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Studies on the Expression and PU.1 Transcription Regulation of Human Zinc Finger Gene, ZNF300

Author: XuJunHua
Tutor: LiWenZuo
School: Wuhan University
Course: Microbiology
Keywords: KRAB-ZFPs ZNF300 Myeloblastic leukemia hematopoietic development PU.1
CLC: Q343
Type: PhD thesis
Year: 2009
Downloads: 21
Quote: 0
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Abstract


Haematopoietic development is an exactly regulated process that haematopoietic stem cells or haematopoietic progenitors differentiate into all kinds of functional haematopoietic cells. And transcription factors play an essential role in haematopoietic differentiation. One kind of transcription repression factors are KRAB zinc finger factors(KRAB-ZFPs), which kinds of genes are a large number of candidates for human diseases on the basis of chromosomal location. Recently, KRAB-ZFPs genes are found to play an important role in the regulation of morphogenesis and development. For example, expression of the KRAB-ZFPs HPF4, HTF10, and HTF34 is down-regulated during myeloid differentiation. The cDNA of KRAB-ZFPs gene, ZNF300, was originally cloned from an early human embryo and was found to encode a KRAB-ZFP with twelve C2H2 zinc finger motifs in the C-terminus. Interestingly, ZNF300 is a nuclear protein expressed in human tissues but not in tissues of lower mammals such as the mouse. Moreover, ZNF300 gene expression was reduced markedly when embryonic stem cells were induced to immature hepatocytes or embryoid bodies to neuroectoderm cells. Furthermore, immunohistochemistry assays revealed that high expression levels of ZNF300 were detected in 5-weeks’human embryonic heart and liver, and that the 5-weeks’human embryonic liver is one of the primary haematopoietic organs of the embryo. Furthermore, ZNF300 gene expression was found to be associated with 5q-syndrome, which is a distinct subtype of primary myelodysplastic syndrome (MDS), defined by the presence of an isolated interstitial deletion of chromosome 5ql3-q33. In this context, we should infer that ZNF300 gene is likely to play an important role in the hematopoietic development. Therefore, we investigate the role of ZNF300 in the process of myeloid differentiation.First, thirty-four bone marrow core biopsy specimens from adult patients with newly diagnosed and untreated AML and CML were evaluated. In situ immunohistochemistry assay demonstrated that ZNF300 gene expression is elevated in the pathological processses of myeloid leukemia, and it is likely that ZNF300 gene plays an important role in the pathogenesis of leukemia and myeloid development. Moreover, it suggests that ZNF300 is possiblely a good candidate of marker for AML and CML diagnosis.Then we studied the transcription regulation modes of ZNF300 gene. Hence we cloned the 5’flanking genomic DNA containing the putative ZNF300 gene promoter and analyzed its function. At first, primer extension assay not only mapped the major transcription start sites of ZNF300 (TG(+1)CGGAA, located at the G (+1) residue 1733 upstream of the ATG of the ZNF300 gene), but also revealed several minor sites. Followingly, deletion assays indicated that the DNA elements located between-624 to-333 bp was responsible for the promoter activity of ZNF300 gene. And TESS online program predicted that there were several hematopoietic transcriptional factor binding sites on the sequences, eg. C/EBPa, PU.1, c-Myb and AML1. Mutagenesis of myeloid-specific PU.1 binding sites (CTTCTC,-602 bp to-597 bp) but not the others significantly reduced ZNF300 promoter activity. Subsequently, gel mobility shift and chromatin immunoprecipitation assays confirmed that PU.1 bound to the ZNF300 promoter in vitro and in vivo. At the end, over-expression of PU.1 led to up-regulation of ZNF300 gene promoter activity in HL60 but not in HeLa and Jurkat cells, whereas silencing of PU.1 expression significantly reduced the promoter activity. These results demonstrate for the first time that human ZNF300 gene expression requires a myeloid-specific factor PU.1 binding site.At last, to further probe into the role of ZNF300 in the myeloid differentiation, in vitro indu(?)ed differentiation of HL60 was employed. Westernblot analysis demonstrated that ZNF300 expression markedly increased synchronously with the expression of PU.1 in HL-60 cells induced with DMSO or TPA. Scince PU.1 plays an important role, especially at the initial stage, in haematopoietic differentiation, it is likely that ZNF300 gene plays an important role in the myeloid differentiation.All together, these results demonstrate for the first time that human ZNF300 gene expressed spcifically in the bone marrow tissues of the AML and CML patients, and its expression elevated in the process of myeloid development. Moreover, ZNF300 gene expression requires a myeloid-specific factor PU.1 binding to its binding site (CTTCTC,-602bp to-597bp). Furthermore, in vitro induced differentiation of HL60 that ZNF300 expression markedly increased synchronously with the expression of PU.1. It suggests that ZNF300 is likely to play an important role in myeloid differentiation and pathological processs of myeloblastic leukemia. And ZNF300 gene is possiblely a good candidate of marker for AML and CML diagnosis and a potential target of medical treatment.

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