Role In DNA Binding Activity Research Articles

Identification of a Novel ENU-Induced Mutation in Mouse Tbx1 Linked to Human DiGeorge Syndrome.

The patients with DiGeorge syndrome (DGS), caused by deletion containing dozens of genes in chromosome 22, often carry cardiovascular problem and hearing loss associated with chronic otitis media. Inside the deletion region, a transcription factor TBX1 was highly suspected. Furthermore, similar DGS phenotypes were found in the Tbx1 heterozygous knockout mice. Using ENU-induced mutagenesis and G1 dominant screening strategy, here we identified a nonsynonymous mutation p.W118R in T-box of TBX1, the DNA binding domain for transcription activity. The mutant mice showed deficiency of inner ear functions, including head tossing and circling, plus increased hearing threshold determined by audiometry. Therefore, our result further confirms the pathogenic basis of Tbx1 in DGS, points out the crucial role of DNA binding activity of TBX1 for the ear function, and provides additional animal model for studying the DGS disease mechanisms.

Substitutional analysis of the C-terminal domain of AbrB revealed its essential role in DNA-binding activity.

The global transition state regulator AbrB controls more than 100 genes of the Bacillus relatives and is known to interact with varying DNA-sequences. The DNA-binding domain of the AbrB-like proteins was proposed to be located exclusively within the amino-terminal ends. However, the recognition of DNA, and specificity of the binding mechanism, remains elusive still in view of highly differing recognition sites. Here we present a substitutional analysis to examine the role of the carboxy-terminal domain of AbrB from Bacillus subtilis and Bacillus amyloliquefaciens. Our results demonstrate that the carboxy-terminal domains of AbrB affect the DNA-binding properties of the tetrameric AbrB. Most likely, the C-termini are responsible for the cooperative character observed for AbrB interaction with some DNA targets like tycA and phyC.

The flexible loop L1 of the H3K4 demethylase JARID1B ARID domain has a crucial role in DNA-binding activity

JARID1B, a member of the JmjC demethylase family, has a crucial role in H3K4me3 demethylation. The ARID domain is a potential DNA-binding domain of JARID1B. Previous studies indicate that a GC-rich DNA motif is the specific target of the ARID domain. However, the details of the interaction between the ARID domain and duplex DNA require further study. Here, we utilized NMR spectroscopy to assign the backbone amino acids and mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to 1H– 15N correlation spectra revealed that the flexible loop L1 of ARID was the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrated that mutations on loop L1 strongly reduced the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms resulted in a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 made a major contribution to sustaining the DNA-binding ability of JARID1B ARID domain.

Gene expression patterns in the histopathological classification of epithelial ovarian cancer

The purpose of this study was to screen cancer-related genes and to identify histopathological gene expression patterns as potential biomarkers in human epithelial ovarian cancer (EOC). Fifty genes were screened by reverse-transcription polymerase chain reaction assay with cDNA from 83 EOC tissues and 48 normal ovarian specimens of ovarian cancer patients and evaluated by gel electrophoresis analysis. Twenty expressed genes were assessed by real-time relative-quantity (RQ)-PCR in 30 EOC specimens for gene signature study. Four genes, TAL2, EGF, ILF3 and UBE2I, were investigated for gene expression patterns in histopathological classification of EOC. RQ-value (Ct, ΔCt, ΔΔCt, RQ and gene expression plots) was generated by ABI 7500 Fast System SDS Software (version 1.4). SPSS 15.0 software was used for statistical analysis. Using real-time RQ-PCR, we found that TAL2, EGF, ILF3 and UBE2I demonstrated distinct expression patterns in histological types of epithelial ovarian cancer. The expression of ILF3 and UBE2I in tumors was significantly higher than in normal tissue, with extremely high expression in serous carcinomas compared to mucinous, endometrium and clear cell carcinomas. In addition, ILF3 and UBE2I were overexpressed in advanced stage and advanced grade ovarian cancer, compared to early stage or well-differentiated ovarian cancer. This is the first report of TAL2 and ILF3 expression in the normal human ovary and epithelial ovarian cancer. Our results indicate that overexpression of ILF3 and UBE2I in advanced stage and advanced grade suggest that these two genes may play an important role in tumorigenesis/tumor progression and pathological differentiation of the disease. Notably, ILF3 plays a role in DNA binding activity and transcriptional and post-transcriptional regulation; UBE2I is required in ubiquitination and sumoylation and is involved in DNA repair and apoptosis of cells. Further investigations to reveal the molecular mechanisms related to the activation of ILF3 and UBE2I in the development of EOC are warranted.

Gene expression patterns in the histopathological classification of epithelial ovarian cancer

The purpose of this study was to screen cancer-related genes and to identify histopathological gene expression patterns as potential biomarkers in human epithelial ovarian cancer (EOC). Fifty genes were screened by reverse-transcription polymerase chain reaction assay with cDNA from 83 EOC tissues and 48 normal ovarian specimens of ovarian cancer patients and evaluated by gel electrophoresis analysis. Twenty expressed genes were assessed by real-time relative-quantity (RQ)-PCR in 30 EOC specimens for gene signature study. Four genes, TAL2, EGF, ILF3 and UBE2I, were investigated for gene expression patterns in histopathological classification of EOC. RQ-value (Ct, ΔCt, ΔΔCt, RQ and gene expression plots) was generated by ABI 7500 Fast System SDS Software (version 1.4). SPSS 15.0 software was used for statistical analysis. Using real-time RQ-PCR, we found that TAL2, EGF, ILF3 and UBE2I demonstrated distinct expression patterns in histological types of epithelial ovarian cancer. The expression of ILF3 and UBE2I in tumors was significantly higher than in normal tissue, with extremely high expression in serous carcinomas compared to mucinous, endometrium and clear cell carcinomas. In addition, ILF3 and UBE2I were overexpressed in advanced stage and advanced grade ovarian cancer, compared to early stage or well-differentiated ovarian cancer. This is the first report of TAL2 and ILF3 expression in the normal human ovary and epithelial ovarian cancer. Our results indicate that overexpression of ILF3 and UBE2I in advanced stage and advanced grade suggest that these two genes may play an important role in tumorigenesis/tumor progression and pathological differentiation of the disease. Notably, ILF3 plays a role in DNA binding activity and transcriptional and post-transcriptional regulation; UBE2I is required in ubiquitination and sumoylation and is involved in DNA repair and apoptosis of cells. Further investigations to reveal the molecular mechanisms related to the activation of ILF3 and UBE2I in the development of EOC are warranted.

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, ɛ and γ. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer–dimer dissociation constant from its normal value of 120–150 nM.

Possible association of thioredoxin and p53 in breast cancer

Expression of thioredoxin (TRX), a dithiol-reducing enzyme, and mutations of p53 have been detected in various cancer tissues. We recently reported that TRX-dependent redox regulation plays a crucial role in DNA binding activity of p53. In this study, we investigated the possibility of functional association between TRX and p53 in breast cancer. First, we examined the expression of TRX and mutated p53 in 100 primary breast cancer tissues by immunohistochemistry. Expression of TRX was detected in cases of 84/100 (84%) and expression of p53, which means existence of mutated p53, in cases of 63/100 (63%). TRX positive cases was 89% (56/63) in mutant p53 positive cases. Next, we examined the expression of TRX and p53 in breast cancer cell line MCF-7 cells after CDDP treatment or irradiation. CDDP treatment or irradiation augmented expression of TRX and p53 in MCF-7 cells by western blotting. Immunofluorescence cell analysis by confocal microscopy showed that CDDP treatment induced translocation of TRX into nuclei. These results suggest the possible association of TRX with p53-dependent function including DNA repair in breast cancer.