Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity

<p>Abstract</p> <p>Background</p> <p>In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before.</p> <p>Results</p> <p>We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (<it>N </it>= 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (<it>N </it>= 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters.</p> <p>Conclusions</p> <p>We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity.</p

Analysis of the Serum Bile Acid Composition for Differential Diagnosis in Patients with Liver Disease

Objectives. We determined the serum bile acid (BA) composition in patients with liver diseases and healthy volunteers to investigate the relationship between the etiologies of liver disease and BA metabolism. Material and Methods. Sera from 150 patients with liver diseases and 46 healthy volunteers were obtained. The serum concentrations of the 16 different BAs were determined according to the LC-MS/MS method and were compared between the different liver diseases. Results. A total of 150 subjects, including patients with hepatitis C virus (HCV) (n=44), hepatitis B virus (HBV) (n=23), alcoholic liver disease (ALD) (n=21), biliary tract disease (n=20), nonalcoholic fatty liver disease (NAFLD) (n=13), and other liver diseases (n=29), were recruited. The levels of UDCA and GUDCA were significantly higher in the ALD group, and the levels of DCA and UDCA were significantly lower in the biliary tract diseases group than in viral hepatitis group. In the UDCA therapy (−) subgroup, a significantly lower level of TLCA was observed in the ALD group, with lower levels of CDCA, DCA, and GLCA noted in biliary tract diseases group compared to viral hepatitis group. Conclusions. Analysis of the BA composition may be useful for differential diagnosis in liver disease

Improved Derivation Efficiency and Pluripotency of Stem Cells from the Refractory Inbred C57BL/6 Mouse Strain by Small Molecules

<div><p>The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK), and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their <i>in vitro</i> characteristics, <i>in vitro</i> differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem (iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.</p></div

Embryoid body (EB) formation and differentiation of C57BL/6 ntES cells after withdrawal of LIF and SC1 from SC1-supplemented medium.

<p>A, B. EBs formation in microwells by C57BL/6 ntES cells 24 h after LIF and SC1 withdraw. C. Morphology of enlarged EBs and cystic EBs after 7 days of culture in suspension. D. RT-PCR analysis of day 10 EBs for markers of the three germ layers.</p

Characterization of C57BL/6 ntES cells cultured in different media.

<p>A. Alkaline phosphatase activity of ntES colonies cultured in different ES media. Scale bar = 50 µm. B. SSEA-1 expression profiles of ntES cells cultured in different ES media and characterized by flow cytometry. Pink lines refer to SSEA-1 profiles; green lines refer to negative isotype control. C. Immunocytochemistry detection of ES specific markers Oct4 (green) and Nanog (red) of C57BL/6 ntES cells cultured in different ES media. Scale bar = 25 µm. D. Immunocytochemistry detection of ES specific markers Sox2 (green) and Nanog (red) of C57BL/6 ntES cells cultured in different ES media. E. Effect of small molecules on ES specific marker expression level determined by real-time RT-PCR. ^a,b,cValues within grouped bar chart with different superscripts differ, <i>P</i><0.05.</p

Alteration in morphology and cell cycle distribution of C57BL/6 nuclear transfer ES (ntES) cells cultured in different media.

<p>A, B, C. Slight differences of C57BL/6 ntES colonies cultured in control ES medium (A), PD- (B) and SC1-supplemented media (C). Scale bar = 50 µm. Arrow indicates the slight differentiation morphology in control ES medium. D, E, F. Histograms of cell cycle distribution of C57BL/6 ntES cells in the present in control ES medium (D), PD- (E) and SC1-supplemented media (F).</p

Morphology of newly derived C57BL/6 fES cells cultured in different ES media.

<p>A. Expanded blastocysts of C57BL/6 mouse. B, C. Embryonic outgrowth from blastocyst after 2 (B) and 9 days (C) of culture on MEF feeder cells, respectively. D, E, F. ES-like colonies appeared after trypsinization in control ES medium (D), PD- (E) and SC1-supplemented media (F). G. Differentiated colony appeared after trypsinization in control ES medium. Scale bar = 50 µm.</p

Derivation of ES cell lines in different ES culture media from fertilized embryos of the C57BL/6 strain.

<p>*the other 6 outgrowths appeared differentiated after trypsinization.</p>a,b<p>Values within columns with different superscripts differ, p<0.05.</p><p>Derivation of ES cell lines in different ES culture media from fertilized embryos of the C57BL/6 strain.</p