1can directly interact with HP1which possesses an NLS, so under the participation of HP1which can recruit Importin α/β nuclear transporter together with KAP1and KRAB Zinc Finger proteins to form a nuclear import complex. Then the complex transport through the NPC into the nuclear.We know more about the function of ZNF268gene while little is known about ZNF300. Based on the previous studies which showed that ZNF300gene may be associated with the occurrence and development of leukemia, we recruited the human erythroleukemia cell line K562as the model to study the function of ZNF300gene in the process of its induced differentiation. The result showed that the expression of ZNF300gene in both mRNA level and protein level was significantly increased when the K562cells were induced differentiation into megakaryocyte or erythropoiesis, respectively. Then we constructed the stable transfectants in which the expression of ZNF300gene was significantly knockdown by RNAi which was mediated by the reconstructed lentivirus vectors. Based on these stable transfectants, we found that the ability of K562cells induced differentiation into megakaryocyte or erythropoiesis was significantly inhibited, while the cell proliferation experiments showed the knockdown of ZNF300expression could significantly promote the proliferation of K562cells. By cell cycle analysis we found that the G0/G1phase was significantly shortened and the S phase was remarkably elongated when ZNF300expression was significantly knockdown, which suggested that knockdown of ZNF300could promote the cell cycle shifting from G0/G1to S phase, then promote the cell proliferation. The molecular mechanism of the cell cycle change in K562cells caused by knockdown of ZNF300gene is probably by inhibiting the expression of tumor suppressor genes p15and p27, at the same time promoting the expression of PCNA. Therefore, we think that ZNF300gene inhibits the induced differentiation of K562cells by promoting cell proliferation.Above all, we found some new features and function of KRAB zinc fingers. Generally we believed that the nuclear transport of the KRAB zinc finger proteins relied on the NLS localized in the zinc finger domain of these proteins. Here we found that the KRAB domain of zinc finger proteins also possessed the ability to be transported through the NPC, and this ability could carry the GFP protein into the nuclear specifically. We also found that the expression of the KRAB zinc finger protein gene ZNF300was upregulated during the induced differentiation of K562cells. And when knockdowned the ZNF300gene expression, we found that the induced differentiation ability of K562cells were inhibited, while the cell proliferation was accelerated. When to study the molecular mechanism of these phenomenons, we found that the expression of some cell cycle regulatory proteins were significantly changed which resulted in the obvious changes of the cell cycle and this may affect the cell differentiation.