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Qiliang Li

Publications and source records attributed to Qiliang Li.

At least 19 recordsLinked to original sources
PubMed2006

Purification and identification of proteins that bind to the hereditary persistence of fetal hemoglobin -198 mutation in the gamma-globin gene promoter.

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

Adult↗
PubMed2006

Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Animals↗
PubMed2006

Transcriptional potential of the gamma-globin gene is dependent on the CACCC box in a developmental stage-specific manner.

To test the role of CACCC box on gamma-globin gene activation, the CACCC box was deleted or mutated and gamma-gene expression was monitored in transgenic mice. Disruption of the CACCC box had no effect on gamma-gene expression in the cells of embryonic erythropoiesis but it strikingly reduced gamma-gene expression in fetal erythropoiesis, and abolished gamma-gene expression in adult erythroid cells. The CACCC mutation diminished HS formation, as well as TBP and polII recruitment at the gamma-gene promoter; however, it only resulted in slight or no effects on histone H3 and H4 acetylation in adult erythropoiesis. Our findings indicate that each basic cis element of the proximal gamma-gene promoter, i.e. CACCC, CCAAT or TATA box, can be disrupted without affecting the activation of gamma gene in embryonic erythroid cells. We propose that the trans factors recruited by the three boxes interact with each other to form a 'promoter complex'. In embryonic erythropoiesis the locus control region enhancer is able to interact with the complex even when components normally binding to one of the motifs are missing, but it can only activate an intact 'promoter complex' in adult erythroid cells.

Animals↗
PubMed2006

Non-coding transcripts far upstream of the epsilon-globin gene are distinctly expressed in human primary tissues and erythroleukemia cell lines.

Non-coding exons of epsilon-globin mRNA originating within the 236 kb upstream region of the epsilon-globin gene were identified in human primary tissues and K562 cells. One predominant type of upstream epsilon mRNA, which originated in the -76 kb region 5' to the epsilon gene, was present in human primary tissues, whereas 11 other isoforms were identified in K562 cells. Fragment from the -76 kb region possessed promoter activity and a prominent DNase I hypersensitive site was formed in the region approximately 2 kb 5' to the -76 kb promoter in human fetal liver, but not in K562 cells. The promoter activity in the -236 kb region resided in a retrotransposon in K562 cells. A DNase I hypersensitive site was formed at the -236 kb promoter in K562 cells, but not in human fetal liver. We discussed these results in the context of intergenic transcription and chromatin opening in the beta-globin gene cluster.

Cell Line, Tumor↗
PubMed2006

Chromatin looping and the probability of transcription.

Recent studies of several multigene clusters have shown that gene activation by a remote enhancer is associated with chromatin loop formation. It is not fully understood how a chromatin loop forms in a nucleus or how it is involved in gene regulation. In this article, we propose that the major feature that determines loop formation is the flexibility of chromatin, and that this flexibility is modulated by histone acetylation (and other modifications). Thus, histone modifications will modulate distribution of the preferential looping site in chromatin, which, in turn, determines the probability of interaction between a remote enhancer and the cognate genes. This model can explain gene expression changes in the Hoxd gene cluster and the beta-globin locus.

Acetylation↗
PubMed2006

Autonomous silencing as well as competition controls gamma-globin gene expression during development.

To investigate the control of the gamma-globin gene during development, we produced transgenic mice in which sequences of the beta-gene promoter were replaced by equivalent sequences of the gamma-gene promoter in the context of a human beta-globin locus yeast artificial chromosome (betaYAC) and analyzed the effects on globin gene expression during development. Replacement of 1,077 nucleotides (nt) of the beta-gene promoter by 1,359 nt of the gamma promoter resulted in striking inhibition of the gamma-promoter/beta-gene expression in the adult stage of development, providing direct evidence that the expression of the gamma gene in the adult is mainly controlled by autonomous silencing. Measurements of the expression of the gamma promoter/beta-globin gene as well as the wild gamma genes showed that gene competition is also involved in the control of gamma-gene expression in the fetal stage of development. We conclude that autonomous silencing is the main mechanism controlling gamma-gene expression in the adult, while autonomous silencing as well as competition between gamma and beta genes contributes to the control of gamma to beta switching during fetal development.

Animals↗
PubMed2006

Investigations of a human embryonic globin gene silencing element using YAC transgenic mice.

A silencing element has been previously located upstream of the human epsilon-globin gene promoter using transient assays and transgenic mice carrying plasmid constructs in which the element has been deleted or its transcriptional motifs have been mutated. To investigate whether this element functions in the context of the whole beta-globin locus, we analyzed epsilon-globin gene expression in transgenic mice carrying a deletion of the silencing element in the context of a 213-kilobase human beta-globin yeast artificial chromosome (beta-YAC). epsilon-Globin gene expression was measured during embryonic and fetal development and in adult mice. epsilon-mRNA levels in embryonic cells in Day 12 blood were as high as those measured in wild-type beta-YAC controls, indicating that the deletion does not affect epsilon gene promoter function. epsilon-Globin gene expression was confined to the embryonic cells, indicating that deletion of this silencing element did not affect epsilon-globin developmental expression in the context of the beta-YAC. These results suggest that in the context of the whole beta-globin locus, other proximal and upstream epsilon gene promoter elements as well as competition by the downstream globin genes contribute to the silencing of the epsilon-globin gene in the cells of definitive erythropoiesis.

Animals↗
PubMed2005

Investigations of the induction of the goat beta(C) globin gene by erythropoietin: studies in transgenic mice.

The genes of the beta globin locus of sheep and goats undergo three developmental switches, from embryonic epsilon to fetal gamma to juvenile beta(C) and from beta(C) to adult beta(A). The juvenile beta(C) gene of adult goats and sheep are strikingly induced by erythropoietin (Epo). To obtain insights on the mechanism of beta(C) induction by Epo, we produced transgenic mice carrying various beta(C) gene constructs and examined the inducibility of the beta(C) gene following administration of high doses of erythropoietin. None of the treatments resulted in reproducible induction of the beta(C) gene by erythropoietin. We conclude that the Epo inducibility elements are not contained in the 4.7 kb (which included 0.88 kb of the beta(C) promoter and 2.3 kb downstream sequence) beta(C) gene we used or that a trans-acting environment specific to the goat and sheep erythroid cell lineage is required for induction of the beta(C) globin gene by erythropoietin.

Animals↗
PubMed2005

Investigations of the effect of DNA size in transient transfection assay using dual luciferase system.

In transient transfection assays deletions, insertions, or truncations are broadly used to define cis regulatory elements, such as promoters, enhancers, and silencers. This application is based on the assumption that size changes of a test fragment have little or no effect on the apparent activity of the reporter genes. While it is known that unusually large size of a construct, such as PAC DNA, substantially reduces transfection efficiency, the size effect of a DNA fragment ranging from a couple of hundred basepairs to a few thousand basepairs, within which most transient transfection assays are performed, has not been rigorously investigated. Here we report a systematic study on the effect of plasmid size on the measurement of promoter/enhancer activity in transient transfection assay based on the dual luciferase reporter system. By inserting various lengths of stuffer DNA into the luciferase parental plasmids, we found an inverse correlation between the relative luciferase activity and the construct size. Furthermore, this size effect can be only partially corrected by inserting the same size of stuffer DNA into both experimental and control plasmids. The results suggest that the effect of DNA size should be taken into consideration for evaluation of transcriptional elements using transient transfection analysis.

DNA↗
PubMed2005

Juxtaposition of the HPFH2 enhancer is not sufficient to reactivate the gamma-globin gene in adult erythropoiesis.

Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal gamma-globin gene results in reactivation of the gamma-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced beta locus YAC transgenic mice carrying an exact beta locus replicate of a deletional HPFH mutation, HPFH 2. Although the gamma-globin gene was expressed in the HPFH 2/beta locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of gamma-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the gamma-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the beta-YAC construct might be involved in gamma-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.

Acetylation↗
PubMed2005

Enhanced channel modulation in dual-gated silicon nanowire transistors.

Dual-gated silicon nanowire (SiNW) field-effect transistors (FETs) have been fabricated by using electron-beam lithography. SiNW devices (W approximately 60 nm) exhibit an on/off current ratio greater than 10(6), which is more than 3 orders of magnitude higher than that of control devices prepared simultaneously having a large channel width (approximately 5 microm). In addition, by changing the local energy-band profile of the SiNW channel, the top gate is found to suppress ambipolar conduction effectively, which is one of the factors limiting the use of nanotube or nanowire FETs for complimentary logic applications. Two-dimensional numerical simulations show that the gate-induced electrostatic control is improved as the channel width of the FETs decreases. Therefore, enhanced channel modulations can be achieved in these dual-gated SiNW devices.

Electric Wiring↗
PubMed2005

A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression.

Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the beta-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-beta-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34(+) erythroleukemic cell line, and in cord blood-derived human CD34(+) cells. Stable transduction of MO7e cells with Ad.LCR-beta-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the beta-globin LCR. Analysis of integration sites indicated that Ad.LCR-beta-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal beta-globin LCR. None of the Ad.LCR-beta-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the beta-globin LCR-containing vector. Infection of primary CD34(+) cells with Ad.LCR-beta-GFP did not affect the clonogenic capacity of CD34(+) cells. Transduction of CD34(+) cells with Ad.LCR-beta-GFP resulted in vector integration and erythroid lineage-specific GFP expression.

Adenoviridae↗
PubMed2005

Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture.

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.

Acetylation↗
PubMed2004

Differences of globin transgene expression in stably transfected cell lines and transgenic mice.

Previous studies demonstrated that DNase I hypersensitive site -40 (HS-40) of the alpha-globin locus is capable of greatly enhancing expression of a hybrid beta/gamma-globin transcriptional unit in plasmid-transfected murine erythroleukemia (MEL) cells. However, as reported here, this same gamma-globin gene expression cassette was only transcribed at trace amounts in erythroid cells of transgenic mice. This lack of expression was not directly attributable to the beta/gamma-globin transcriptional unit, since this same unit linked to a composite beta-globin locus control region was expressed at high levels in transgenic mice. This lack of expression was also not directly attributable to chromosomal position effects, since addition of chromatin insulators failed to increase the frequency of expression. DNase I hypersensitivity and chromatin immunoprecipitation assays demonstrated that the lack of expression was correlated with a closed chromatin structure. We hypothesize that transgenes undergo dynamic changes in chromatin conformation following chromosomal integration and that the discrepant results reported here can be attributed to the relatively high level of chromatin remodeling that occurs in the transgenic mouse model, coupled with the relative inability of the HS-40 element to maintain an open chromatin state under such conditions.

Animals↗
PubMed2004

High-throughput localization of functional elements by quantitative chromatin profiling.

Identification of functional, noncoding elements that regulate transcription in the context of complex genomes is a major goal of modern biology. Localization of functionality to specific sequences is a requirement for genetic and computational studies. Here, we describe a generic approach, quantitative chromatin profiling, that uses quantitative analysis of in vivo chromatin structure over entire gene loci to rapidly and precisely localize cis-regulatory sequences and other functional modalities encoded by DNase I hypersensitive sites. To demonstrate the accuracy of this approach, we analyzed approximately 300 kilobases of human genome sequence from diverse gene loci and cleanly delineated functional elements corresponding to a spectrum of classical cis-regulatory activities including enhancers, promoters, locus control regions and insulators as well as novel elements. Systematic, high-throughput identification of functional elements coinciding with DNase I hypersensitive sites will substantially expand our knowledge of transcriptional regulation and should simplify the search for noncoding genetic variation with phenotypic consequences.

Algorithms↗
PubMed2004

Butyrate increases the efficiency of translation of gamma-globin mRNA.

Fetal hemoglobin (Hb F) levels increase in most patients with sickle cell disease following intermittent butyrate therapy. Although the full effects of butyrate on Hb F levels usually require multiple treatment cycles, in some patients a peak level is achieved after a few days of butyrate therapy. Our investigation of the mechanism(s) responsible for this rapid induction of Hb F by butyrate showed that reticulocyte gamma-globin chain synthesis markedly increased within 24 hours of butyrate exposure, without concomitant changes in reticulocyte gamma-globin mRNA levels. This suggests that butyrate might induce Hb F by increasing the efficiency of translation of gamma-globin mRNA. This hypothesis was confirmed by ribosome loading studies that demonstrated enrichment of the polysomal fraction of reticulocytes with gamma-globin mRNA following butyrate exposure. Thus, the induction of Hb F by butyrate may be mediated by translational effects in addition to its well-known effects on transcription of the gamma-globin genes.

Adult↗
PubMed2004

Synthesis of porphyrins bearing hydrocarbon tethers and facile covalent attachment to si(100).

The use of redox-active molecules as the active storage elements in memory chips requires the ability to attach the molecules to an electroactive surface in a reliable and robust manner. To explore the use of porphyrins tethered to silicon via carbosilane linkages, 17 porphyrins have been synthesized. Fourteen porphyrins bear a tether at a single meso site, and three porphyrins bear functional groups at two beta sites for possible two-point attachment. Two high-temperature processing methods (400 degrees C under inert atmosphere) have been developed for rapid (minutes), facile covalent attachment to Si platforms. The high-temperature processing conditions afford attachment either by direct deposition of a dilute solution (1 microM-1 mM) of the porphyrin sample onto the Si substrate or sublimation of a neat sample onto the Si substrate. The availability of this diverse collection of porphyrins enables an in-depth examination of the effects of the tether (length, composition, terminal functional group, number of tethers) and steric bulk of nonlinking substituents on the information-storage properties of the porphyrin monolayers obtained upon attachment to silicon. Attachment proceeds readily with a wide variety of hydrocarbon tethers, including 2-(trimethylsilyl)ethynyl, vinyl, allyl, or 3-butenyl directly appended to the porphyrin and iodo, bromomethyl, 2-(trimethylsilyl)ethynyl, ethynyl, vinyl, or allyl appended to the 4-position of a meso-phenyl ring. No attachment occurs with substituents such as phenyl, p-tolyl, mesityl, or ethyl. Collectively, the studies show that the high-temperature attachment procedure (1) has broad scope encompassing diverse functional groups, (2) tolerates a variety of arene substituents, and (3) does not afford indiscriminate attachment. The high-temperature processing conditions are ideally suited for use in fabrication of hybrid molecular/semiconductor circuitry.

Journal Article↗