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In β-thalassemia, free α-globin chains are unstable and tend to aggregate or degrade, releasing toxic heme, porphyrins and iron, which produce reactive oxygen species (ROS). α-Hemoglobin-stabilizing protein (AHSP) is a potential modifier of β-thalassemia due to its ability to escort free α-globin and inhibit the cellular production of ROS. The influence of AHSP on the redox equilibrium raises the question of whether AHSP expression is regulated by components of ROS signaling pathways and/or canonical redox proteins. Here, we report that AHSP expression in K562 cells could be stimulated by NFE2-related factor 2 (Nrf2) and its agonist tert-butylhydroquinone (tBHQ). This tBHQ-induced increase in AHSP expression was also observed in Ter119+ mouse erythroblasts at each individual stage during terminal erythroid differentiation. We further report that the AHSP level was elevated in α-globin-overexpressing K562 cells and staged erythroblasts from βIVS-2-654 thalassemic mice. tBHQ treatment partially alleviated, whereas Nrf2 or AHSP knockdown exacerbated, α-globin precipitation and ROS production in fetal liver-derived thalassemic erythroid cells. MafG and Nrf2 occupancy at the MARE-1 site downstream of the AHSP transcription start site was detected in K562 cells. Finally, we show that MafG facilitated the activation of the AHSP gene in K562 cells by Nrf2. Our results demonstrate Nrf2-mediated feedback regulation of AHSP in response to excess α-globin, as occurs in β-thalassemia.

The oncogenic mechanisms of overnutrition, a confirmed independent cancer risk factor, remain poorly understood. Herein, we report that enoyl-CoA hydratase-1 (ECHS1), the enzyme involved in the oxidation of fatty acids (FAs) and branched-chain amino acids (BCAAs), senses nutrients and promotes mTOR activation and apoptotic resistance. Nutrients-promoted acetylation of lys101 of ECHS1 impedes ECHS1 activity by impairing enoyl-CoA binding, promoting ECHS1 degradation and blocking its mitochondrial translocation through inducing ubiquitination. As a result, nutrients induce the accumulation of BCAAs and FAs that activate mTOR signaling and stimulate apoptosis, respectively. The latter was overcome by selection of BCL-2 overexpressing cells under overnutrition conditions. The oncogenic effects of nutrients were reversed by SIRT3, which deacetylates lys101 acetylation. Severely decreased ECHS1, accumulation of BCAAs and FAs, activation of mTOR and overexpression of BCL-2 were observed in cancer tissues from metabolic organs. Our results identified ECHS1, a nutrients-sensing protein that transforms nutrient signals into oncogenic signals., Overnutrition has been linked to increased risk of cancer. Here, the authors show that exceeding nutrients suppress Enoyl-CoA hydratase-1 (ECHS1) activity by inducing its acetylation resulting in accumulation of fatty acids and branched-chain amino acids and oncogenic mTOR activation.

Mitofilin was originally described as a heart muscle protein because of its abundance in the heart tissue; however, its function in the heart is still to be elucidated. Thus, this study aims at investigating the role of mitofilin in the heart in response to hypertrophic stimuli.In this study, a significant increase in mitofilin expression was observed in the hearts of patients with hypertrophic cardiomyopathy. Transgenic (TG) mice with cardiomyocyte-specific overexpression of mitofilin were generated, and cardiac hypertrophy was introduced by transverse aortic constriction (TAC) or chronic infusion of isoproterenol (ISO). In TG mice overexpressing mitofilin, the level of cardiac hypertrophy was significantly greater than that in wild-type (WT) mice after TAC and ISO stimulation. A detailed analysis showed that compared with WT mice, the level of reactive oxygen species was increased after TAC and ISO induction and mitochondrial oxidative phosphorylation (OXPHOS) activity in the TG hearts was lower. These alterations may contribute to the aggravated cardiac hypertrophy observed in response to TAC and ISO stimulation.Over-expression of mitofilin promotes cardiac hypertrophy under pathological conditions both in vivo and in vitro.Mitofilin, a mitochondria protein, is shown to be related to cardiac hypertrophy for the first time, which enhances our understanding of the role of mitochondria in cardiac hypertrophy.

The homeodomain transcription factor Nkx2.5/Csx is critically essential for heart specification, morphogenesis, and homeostasis. Acetylation/deacetylation is important for the localization, stability and activation of transcription factors. It remains unknown how Nkx2.5 is deacetylated and how Nkx2.5 acetylation determines its activity. In this study, we provide evidence that the NAD+-dependent class III protein deacetylase SIRT1 deacetylates Nkx2.5 in cardiomyocytes and represses the transcriptional activity of Nkx2.5. We show that SIRT1 interacts with the C-terminus of Nkx2.5 and deacetylates Nkx2.5 at lysine 182 in the homeodomain. The mutation of Nkx2.5 at lysine 182 reduces its transcriptional activity. Furthermore, SIRT1 inhibits the transcriptional activity of Nkx2.5 and represses the expression of its target genes partly by reducing Nkx2.5 binding to its co-factors, including SRF and TBX5. Taken together, these findings demonstrate that SIRT1 deacetylates Nkx2.5 and inhibits the transcriptional activity of Nkx2.5.

Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology. However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog of human FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restricted to the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression of the cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulated expression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns at the 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domain in embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes in the number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause between heartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals. Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed the expression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthase inhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterations in the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RA signaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducing the number of cardiac cells.

Aging is one of the greatest risk factors in vascular diseases (VDs). During aging, there are structural and functional changes in the vasculature, including dilated lumen, altered intimal-medial thickness (IMT), vascular stiffness, endothelial dysfunction, increased endothelial apoptosis, matrix metalloproteinase (MMP) dysregulation, increased expression of inflammatory molecules, aggravated oxidative stress and shortened telomere length. These changes leave the body more susceptible to primary hypertension, stroke and coronary artery disease. Molecules that suppress these age-related changes would provide an excellent medical intervention for VDs. Mammalian Sir2 (SIRT1, a NAD+-dependent deacetylase), previously shown to extend the lifespan of lower organisms, is a promising target molecule to influence some aspects of vascular aging. In this review, we summarized roles of SIRT1 in various pathophysiological processes of vascular aging and proposed that SIRT1 and its activators can become novel therapeutic targets for age-related VDs.

The adeno-associated virus (AAV) genome can be stably integrated into the AAVS1 region of human chromosome 19 (19q13.4-qter) with the assistance of Rep68/78 protein. In the current models of AAV integration in a locus-specific manner, the foreign genes were randomly inserted into the AAVS1 region, which contains several functional genes. As random integration in this region may lead to insertion mutations and disrupt normal gene expression or critical signaling pathways of the host cells, it is necessary to find a precise insertion site in the AAVS1 region. Homologous recombination is the most accurate and versatile mechanism for such site-specific integration. To investigate site-specific integration in the AAVS1 region, a targeted vector containing two homologous arms derived from AAVS1 and a reporter gene was transfected into HeLa cells with or without Rep68/78 mRNA. The results indicated that transient expression of Rep68/78 in HeLa cells improved integration of the gene of interest at the AAVS1 locus in a site-specific manner. Compared with locus-specific integration reported in previous studies, site-specific integration may minimize the risk associated with random DNA integration in the AAVS1 region, which might be helpful for gene therapy.

Matrix attachment region (MAR)-binding protein (MARBP) has profound influence on gene transcriptional control by tethering genes to the nuclear scaffold. MARBP SATB2 is recently known as a versatile regulator functioning in the differentiation of multiple cell types including embryonic stem cells, osteoblasts and immunocytes. Roles of SATB2 in erythroid cells and its working mechanism in orchestrating target gene expression are largely unexplored. We show here that SATB2 is expressed in erythroid cells and activates γ-globin genes by binding to MARs in their promoters and recruiting histone acetylase PCAF. Further analysis in higher-order chromatin structure shows that SATB2 affects physical proximity of human (G)γ- and (A)γ-globin promoters via self-association. We also found that SATB2 interacts with SATB1, which specifically activates ε-globin gene expression. Our results establish SATB2 as a novel γ-globin gene regulator and provide a glimpse of the differential and cooperative roles of SATB family proteins in modulating clustered genes transcription and mediating higher-order chromatin structures.

The c-Myc oncoprotein plays critical roles in multiple biological processes by controlling cell proliferation, apoptosis, differentiation, and metabolism. Especially, c-Myc is frequently overexpressed in many human cancers and widely involved in tumorigenesis. However, how the post-translational modifications, especially acetylation of c-Myc, contribute to its activity in the leukemia cells remains largely unknown. Sirt1, a NAD-dependent class III histone deacetylase, has a paradoxical role in tumorigenesis by deacetylating several transcription factors, including p53, E2F1 and forkhead proteins. In this study, we show that Sirt1 interacts physically with the C-terminus of c-Myc and deacetylates c-Myc both in vitro and in vivo. Moreover, the deacetylation of c-Myc by Sirt1 promotes its association with Max, a partner essential for its activation, thereby facilitating c-Myc transactivation activity on hTERT promoter. Finally, inhibition of endogenous Sirt1 in K562 cells by either RNAi or its inhibitor NAM causes the overall decrease of c-Myc target genes expression, including hTERT, cyclinD2 and LDHA, which further suppress cell proliferation and arrest cell cycle at G1/S phase. Thus, our results demonstrate the positive effect of Sirt1 on c-Myc activity by efficiently enhancing c-Myc/Max association in human leukemia cell line K562, suggesting a potential role of Sirt1 in tumorigenesis.

To verify the regulation of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT 2), which is associated with cholesterol metabolism, by saturated fatty acids (SFAs).Palmitic acid (PA), the most abundant saturated fatty acid in plasma, and oleic acid (OA), a widely distributed unsaturated fatty acid, were used to treat hepatic cells HepG2, HuH7, and mouse primary hepatocytes. In addition, PA at different concentrations and PA treatment at different durations were applied in HepG2 cells. In in vivo experiment, three-month male C57/BL6 mice were fed with control diet and SFA diet containing hydrogenated coconut oil rich of SFAs. The mRNA level of ACAT2 in those hepatic cells and the mouse livers was detected with real-time polymerase chain reaction (PCR).In the three types of hepatic cells treated with PA, that SFA induced significant increase of ACAT2 expression (Pü0.01), whereas treatment with OA showed no significant effect. That effect of PA was noticed gradually rising along with the increase of PA concentration and the extension of PA treatment duration (both Pü0.05). SFA diet feeding in mice resulted in a short-term and transient increase of ACAT2 expression in vivo, with a peak level appearing in the mice fed with SFA diet for two days (Pü0.05).SFA may regulate ACAT2 expression in human and mouse hepatic cells and in mouse livers.

Glioma-specific transcription of tumor-killing genes has been exploited as a promising gene therapeutic modality in glioma patients. Musashi1 (Msi1) and GFAP gene promoters are both cancer-specific promoters. Optimized HIF-binding site (optHBS) sequence was newly found as efficient as EPO HREs used as enhancer in cancer gene therapy. We constructed 4optHBS-Msi1/GFAP promoters and tested their ability to mediate BAX expression to induce apoptosis in glioma cell lines. Our results demonstrated that 4optHBS-Msi1/GFAP promoters are apparently strong and glioma-selective promoters with potential application in targeted glioma gene therapy, and 4optHBS-Msi1/GFAPBAXa are valuable tools for glioma gene therapy.

SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E(2) (PGE(2)) production of peritoneal macrophages (pMPhis). pMPhis with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE(2). Furthermore, SIRT1 protein level was up-regulated in CR mouse pMPhis, whereas elevated SIRT1 decreased COX-2 expression and improved PGE(2)-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function.

The nuclear location and relocation of genes play crucial regulatory roles in gene expression. SATB1, a MAR-binding protein, has been found to regulate beta-like globin genes through chromatin remodeling. In this study, we generated K562 cells over-expressing wild-type or nuclear matrix targeting sequences (NMTS)-deficient SATB1 and found that like wild-type SATB1, NMTS-deficient SATB1 induces out loop of beta-globin cluster from its chromosome territory (CT), while it is unable to associate the cluster with the nuclear matrix as wild-type SATB1 does and had no regulatory functions to the beta-globin cluster. Besides, our data showed that the transacting factor occupancies and chromatin modifications at beta-globin cluster were differentially affected by wild-type and NMTS-deficient SATB1. These results indicate that SATB1 regulates beta-like globin genes at the nuclear level interlaced with chromatin and DNA level, and emphasize the nuclear matrix binding activity of SATB1 to its regulatory function.

The apolipoprotein (apo) AI/CIII/AIV/AV cluster genes are expressed at different levels in the liver and intestine. The apoCIII enhancer, a common regulatory element, regulates the tissue-specific expression of apoAI, apoCIII, and apoAIV but not apoAV. To study this regulation at the chromatin level, the histone modifications and intergenic transcription in the human apoAI/CIII/AIV/AV cluster were investigated in HepG2 and Caco-2 cells and in the livers of transgenic mice carrying the human gene cluster constructs with or without the apoCIII enhancer. We found that both the promoters and the intergenic regions of the apoAI/CIII/AIV genes were hyperacetylated and formed an open subdomain that did not include the apoAV gene. Hepatic and intestinal intergenic transcripts were identified to transcribe bidirectionally with strand preferences along the cluster. The deletion of the apoCIII enhancer influenced both histone modification and intergenic transcription in the apoAI/CIII/AIV gene region. These results demonstrate that the apoCIII enhancer contributes to the maintenance of an active chromatin subdomain of the apoAI/CIII/AIV genes, but not apoAV.

Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse alpha-globin gene cluster and proved the extensive interaction between HS26 and alpha-globin genes. In addition, we have identified a candidate alpha1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.

Nonsense-mediated RNA decay (NMD), an mRNA quality control mechanism, triggers degradation of mRNAs that contain premature termination codon (PTC) within their coding regions. NMD is a relatively conservative process that involves many trans-acting factors. The key domains for their function in NMD are conserved in evolution. These trans-acting factors are classified as different groups by their function in NMD. In addition, the mRNP formation is dynamic in NMD process because of sequential recruitment and interaction of these factors. To gain an insight into the mechanism of NMD, we dissect the mechanism of NMD based on the information on the structure, the regulation and interaction of these factors.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA (shRNA) transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.

Higher eukaryote contains several hundreds of different cell types, each with a distinctive set of property defined by a unique gene expression pattern, even though every cell (with minor exception) shares the common genome. During cellular differentiation, the committed gene expression pattern is set up and propagated through numerous cell divisions. Therefore, cells must have evolved some elegant and inherent mechanisms to remember their expression states for the requirement of the stability of differentiation and development. Here we speculate a hypothetically cellular memory mechanism. In this hypothesis, the cell-cell variation during cellular differentiation may result from the inherent stochastic gene expression. The evolution of histone and distant regulatory sequences change the parameters of expression stochasticity. S-phase-dependent gene activation and epigenetic marks on chromatin provide means to discriminate transcriptionally active and repressive states. Eventually, mitotic memory mechanisms have been developed through which these expression states are transmitted through numerous cell divisions.

Eukaryotic genomes are packaged into a dynamic hierarchy chromatin structure. In such a particular context, the transition from a repressed compacted chromatin to a rather extended fiber is necessary for transcription. The chromatin opening includes three events, the initial factor getting access to nucleosome DNA, local chromatin opening mediated by activator/coactivator, and transcription associated with extensive chromatin opening. Chromatin dynamics, which is DNA sequence dependent, and also occurs in condensed fiber, provides the opportunity for activators binding to DNA. Coactivators recruited by the activator open the chromatin locally. However, it appears that genes adopt distinct chromatin opening mechanisms according to whether the gene is induced expression, developmental and tissue-specific expression, or constitutive expression. In contrast to transcription initiation-related local chromatin opening, large scale of chromatin opening is associated with a functional enhancer as well as high transcription rate. How the transcription initiated from an enhancer or enhancer like modules, i.e. intergenic transcription, conducts the extensive chromatin opening is discussed. A model for long-range interaction that non-coding transcripts from enhancers may promote efficient communication with promoters is proposed.

RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21-23 nucleotides synthesized in vitro or expressed by DNA-based vector. However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo. Virus vector-mediated RNAi provides an alternative to transfection. In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult beta-globin gene in MEL cells. The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy.

microRNAs (miRNAs) are a newly identified and surprisingly large class of endogenous tiny regulatory RNAs. They exhibit various expressional patterns and are highly conserved across species. Recently, several regulatory targets of miRNAs have been predicted. Functional analysis of the potential targets indicated that miRNAs may be involved in a wide range of pivotally biological events. The nature of miRNAs and their intersection with small interfering RNAs endow them with many regulatory advantages over proteins and make them a potent and novel means to regulate gene expression at almost all levels. Here we argue that miRNAs are key participants in gene regulatory network.

β-Thalassemia is the most common single gene disorder in the world, which is caused by the imbalance between α-globin chain and β-globin chain synthesis. Several medicines, such as 5-azacytidine, hydroxyurea, cytarabine, vinblatine, butyrate, and myleran, have been shown to be able to reactivate γ-globin chain synthesis during the adult stage, and some of them (5-azacytidine, hydroxyurea, myleran, and butyrate) have been used clinically to treat thalas semia and sickle cell disease. Much research efforts are focusing on the determination of the underlying mechanisms of medicine action. In this experiment, as an effort to probe the underlying mechanism of medicine action, we used ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the DNA-protein interaction at critical erythroid regulatory elements after hydroxyurea or myleran administration to mice. Our results showed that the patterns of in vivo footprints at both the hypersensitive site 2 of the locus control region and the β-globin gene promoter were changed after medicine treatment. We proposed based on these results that the medicines' administration might result in a change in the interaction between trans-acting factors and cis-acting elements at these regions. These changes might influence the assembly of the transcription complex and, lastly, influence the expression of the β-globin gene.Key words: hydroxyurea, in vivo footprinting, ligation-mediated PCR, LCR, β-globin.

Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.

In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.Key words: gene expression regulation, bacterial artificial chromosome, alpha- and beta-globin gene clusters.

Isolation of regulatory DNA fragments is the basis of the identification of DNA binding proteins and the study of the regulation of gene expression. Presently there is a lack of efficient methods to broadly isolate and identify DNA regulatory fragments. We developed an efficient method to isolate regulatory DNA sequences from both genome and bacterial artificial chromosome (BAC) based on electrophoretic mobility shift assay and PCR techniques without purified transcription factors. Twenty-nine DNA fragments were isolated from human genome and 24 from BAC DNA containing human apolipoprotein AI gene cluster. Transient transfection assay showed that some fragments could enhance the transcription of reporter gene.

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD+-dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming. Stem Cells 2015;33:2135–2147

Objective: To establish a cell-based drug screening system for discovering up-regulators of SIRT1 and confirmed their biological activities in rat aortic VSMCs. Methods and Results: A stable transf...

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA–protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA–protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor – DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the β-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic ε-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal β-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of β-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.Key words: β-globin gene, hypersensitive site, phylogenetic footprint, differential phylogenetic footprint, in vivo footprinting, developmental regulation.

Promyelocytic leukemia protein (PML) has been implicated as a participant in multiple cellular processes including senescence, apoptosis, proliferation, and differentiation. Studies of PML function in hematopoietic differentiation previously focused principally on its myeloid activities and also indicated that PML is involved in erythroid colony formation. However, the exact role that PML plays in erythropoiesis is essentially unknown. In this report, we found that PML4, a specific PML isoform expressed in erythroid cells, promotes endogenous erythroid genes expression in K562 and primary human erythroid cells. We show that the PML4 effect is GATA binding protein 1 (GATA-1) dependent using GATA-1 knockout/rescued G1E/G1E-ER4 cells. PML4, but not other detected PML isoforms, directly interacts with GATA-1 and can recruit it into PML nuclear bodies. Furthermore, PML4 facilitates GATA-1 trans-activation activity in an interaction-dependent manner. Finally, we present evidence that PML4 enhances GATA-1 occupancy within the globin gene cluster and stimulates cooperation between GATA-1 and its coactivator p300. These results demonstrate that PML4 is an important regulator of GATA-1 and participates in erythroid differention by enhancing GATA-1 trans-activation activity.

Using ligation-mediated PCR and in vivo footprinting methods to study the status of DNA-protein interaction at hypersensitive site 2 of locus control region and βmaj promoter of erythroid cells of fetal liver and adult bone marrow, we found that during different developmental periods, the status of DNA-protein interaction at both hypersensitive site 2 and βmaj promoter changed significantly, and indicated that locus control region might function through a looping mechanism to regulate the expression of downstream genes, and that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β-globin gene cluster participate in the regulation of developmental specificity. J. Cell. Biochem. 76:376–385, 2000. © 2000 Wiley-Liss, Inc.

Accumulating evidence indicates that oxidative stress can occur through overproduction of reactive oxygen species (ROS) and/or reduced anti-oxidant potentials under pathophysiological conditions and plays an important role in the development of cardiovascular diseases (CVDs). Adapter protein p66Shc has the property to directly stimulate mitochondrial ROS generation by an oxidoreductase activity. A growing body of evidence implies that p66Shc plays a critical role in the pathophysiology of age-related vascular diseases. Silent mating type information regulator 2 homolog 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase (HDAC), has also been implicated in protection against vascular aging and age-related vascular diseases. Recently, we demonstrated that SIRT1 protects blood vessels from hyperglycemia-induced endothelial dysfunction through a novel mechanism involving the downregulation of p66Shc expression. In this review, we discuss the cross-talk between these two longevity genes as a mechanism of preventing vascular diseases by involving anti-oxidative stress responses and inhibiting endothelial senescence.

Mitochondria amplify caspase-dependent apoptosis by releasing proapoptotic proteins, especially cytochrome c. This process is accompanied by mitochondrial cristae remodeling. Our studies demonstrated that mitofilin, a mitochondrial inner membrane protein, acted as a cristae controller to regulate cytochrome c release during apoptosis. Knockdown of mitofilin in HeLa cells with RNAi led to fragmentation of the mitochondrial network and disorganization of the cristae. Mitofilin-deficient cells showed cytochrome c redistribution between mitochondrial cristae and the intermembrane space (IMS) upon intrinsic apoptotic stimuli. In vitro cytochrome c release experiments further confirmed that, compared with the control group, tBid treatment led to an increase in cytochrome c release from mitofilin-deficient mitochondria. Furthermore, the cells with mitofilin knockdown were more prone to apoptosis by accelerating cytochrome c release upon the intrinsic apoptotic stimuli than controls. Moreover, mitofilin deficiency did not interfere with the activation of proapoptotic member Bax upon intrinsic apoptotic stimuli. Thus, mitofilin distinctly functions in cristae remodeling and controls cytochrome c release during apoptosis.

Myocardial infarction (MI) is a restrictive phenotype of coronary artery disease. To date, a group of genes and genetic loci have been associated to MI. However, the genetic causes and underlying molecular mechanisms for MI remain largely unknown. SIRT1, one of highly conserved NAD-dependent class III deacetylases, has been involved in several cellular processes and implicated in human diseases. Autophagy is one of major cellular degradative pathways, which plays important roles in lipid metabolism. Recent studies have shown that SIRT1 deacetylates autophagy-related genes, and the expressions of autophagic genes are altered in MI patients. Accordingly, we hypothesized that SIRT1 may be linked to the MI pathogenesis. In this study, the SIRT1 gene promoter were genetically analyzed in large cohorts of MI patients (n = 327) and controls (n = 358). The results showed that six single-nucleotide polymorphisms and 14 sequence variants were identified. Among these, five novel heterozygous variants (g.69643743Ins, g.69643840Ins, g.69643903G > C, g.69644235G > C and g.69644353G > T) and one single-nucleotide polymorphism (rs35706870) were identified in MI patients, but in none of controls. Moreover, five novel heterozygous variants (g.69643672G > A, g.69644226C > T, g.69644278A > G, g.69644408G > A and g.69644408G > T) were only found in controls. The rest variants were found in MI patients and controls with similar frequencies. Taken together, the variants identified in MI patients may alter the transcriptional activities of SIRT1 gene promoter, which may change SIRT1 levels, contributing to the MI pathogenesis as a risk factor.

Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) is important for cholesterol ester synthesis and secretion. A previous study revealed that ACAT2 gene promoter activity was upregulated by hepatocyte nuclear factor 4α (HNF4α) through two sites around -247 and -311 of ACAT2 gene promoter. Here, we identified two novel cis-elements, site I (-1006 to -898) and site II (-38 to -29), which are important for HNF4α effect. In HepG2 cells, mutation of site I decreased ACAT2 gene promoter activity to one-fifth of that of the wild type, while mutation of site II reduced promoter activity to less than one-tenth of that of the wild type. In 293T cells, mutation of these two cis-elements profoundly impaired the HNF4α induction effect. When either of these two elements was inserted into pGL3-promoter, HNF4α induced promoter activity through the inserted element, while mutation of the element impaired HNF4α induction effect. In electrophoretic mobility shift assay and chromatin immunoprecipitation experiment, HNF4α bound to these two elements. Thus, the two cis-elements are important for HNF4α effect on ACAT2 gene transcription. We also showed that HNF4α positively regulates ACAT2 gene expression at mRNA level. Overexpression of HNF4α increased ACAT2 expression, whereas knockdown of HNF4α decreased ACAT2 expression. Peroxisome proliferator-activated receptor gamma coactivator 1α (PCG1α), a coactivator of HNF4α, increased ACAT2 expression, while small heterodimer partner (SHP), a corepressor of HNF4α, decreased ACAT2 expression. These results provide more insights into transcriptional regulation of ACAT2 expression.

The paraoxonase (PON) gene cluster contains three adjacent gene members, PON1, PON2, and PON3. Originating from the same fungus lactonase precursor, all of the three PON genes share high sequence identity and a similar β propeller protein structure. PON1 and PON3 are primarily expressed in the liver and secreted into the serum upon expression, whereas PON2 is ubiquitously expressed and remains inside the cell. Each PON member has high catalytic activity toward corresponding artificial organophosphate, and all exhibit activities to lactones. Therefore, all three members of the family are regarded as lactonases. Under physiological conditions, they act to degrade metabolites of polyunsaturated fatty acids and homocysteine (Hcy) thiolactone, among other compounds. By detoxifying both oxidized low-density lipoprotein and Hcy thiolactone, PONs protect against atherosclerosis and coronary artery diseases, as has been illustrated by many types of in vitro and in vivo experimental evidence. Clinical observations focusing on gene polymorphisms also indicate that PON1, PON2, and PON3 are protective against coronary artery disease. Many other conditions, such as diabetes, metabolic syndrome, and aging, have been shown to relate to PONs. The abundance and/or activity of PONs can be regulated by lipoproteins and their metabolites, biological macromolecules, pharmacological treatments, dietary factors, and lifestyle. In conclusion, both previous results and ongoing studies provide evidence, making the PON cluster a prospective target for the treatment of atherosclerosis. Antioxid. Redox Signal. 16, 597–632.

As a new developmental vector system, the bacterial artificial chromosome (BAC) has been used widely in constructing genomic libraries and in generating transgenic animals. Isolation of the BAC insert end is useful to analyze the BAC clone. Here, we describe a fast and efficient method to obtain the BAC end by ligating the BAC fragments digested with Not I and another selected restriction enzyme into universal cloning vector, followed by determining the correct clones with HindIII digestion. Further DNA sequencing analysis verified the results mentioned above.

Angiotensin II (Ang II) stimulates vascular smooth muscle cell (VSMC) hypertrophy as a critical event in the development of vascular diseases such as atherosclerosis. Sirtuin (SIRT) 1, a nicotinamide adenine dinucleotide dependent deacetylase, has been demonstrated to exert protective effects in atherosclerosis by promoting endothelium-dependent vascular relaxation and reducing macrophage foam cell formation, but its role in VSMC hypertrophy remains unknown. In this study, we tried to investigate the effect of SIRT1 on Ang II-induced VSMC hypertrophy. Results showed that adenoviral-mediated over-expression of SIRT1 significantly inhibited Ang II-induced VSMC hypertrophy, while knockdown of SIRT1 by RNAi resulted in an increased [(3)H]-leucine incorporation of VSMC. Accordingly, nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1) expression induced by Ang II was inhibited by SIRT1 in VSMCs. SIRT1 activator resveratrol decreased, whereas endogenous SIRT1 inhibitor nicotinamide increased Nox1 expression in A7r5 VSMCs. Furthermore, transcription factor GATA-6 was involved in the down-regulation of Nox1 expression by SIRT1. These results provide new insight into SIRT1's anti-atherogenic properties by suppressing Ang II-induced VSMC hypertrophy.

The proinflammatory cytokine TNF-alpha plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-alpha-treated VSMCs. Overexpression of the p65/RelA subunit of NF-kappaB, a TNF-alpha-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/RelA expression by RNAi not only led to a decrease in SIRT1's basal protein expression and promoter activity, but almost abolished the TNF-alpha-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-kappaB binding site. Our study indicates that p65/RelA mediates the TNF-alpha-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions.

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.

Evidences indicate that locus control region (LCR) of beta-globin spatially closes to the downstream active gene promoter to mediate the transcriptional activation by looping. DNA binding proteins may play an important role in the looping formation. NF-E2 is one of the key transcription factors in beta-globin gene transcriptional activation. To shed light on whether NF-E2 is involved in this process, DS19MafKsiRNA cell pools were established by specifically knocked down the expression of MafK/NF-E2 p18, one subunit of NF-E2 heterodimer. In the above cell pools, it was observed that the occupancy efficiency of NF-E2 on beta-globin gene locus and the expression level of beta-globin genes were decreased. H3 acetylation, H3-K4 methylation and the deposition of RNA polymerase II, but not the recruitment of GATA-1, were also found reduced at the beta-globin gene cluster. Chromosome Conformation Capture (3C) assay showed that the cross-linking frequency between the main NF-E2 binding site HS2 and downstream structural genes was reduced compared to the normal cell. This result demonstrated that MafK/NF-E2 p18 recruitment was involved in the physical proximity of LCR and active beta-globin genes upon beta-globin gene transcriptional activation.

Histone deacetylase (HDAC) inhibitors are one of promising drugs to induce fetal hemoglobin (HbF) for treatment of sickle cell disease (SCD) and beta-thalassemia. The HDAC inhibitor apicidin was recently reported as a powerful inducer of HbF via a mechanism involving p38 signaling. In this study, we further investigated the signaling effects on the transcriptional activation of gamma-globin gene. First, we compared histone 3 (H3) acetylation patterns of approximately 70kb beta-globin loci in K562 erythroid versus HeLa cells upon apicidin treatment by chromatin immunoprecipitation assays. The results showed that the level of H3 acetylation was globally increased from the LCR to the promoter of gamma-globin gene in K562 cells, but not in non-erythroid, HeLa cells. Inhibition of p38 signaling blocks the effects of apicidin-induced gamma-globin expression and H3 acetylation. In parallel, we assessed the recruitment of transcriptional complex to beta-globin locus following apicidin treatment. The binding of GATA-1, Sp1 and RNA polymerase II (pol II) were observed to increase over several regulatory regions of beta-globin locus. Inhibitor study revealed that p38 pathway was not involved in their recruitments by apicidin. Collectively, our results provide a molecular basis to elucidate the underlying mechanisms involving p38 signaling pathway in the inducement of gamma-globin transcriptional expression by apicidin.

The lambda-Red-mediated recombination has been exploited as an efficient means for DNA manipulation. We previously reported that replication plays a pivotal role during this process. Replication direction dictates strand bias, such that single stranded oligonucleotide (SSO) with sequence corresponding to the nascent lagging strand directs higher levels of recombinant formation compared to its complementary SSO. In addition, the Escherichia coli methyl-directed mismatch repair system impedes efficient SSO-mediated site-specific gene repair. However, the role of transcription in determining strand bias and recombination efficiency is unclear. To address the potential role of transcriptional processes, we constructed plasmid substrates that harbor a mutant antibiotic reporter under the control of an inducible promoter. We found that transcription activation can promote recombinant formation to more than 10-folds whilst it has negligible effect on strand bias. Our findings provide evidence for a role of transcription in SSO-mediated gene repair process.

Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. Recent in vitro and in vivo studies have suggested that reactive oxygen species (ROS) may play an important role in cardiac hypertrophy. It was therefore thought to be of particular value to examine the effects of antioxidants on cardiac hypertrophy. Epigallocatechin-3-gallate (EGCG) is a major bioactive polyphenol present in green tea and a potent antioxidant. The current study was designed to test the hypothesis that EGCG inhibits cardiac hypertrophy in vitro and in vivo. In this study, we investigated the effects of EGCG on angiotensin II- (Ang II) and pressure-overload-induced cardiac hypertrophy. Our results showed that EGCG attenuated Ang II- and pressure-overload-mediated cardiac hypertrophy. Both reactive oxygen species generation and NADPH oxidase expressions induced by Ang II and pressure overload were suppressed by EGCG. The increased hypertension by pressure overload was almost completely blocked after EGCG treatment. Further studies showed that EGCG inhibited Ang II-induced NF-κB and AP-1 activation. Inhibition of the activity of NF-κB was through blocking ROS-dependent p38 and JNK signaling pathways, whereas inhibition of AP-1 activation was via blocking EGFR transactivation and its downstream events ERKs/PI3K/Akt/mTOR/p70S6K. The combination of these actions resulted in repressing the reactivation of ANP and BNP, and ultimately preventing the progress of cardiac hypertrophy. These findings indicated that EGCG prevents the development of cardiac hypertrophy through ROS-dependent and -independent mechanisms involving inhibition of different intracellular signaling transductional pathways.

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

Synthetic oligonucleotides had been employed in DNA repair and promised great potentials in gene therapy. To test the ability of single-stranded oligonucleotide (SSO)-mediated gene repair within a chromosomal site in human cells, a HeLa cell line stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome was established. Transfection with specific SSOs successfully repaired the mEGFP gene and resulted in the expression of functional fluorescence proteins, which could be detected by fluorescence microscopy and FACS assay. Western blot showed that EGFP was only present in the cells transfected with correction SSOs rather than the control SSOs. Furthermore, DNA sequencing confirmed that phenotype change resulted from the designated nucleotide correction at the target site. Using this reporter system, we determined the optimal structure of SSO by investigating the effect of length, modifications, and polarities of SSOs as well as the positions of the mismatch-forming nucleotide on the efficiency of SSO-mediated gene repair. Interestingly, we found that SSOs with mismatch-forming nucleotide positioned at different positions have varying potencies that homology at the 5'-end of SSOs was more crucial for the SSO's activity. These results provided guidance for designing effective SSOs as tools for treating monogenic inherited diseases.

The apoAI/CIII/AIV gene cluster is involved in lipid metabolism and has a complex pattern of gene expression modulated by a common regulatory element, the apoCIII enhancer. A new member of this cluster, apolipoprotein (apo) AV, has recently been discovered as a novel modifier in triglyceride metabolism. To determine the expression of all four apo genes in combination and, most importantly, whether the transcription of apoAV is coregulated by the apoCIII enhancer in the cluster, we generated an intact transgenic line carrying the 116-kb human apoAI/CIII/AIV/AV gene cluster and a mutant transgenic line in which the apoCIII enhancer was deleted from the 116-kb structure. We demonstrated that the apoCIII enhancer regulated hepatic and intestinal apoAI, apoCIII, and apoAIV expression; however, it did not direct the newly identified apoAV in the cluster. Furthermore, human apo genes displayed integrated position-independent expression and a closer approximation of copy number-dependent expression in the intact transgenic mice. Because apoCIII and apoAV play opposite roles in triglyceride homeostasis, we analyzed the lipid profiles in our transgenic mice to assess the effects of human apoAI gene cluster expression on lipid metabolism. The triglyceride level was elevated in intact transgenic mice but decreased in mutant ones compared with nontransgenic mice. In addition, the expression of human apoAI and apoAIV elevated high density lipoprotein cholesterol in transgenic mice fed an atherogenic diet. In conclusion, our studies with human apoAI/CIII/AIV/AV gene cluster transgenic models showed that the apoCIII enhancer regulated expression of apoAI, apo-CIII, and apoAIV but not apoAV in vivo and showed the influences of expression of the entire cluster on lipid metabolism.