Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1ß is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

Direct and simultaneous identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by rapid multiplex nested PCR-ICT assay.

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application.

Functional interaction between nuclear matrix-associated HBXAP and NF-kappaB.

Hepatitis B virus X-associated protein (HBXAP) is a plant homeodomain (PHD) finger-containing protein implicated in transcription regulation. However, the underlying molecular mechanism remains to be defined. Here, we show that HBXAP represses NF-kappaB-mediated gene activation in a dose-dependent manner. Our results showed that HBXAP and NF-kappaB colocalize to the nuclear matrix with specific physical interaction between them. HBXAP may depend on its nuclear matrix localization for its repression of NF-kappaB-mediated gene repression. A specific nuclear matrix targeting sequence of HBXAP was identified. The sequence is included in a region encompassing amino acids 688-722 that could form a coiled-coil structure. The 18-amino acid stretch lies at the core of that structure. The present results showed that either the coiled-coil conformation or the PHD finger domain is crucial for the transcription repression activity of HBXAP on NF-kappaB-mediated gene activation. Taken together, our results suggest that HBXAP may function as a negative regulator for TNF-alpha-induced, NF-kappaB-mediated gene activation.

Transcriptional activation of C/EBPbeta gene by c-Jun and ATF2.

C/EBPbeta is one of the key transcription factors responsible for the induction of a wide array of genes. Like many proto-oncogenes and transcription factors, transcription of C/EBPbeta gene can be induced by multiple extracellular signals. Using nuclear extracts from lipopolysaccharide (LPS)-stimulated mouse liver, five trans-acting factor-binding motifs, URE1 (-376 to -352), URE2 (-253 to -223), URE3 (-220 to -190), URE4 (-123 to -103), and URE5 (-72 to -45) were identified by DNAse I footprinting assays. Competition and supershift analysis of the complexes formed at the URE2 and URE4 indicated that they contain CREB/ATF and AP-1 family factors. Furthermore, recombinant ATF2 and c-Jun proteins from mammalian and bacterial cells can bind to URE2 and URE4 but not URE1. Cotransfection experiments showed that ATF2 and c-Jun activate the C/EBPbeta gene expression cooperatively through URE2 and URE4, and this activation was greatly increased under the treatment of low concentration of anisomycin. During acute phase response, the phosphorylation of c-Jun and ATF2 was found to correlate with C/EBPbeta gene expression. Taken together, our results provide the evidences that both c-Jun and ATF2 are the regulators of C/EBPbeta gene.