Search

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions. Disruption of one allele of the Sult2a8 gene in male HT mice resulted in losing ~ 50% of the 7α-OH sulfonating activity compared to wild-type (WT) control, but no significant change in female HT mice. Under the fed condition comparing the levels of hepatic and biliary bile acids as well as plasma/serum energy metabolites, HT mice displayed a profile similar to that of WT mice, suggesting that the Sult2a8-haplodeficient mice conducted normal energy metabolism. However, after 48-h fasting, a significant decrease in plasma cholesterol level was found in male HT mice but without any significant reduction in female HT mice. Of interest, in male Sult2a8-haplodeficient mice, an increase of the hepatic taurine-conjugated cholic acid level was noted but no noticeable change in other tested bile acids after fasting. Taken together, SULT2A8 is a male-specific and key hepatic SULT in metabolizing 7α-OH primary bile acids. During energy deprivation, SULT2A8 is required to maintain the bile acid and cholesterol metabolism, suggesting SULT is a potential therapeutic target for controlling metabolic diseases.

The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown. We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis. These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.

SummaryThe mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects β-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

The transcription factor Runx1 is a pivotal regulator of definitive hematopoiesis in mouse ontogeny. Vertebrate Runx1 is transcribed from 2 promoters, the distal P1 and proximal P2, which provide a paradigm of the complex transcriptional and translational control of Runx1 function. However, very little is known about the biologic relevance of alternative Runx1 promoter usage in definitive hematopoietic cell emergence. Here we report that both promoters are active at the very onset of definitive hematopoiesis, with a skewing toward the P2. Moreover, functional and morphologic analysis of a novel P1-null and an attenuated P2 mouse model revealed that although both promoters play important nonredundant roles in the emergence of definitive hematopoietic cells, the proximal P2 was most critically required for this. The nature of the observed phenotypes is indicative of a differential contribution of the P1 and P2 promoters to the control of overall Runx1 levels, where and when this is most critically required. In addition, the dynamic expression of P1-Runx1 and P2-Runx1 points at a requirement for Runx1 early in development, when the P2 is still the prevalent promoter in the emerging hemogenic endothelium and/or first committed hematopoietic cells.

This study investigated the effect of fosinopril (Fos), valsartan (Val), and combination of both drugs (Fos + Val) on the cardiac and renal expression of aquaporin-1 (AQP1) and aquaporin-2 (AQP2) in congestive heart failure (CHF). A rat model of CHF was created by ligating the left anterior descending coronary artery to induce acute myocardial infarction (AMI). Rats were treated by Fos, Val, or Fos + Val for 4 weeks. In renal medulla and cortex, AMI was associated with 2.2- and 1.8-fold increase in AQP2 mRNA expression when compared with Sham-operated rats (medulla: 23.6 +/- 2.8 vs. 52.3 +/- 8.7%; P0.001; cortex: 19.4 +/- 3.9 vs. 35.5 +/- 7.1%; P0.05). All the treatment regimens were able to normalize AQP2 transcription in the renal medulla (Fos, 19.9 +/- 4.9%; Val, 22.8 +/- 4.9%; Fos + Val, 20.1 +/- 5.1%; P=NS vs. Sham) and in the cortex (Fos, 21.2 +/- 6.7%; Val, 20.4 +/- 6.0%; Fos + Val, 18.9 +/- 7.5%; P=NS vs. Sham). Similarly, the AQP2 protein expression increased by 2.1-fold after CHF (P0.05), and was normalized by the treatment regimens (Sham, 0.57 +/- 0.19%; CHF, 1.22 +/- 0.45%; Fos, 0.39 +/- 0.36%; Val, 0.46 +/- 0.34%; Fos + Val, 0.36 +/- 0.15%; all P0.05 vs. CHF). These treatment regimens also prevented the increase in body weight as found in untreated CHF rats (analysis of variance P0.05). The renal and cardiac AQP1 gene and protein expressions were unaltered in CHF or by medical therapy. There was no observed cardiac AQP2 expression in all the study groups. Treatment with Fos, Val, or combination therapy was effective in preventing the upregulation of renal AQP2 gene and protein expressions in CHF rats caused by AMI.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

Ig genes undergo hypermutation with a nucleotide preference of A over T for mutation on the coding strand. As only with concomitant strand bias can such nucleotide bias be observed, Ig gene hypermutation is generally accepted as a strand-specific process, for which the mechanistic basis remains unknown. It has previously been shown that different non-Ig sequences replacing the LVJ region of an Ig transgene to various extents are targeted for hypermutation with similar mutation frequencies. However, the nucleotide bias characteristic of Ig hypermutation was not found in two of the three such sequences studied. To test whether it is the DNA sequences of the non-Ig substrates that determine the pattern of nucleotide bias in hypermutation or whether the LVJ sequence may contain element(s) that confer strand bias, we have added back all the replaced LVJ sequences to one of the transgenes, L(kappa)-Vgpt*, that expresses no strand bias in hypermutation and studied the outcome. The results show that the gpt sequence in the presence of the complete LVJ sequence hypermutates differently from the same sequence in L(kappa)-Vgpt* where 84% of the LVJ was replaced. The main difference is the resumption of strand bias characteristic of Ig hypermutation. Thus, whether or not a substrate sequence manifests strand bias in hypermutation is not inherently determined by the substrate DNA sequence. This indicates the presence of special element(s) within the LVJ that confer strand bias.