Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M710454200 on February 13, 2008

J. Biol. Chem., Vol. 283, Issue 16, 10881-10891, April 18, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/16/10881    most recent
M710454200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smyth, V. A.
Right arrow Articles by Kennedy, B. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smyth, V. A.
Right arrow Articles by Kennedy, B. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Novel, Evolutionarily Conserved Enhancer of Cone Photoreceptor-specific Expression*Formula

Vincent A. Smyth, David Di Lorenzo1, and Breandán N. Kennedy2

From the School of Biomolecular & Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

Received for publication, December 21, 2007 , and in revised form, February 11, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha} subunit of cone transducin (T{alpha}C) is expressed exclusively in cone photoreceptors of the eye and pineal. T{alpha}Cisakey phototransduction protein, and inherited mutations in T{alpha}C cause total color blindness in humans. We use transgenic zebrafish to identify and characterize cone photoreceptor regulatory element 1 (CPRE-1) a novel 20-bp enhancer element in the T{alpha}C promoter (T{alpha}CP). CPRE-1 is located ~2.5 kb upstream of the translation start site and is necessary for strong cone photoreceptor-specific expression in vivo. CPRE-1 comprises of a modular arrangement of two 10-bp elements that have separate, but co-dependent transcriptional activities. In vitro, CPRE-1 specifically binds nuclear factors that are enriched in ocular tissue. Bioinformatic alignments reveal that CPRE-1 sites are evolutionarily conserved in the promoter regions of fish, rodent, and mammalian T{alpha}C orthologues and identify a 5'-CTGGAGTG(A/T)TGGA(G/A)GCAGGG(G/C)T-3' consensus sequence.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The vertebrate retina contains distinct cone and rod photoreceptors that mediate scotopic and photopic vision, respectively (1). Although cones and rods originate from the same population of retinal progenitor cells, they have unique gene expression profiles that account for their differential cell fate, morphology, and signal transduction mechanisms. For example, many components of the G protein-coupled receptor phototransduction cascade are encoded by cone- or rod-specific genes (1). The molecular genetics underpinning cone photoreceptor-specific gene expression remain poorly defined.

Several transcription factors, including Crx, Nr2e3, Nrl, and Trβ2, regulate photoreceptor-specific gene expression. Cone rod homeobox is expressed in photoreceptors, and cone rod homeobox depletion leads to a developmental loss of both cone and rod photoreceptors in mice and zebrafish and to blindness in humans (2-4). Nr2e3 and Nrl are expressed in rods where they repress expression of cone-specific genes (5-10). Mutations in Nr2e3 or Nrl lead to rods inappropriately expressing cone-specific markers and to the human retinal disease enhanced S-cone syndrome (5-12). Thyroid hormone receptor β2 is a nuclear receptor required for M-cone development (13, 14). Targeted deletion of thyroid hormone receptor β2 leads to a loss of M-cone function with an increase in functional S-cones (13, 14). Although these transcription factors are known to regulate photoreceptor genes, the molecular regulators of cone-specific gene expression are not well defined.

One method of deciphering the molecular mechanisms regulating cone-specific expression is to characterize the cis-elements in a cone-specific promoter. Several studies have identified large promoter regions sufficient to direct transgene expression in cones (15-23). However, many of these promoter regions have weak activity, exhibit ectopic, non-cone expression, or require a heterologous enhancer element (16-19, 21-23). With the exception of a cone rod homeobox-binding element, none of these studies have characterized individual cis-elements that direct cone-specific gene expression in vivo (16).

GNAT2 encodes the cone-specific {alpha}-subunit of cone transducin (T{alpha}C)3 that is expressed in all cone types and binds to activated cone opsin receptors (24). Missense mutations in the human T{alpha}C gene are linked with achromatopsia, a recessive disease leading to total loss of color vision (24-26). Similar mutations in the zebrafish T{alpha}C gene result in loss of visual behavior (27). The cone-specific expression of T{alpha}C makes its promoter an ideal candidate for identifying cis-elements controlling cone-specific expression. Previous studies determined that a 277-bp fragment of the human T{alpha}C promoter combined with a heterologous 214-bp interphotoreceptor retinoid binding protein enhancer element is sufficient to drive expression in murine cones (18). However, characterizing functional T{alpha}C cis-elements in mice is limited by the costs and technical expertise associated with transgenics and the limited number of cones in mice. The cost-effective and genetically tractable zebrafish is an attractive alternative model to characterize in vivo, cis-elements controlling cone-specific expression (28, 29).

Like humans, zebrafish have unique spectral cone subtypes with distinct cytology and synaptic connectivity that mediate color vision (28, 30). Zebrafish cones are divided into three morphological types; the UV-sensitive long single cones, the blue-sensitive short single cones, and the red-sensitive and green-sensitive double cones (30, 31). In vivo studies in zebrafish have identified large regulatory regions that direct cone photoreceptor gene expression (15, 19, 32). Transient transgenic studies identified a 105-bp cis-region of the zebrafish UV opsin promoter that can alter a rod-specific promoter to express in rod and UV cones (15). Recently a 500-bp region directing M-cone specific expression was identified that is sufficient to induce a non-retinal keratin 8 promoter to express in zebrafish M-cones (32). Previously we reported that a 3.2-kb promoter fragment of the zebrafish T{alpha}C gene is sufficient to drive strong EGFP expression in zebrafish cone photoreceptors and that the most distal 1.2-kb sequence contains an enhancer-like activity (19).


Figure 1
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 1.
A 55-bp T{alpha}C promoter fragment enhances EGFP expression in cone photoreceptors. A, schematic of the promoter-reporter portion of the 5' zebrafish T{alpha}CP deletion constructs and corresponding activity in vivo. The number of transgenic fish scored for each construct is indicated at the right of the histograms. Enhancer-1 activity localizes to a 55-bp region (highlighted in red) between -2521 and -2466 bp of T{alpha}CP. B, examples of the activity levels used to determine the activity of 5' zebrafish T{alpha}CP deletion constructs. C-G, confocal micrographs of retinal sections from 5-dpf embryos injected with -2521 T{alpha}CP. C-E, optical sections demonstrate that the EGFP-expressing cells generated co-localize with double cones (Zpr-1), UV (UV opsin), and blue cone (blue opsin) photoreceptors (highlighted by white arrows). F, EGFP-expressing cells do not co-localize with rod photoreceptors (rhodopsin). G, all EGFP-positive cells are situated in the outer nuclear layer (ONL). INL, inner nuclear layer; GCL, ganglion cell layer.

 
To identify this enhancer of cone-specific expression, we performed an in vivo analysis of the 3.2-kb zebrafish T{alpha}C promoter using deletion, mutant and chimeric reporter constructs, and in vitro characterization of binding to eye nuclear protein. Here we describe a novel, evolutionarily conserved 20-bp enhancer of the zebrafish T{alpha}C promoter that preferentially binds eye nuclear protein, is active upstream of a heterologous promoter, and is necessary for driving strong cone-specific expression.


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 2.
Enhancer 1 preferentially binds eye nuclear protein. A, schematic of overlapping 42-bp probes within the 55-bp enhancer-1 sequence. B, electrophoretic mobility shift assay demonstrates that Probes 1 and 2 bind nuclear protein isolated from adult zebrafish eyes. C, electrophoretic mobility shift assay with probe 1 shows a larger quantity of radiolabeled probe 1 is bound by eye nuclear protein (ENP) than body nuclear extract (BNP) suggesting that binding of the trans-factor(s) is more highly expressed in ocular tissue. This interaction is specific, because unlabeled probe 1 at 50- or 100-fold molar excess is able to compete away binding of eye nuclear protein to labeled probe 1, whereas a non-related cold competitor for clusterin at 50- or 100-fold molar excess is not. Eye nuclear protein (ENP), body nuclear protein (BNP), cold competitor (CC).

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Reporter Constructs—The -3173-bp T{alpha}CP-EGFP-1 plasmid based on the pEGFP-1 plasmid vector (Clontech) was used as the initial template for all constructs. The -3173({Delta}-2698/-2300) deletion was constructed by removing a 397-bp ScaI-BmgBI fragment and re-ligation. The 5' deletions, internal mutants, -2510/-2501 "RR" repeat and "LR" inversion were generated by introducing PCR fragments into -3173 T{alpha}CP-EGFP-1 digested with PstI and EcoRI. The UV zebrafish opsin promoter (zUVOP) was PCR-amplified from a zebrafish genomic library clone (Stratagene) and inserted into the pEGFP-1 vector. Chimeric constructs were engineered by introducing PCR fragments upstream of the -897-bp zUVOP minimal promoter. The 3 and 6 copy repeats of "LR" were generated by cloning synthetic oligonucleotides upstream of the -897-bp zUVOP. A spacer sequence was introduced by PCR-amplifying the kanamycin cassette from the EGFP-1 vector and cloning between the -897-bp zUVOP and enhancer repeat sequence using SpeI restriction sites. The (-2521/-890-bp zT{alpha}CP)-(-836-bp human T{alpha}C promoter)-EGFP-1 construct was generated by digesting (-2521/-890-bp zT{alpha}CP)-(-897-bp zUVOP)-EGFP-1 with SpeI and BamHI and cloning a -836-bp human T{alpha}C promoter fragment PCR-amplified from genomic clone pCYPAC2 (RZPD GmBH). All constructs were confirmed by DNA sequencing.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 3.
Refinement of the enhancer 1 sequence to CPRE-1 a 20-bp region containing separate, but dependent, functional domains. A, probes containing 10-bp mutations in probe 1 were designed to further narrow the trans-factor(s) binding site. Introduced mutations are highlighted by boxes, and the relative position of the deletion constructs are illustrated by arrows. B, electrophoretic mobility shift assay of probes 1, 1M L, 1M R, and 1M D with eye nuclear extract. All three mutant probes are unable to form a complex with eye nuclear extract, and probes 1M L and 1M R are unable to compete away binding of eye nuclear extract to labeled probe 1. C, to test the functional significance of the 10-bp mutations on CPRE-1 activity in vivo, constructs containing corresponding 10-bp deletions were tested. In agreement with the in vitro assay deletions of 10-bp regions from -2521 to -2510 bp (L) and -2510 to -2501 bp (R) of T{alpha}CP resulted in a significant loss of functional activity in vivo. A significant reduction in EGFP expression levels is also observed when R is mutated. When L is mutated a statistically significant reduction in EGFP expression is observed (C and D). A duplicate RR combination is insufficient to recapitulate activity to that of the wild type LR arrangement. The enhancer activity is also partially dependent on orientation, because when the LR was inverted and reversed there is a significant loss of EGFP expression. (All constructs were injected as covalently closed plasmids.) D, p values for comparisons of construct activities (*, p < 0.05; **, p < 0.005; ***, p < 0.0005). NSS, no statistical significance.

 
Embryo Microinjections—Covalently closed circular plasmids were suspended in 0.1 M Tris (pH 7.9) plus 0.1% Phenol Red (Sigma) at a final concentration of 25 ng/µl. The pressure injector was regulated so that ~300 pl (~7.5 pg) of each reporter construct was injected per embryo. The covalently closed plasmids were injected into the cytoplasm of 1-2 cell stage zebrafish embryos using an air pressure-regulated Pico Pump (World Precision Instruments) attached to a Narishige micromanipulator and needle holder. Injected embryos were transferred to water containing 0.003% 1-phenyl-2-thiourea 24 h post fertilization to inhibit melanin production. Animal studies were sanctioned by the UCD Animal Research Ethics Committee (Ref: P04-18) and licensed by the Department of Health and Children, Rep. of Ireland (License Ref: B100/3358).

Screening for EGFP Expression—Injected embryos were anesthetized and analyzed by fluorescence microscopy using a Zeiss Axioplan 2 fluorescence microscope at 5 days post fertilization (dpf). Construct activity was scored on the basis of a semiquantitative assay, where one of three ordinal activity levels was assigned to an embryo based on the number of fluorescent cells within the eye. Embryos with >50 GFP+ cells in the eye were assigned an activity level of +++, fish with 5-50 GFP+ cells were assigned as ++, 1-5 fluorescent cells as +, and fish with no GFP+ cells were assigned an activity level of - (15). The sum of each ordinal level was divided by the total number of embryos to give relative activity. Statistical analysis was performed using Student's t tests, and p values of ≤0.05 were considered significant.

Immunostaining—Whole mount immunostaining was performed on 5 dpf larvae as described previously (33) and analyzed by confocal microscopy (LSM510 Zeiss). For retinal sections, 5-dpf transgenic larvae were fixed overnight at 4 °C in 4% paraformaldehyde/5% sucrose/0.1 M phosphate buffer (pH 7.4). Following fixation fish were cryoprotected in 20% sucrose/0.1 M phosphate buffer (pH 7.4) then infiltrated with OCT (Tissue-TeK) as previously described (33). 12-µm sections were thaw-mounted to Superfrost slides (BDH Chemical Ltd.). Sections were rehydrated in TBS for 30 min, and then incubated for 60 min in blocking buffer (5% normal goat serum/1% bovine serum albumin/1% Triton X-100/TBS). Sections were incubated overnight with primary antibodies diluted in blocking buffer at the following ratios: zpr-1 (1:200, Oregon Monoclonal Bank), UV opsin (1:400), Blue opsin (1:400), and rod opsin (1:200), cone and rod opsin antibodies were generously donated by Profs. D. Hyde and T. Vihtelic (34). Following three 5-min washes in TBS/0.1% Tween, the sections were incubated at room temperature for 1 h in secondary antibodies diluted 1:200 in blocking buffer. Prior to mounting in Anti-Fade (Molecular Probes), the sections were washed three times for 5 min in TBS. Retinal sections were analyzed by confocal microscopy (LSM510, Zeiss).

Nuclear Protein Extract Preparation and Electrophoretic Mobility Shift Assay—Nuclear protein extracts were prepared from adult zebrafish eyes. Adult zebrafish eyes were dissected and homogenized in 4 ml of homogenization buffer (50 mM Hepes/pH 7.9, 25 mM KCl/5 mM MgCl2/200 mM sucrose/0.5 mM dithiothreitol/0.25 mM phenylmethylsulfonyl fluoride (Sigma)), centrifuged at 2000 x g for 10 min at 4 °C. The pellet was re-suspended in 600 µl of Chelsky buffer (10 mM Tris, pH 7.0/10 mM NaCl/3 mM MgCl2/30 mM sucrose/1% Nonidet P-40), incubated on ice for 10 min, and centrifuged at 3000 x g for 10 min at 4 °C. Pellets were re-suspended in 600 µl of Chelsky buffer plus 10 mM CaCl2, layered on sucrose cushion (50 mM Hepes, pH 7.9/25 mM KCl/5 mM MgCl2/876 mM sucrose/0.2 mM EDTA/0.5 mM dithiothreitol/0.25 mM phenylmethylsulfonyl fluoride), and centrifuged at 3000 x g for 10 min at 4 °C. Nuclei were re-suspended in Chelsky buffer c-low salt (20% glycerol/20 mM Tris-Cl pH 7.9/25 mM KCl/0.2 mM EDTA/0.5 mM dithiothreitol/0.25 mM phenylmethylsulfonyl fluoride), lysed by the dropwise addition of a high salt Chelsky buffer c-low salt plus 1.2 M KCl, and homogenized. Samples were incubated on ice for 30 min and centrifuged at 13,000-25,000 x g for 10 min at 4 °C. Supernatant was removed and concentrated by ultra centrifugal filter devices (Amicon ultra 4). Protein content was determined by using the BCA Protein Assay Reagent (Pierce). Oligonucleotide probes (supplemental Table S1) were synthesized with 5' overhangs and radioactively labeled with [{alpha}-32P]dCTP using Klenow Exo- (New England Biolabs). Nuclear extracts were incubated with a 200-fold molar excess of poly(dI-dC) at 4 °C for 10 min. Labeled probe was added, and the mixture was incubated at room temperature for 30 min. Reactions were stopped and loaded on 5% non-denaturing polyacrylamide gels, followed by autoradiography. For the competition assay, cold competitor was added prior to addition of labeled probe, in 50-, 100-, and 200-fold molar excess.

Sequence Analysis—The 10-kb 5' promoter sequence of GNAT2 genes were obtained from ENSEMBL and aligned with the 20-bp CPRE-1 sequence using ClustalW (35). Only sequences with a pairwise alignment score greater than 0.61 were considered significant. The consensus CPRE-1 sequences was generated using the online Weblogo server (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A 55-bp Distal Promoter Region Is Necessary for Strong EGFP Expression in Cone Photoreceptors—In transient transgenic assays, the -3173-bp fragment of the zebrafish T{alpha}C promoter (T{alpha}CP) region drives robust EGFP expression specifically in retinal cone photoreceptors and in the pineal (Fig. 1, data not shown). The expression pattern is identical to that observed in the Tg(3.2T{alpha}CP-EGFP-1) transgenic line generated using the same promoter fragment (19). To identify cis-elements controlling this cone photoreceptor-specific expression, we tested the reporter activity of a series of 5' deletions of the -3173-bp T{alpha}CP in vivo.

Deletion of the region between -3173 and -2749-bp of zebrafish T{alpha}CP results in a significant reduction of reporter activity, revealing a potential enhancer element, designated "enhancer region 2," within this 424-bp region (Fig. 1A). However, deletion of the 283-bp region between -2749 and -2466-bp of the T{alpha}CP, designated "enhancer region 1," practically abolishes EGFP expression (Fig. 1A). An internal deletion of enhancer region 1, within the -3173 bp T{alpha}CP construct, results in minimal reporter activity (Fig. 1A). These results suggest that enhancer region 1 is necessary for high level cone-specific expression and that enhancer region 2 is not sufficient to compensate for loss of enhancer region 1.

We focused on refining the enhancer element (enhancer-1) in enhancer region 1. Analyses of additional 5' deletions initially narrowed enhancer-1 to the 55-bp region, between -2521 and -2466 bp of T{alpha}CP (Fig. 1A). The -2521-bp T{alpha}CP construct results in robust expression, with >34% of injected embryos expressing EGFP in the retina. The -2521-bp T{alpha}CP-EGFP construct confines transgene expression to all cone photoreceptor types based on expression in the outer nuclear layer and co-immunolocalization of EGFP with markers for double, short single, and long single cones, but not with a rod photoreceptor marker (Fig. 1, C-F, and Table 1). Deletion of the 55 bp between -2521 and -2466 bp of T{alpha}CP results in a significant loss of EGFP expression, with <10% of injected embryos expressing EGFP in only in one to five cells per eye (Fig. 1A).


View this table:
[in this window]
[in a new window]

  		TABLE 1
Number of EGFP+ cells from transient transgenic fish sections that label with rod or cone opsin antibodies

Parentheses indicated number of EGFP+ cells and the number that labeled with cone or rod opsin antibodies is highlighted in bold.

 
Eye Nuclear Protein Binds Specifically to Enhancer-1—The ability of enhancer-1 to bind eye nuclear protein was evaluated by testing three overlapping oligonucleotide probes that span the 55-bp sequence of -2521 to -2466 bp of T{alpha}CP (Fig. 2A). Probe 1 and Probe 2 form distinct complexes with eye nuclear protein, but Probe 1 became the focus of our studies as it generates a more intense complex (Fig. 2B).

The specificity of eye nuclear protein binding to Probe 1 was analyzed. Addition of Probe 1 as a cold competitor at 50 and 100 M excess demonstrates a specific ability of unlabeled Probe 1 to compete for nuclear proteins binding to Probe 1 in a concentration-dependent manner (Fig. 2C). An unrelated competitor is unable to compete away binding of nuclear protein to Probe 1 (Fig. 2C). In addition, non-retinal nuclear protein isolated from zebrafish body is capable of binding to Probe 1, although at a much reduced intensity compared with eye nuclear protein (Fig. 2C). This suggests an enrichment of trans-factors in the eye that specifically bind to enhancer-1.

CPRE-1 Comprises Two 10-bp Functional cis-Elements—To narrow the minimal sequence of enhancer-1, three mutant probes containing sequential 10-bp mutations were analyzed by electrophoretic mobility shift assay (Fig. 3A). The three mutant probes fail to form a complex with eye nuclear protein (Fig. 3B). However, when used as cold competitors, Probe 1M R is unable to compete, Probe 1M L partially competes, and Probe 1M D efficiently competes away binding of eye nuclear protein to wild-type Probe 1 (Fig. 3B). Thus, the 20 bp spanning Probes 1M L and 1M R, designated "cone photoreceptor regulatory element 1" (CPRE-1) appear essential for trans-factor(s) binding to enhancer-1 (Fig. 3A). To test the significance of CPRE-1 for transcriptional activity in vivo, constructs containing complete or partial deletions of CPRE-1 were tested (Fig. 3C). The -2501-bp T{alpha}CP-EGFP construct had limited activity compared with the -2521 bp T{alpha}CP-EGFP construct demonstrating that CPRE-1 is necessary for robust cone-specific expression (Fig. 3C). 5' deletion of the sequence from -2521 and -2510 bp of T{alpha}CP resulted in a significant loss of EGFP expression, and deletion of the sequence from -2510 and -2501 bp of T{alpha}CP results in a further statistically significant loss of EGFP expression (Fig. 3, C and D). The in vitro and in vivo assays suggest that CPRE-1 comprises two separate cis-elements, "R" located between -2521 and -2510 bp of T{alpha}CP and "L" located between -2510 and -2501 bp of T{alpha}CP. To identify the functional importance of the L and R cis-elements, they were mutated independently in the -2521-bp background and tested in vivo. Mutation of either L or R results in a significant loss of transcriptional activity compared with the -2521-bp T{alpha}CP construct, with mutation of R having a more pronounced affect (Fig. 3, C and D). However, duplication of R is insufficient to recapitulate the transgene expression levels of the L plus R arrangement, demonstrating a co-dependence of the modules for enhanced transcriptional activation (Fig. 3, C and D). The ability of CPRE-1 to increase transcription appears orientation-dependent as inverting and reversing the L and R modules results in a significant reduction in transgene expression, compared with their wild-type orientation (Fig. 3, C and D).

Identification of L and R Sequences Essential for trans-Factor(s) Binding—To identify the base pairs necessary for binding of trans-factor(s) to CPRE-1, the ability of L and R probes, with consecutive 3-bp mutations, to bind eye nuclear protein was tested (Fig. 4, A and B). The 6-bp sequence from -2518 to -2512 bp (5'-GAGTGT) of L is essential for binding, because mutation of these residues in Probes 1M L.2 and 1M L.3 abolishes the ability to form a complex with eye nuclear protein and to compete with binding to wild-type Probe 1 (Fig. 4C and Table 2). For R, only the 3-bp sequence from -2507 to -2504 bp (5'-GGC), mutated in Probe 1M R.2, is essential for binding (Fig. 4D and Table 2). The sequences mutated in Probes 1M L.4 and 1M D.1, although not essential for binding, result in less intense complexes suggesting a role in stabilizing the complex. A significant role for the thymidine nucleotide at position -2511 bp is demonstrated as Probe 1M L.4 forms complexes much less efficiently than overlapping Probe 1M R.1, and this nucleotide is the distinguishing mutation (Fig. 4, C and D, and Table 2).


View this table:
[in this window]
[in a new window]

  		TABLE 2
The position of 3-bp mutations on mutant probes in relation to enhancer-1 sequence

In Probe 1 the L cis-element is in bold, and the R is in italic. Altered bases base pairs are underlined; engineered overhang sequences are shown in lowercase italic letters.

 
CPRE-1 Is Necessary but Not Sufficient to Drive Expression of a Heterologous Promoter in Vivo—The ability of CPRE-1 to drive the expression of a heterologous zebrafish UV opsin promoter (zUVOP) was characterized (Fig. 5A). We identified a -800-bp fragment of the zebrafish UV opsin proximal promoter region that directs low but specific EGFP expression in UV cones (Fig. 5A, data not shown). Chimeric constructs comprising of the heterologous UV opsin promoter with either the -3173/-890-bp or -2521/-890-bp regions of the distal T{alpha}CP upstream of the UV opsin promoter are sufficient to enhance levels of EGFP expression (Fig. 5, A and B-F). Although the -2521/-890-bp T{alpha}CP fragment originates from a promoter expressing in all cones, in the chimera it is not sufficient to override the UV cone specificity of the heterologous promoter nor to direct expression of the ubiquitous SV40 minimal promoter to cone photoreceptors (Fig. 5, A and B). However, when CPRE-1 is removed (-2501/-890 bp) from the chimeric construct a significant loss in transgene expression was observed, confirming that CPRE-1 is necessary to drive expression from the heterologous zUVOP promoter (Fig. 5A).


Figure 4
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 4.
3-bp mutations in CPRE-1 eliminate trans-factor binding. A and B, to identify the binding sequence for CPRE-1, mutant probes containing 3-bp mutations in the 20-bp sequence of probe 1M L and 1M R were designed. Mutations are highlighted by boxes, and the relative position of the deletion constructs are illustrated by arrows. Red boxes indicate 3-bp mutations that diminish trans-factor(s) binding. C, electrophoretic mobility shift assays demonstrate mutant that probes 1M L.2, 1M L.3, and 1M L.4 are unable to bind eye nuclear extract and can only partially compete away binding of eye nuclear extract to radiolabeled probe 1. D, electrophoretic mobility shift assay demonstrates that mutant probes 1M R.2 and 1M D.1 are unable to bind eye nuclear extract or compete away binding of eye nuclear extract to labeled wild-type probe 1.

 
CPRE-1 is not sufficient to enhance expression from the heterologous promoter, because single or multiple copies cloned upstream of the minimal UV opsin promoter do not increase expression from the minimal UV opsin promoter (Fig. 5A). To determine if this insufficiency relates to spatial context, six repeats of the 20-bp "LR" CPRE-1 sequence were placed 1721 bp upstream of the UV opsin promoter by introducing an unrelated spacer sequence. No increase in expression from the heterologous promoter was observed (Fig. 5A), suggesting that the LR CPRE-1 sequence requires interaction with specific cis-element(s) in the -2501/-890-bp T{alpha}C distal promoter. These dependent elements are not functionally or spatially conserved in the human T{alpha}C proximal promoter, because replacement of the spacer sequence with the -2453/-847 bp of the human T{alpha}C proximal promoter did not increase transgene expression (Fig. 5A).


Figure 5
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 5.
CPRE-1 is necessary but not sufficient to drive expression of a heterologous promoter. A, schematic of the promoter-reporter portion of chimeric constructs containing CPRE-1 upstream of an 800-bp minimal zebrafish UV opsin promoter (zUVOP). Blue rectangles represent the zUVOP 800-bp proximal promoter sequence, green rectangles represent EGFP coding sequences, orange rectangles represent T{alpha}CP distal sequence, and the 20-bp CPRE-1 coding sequence is represented by a red rectangle. A chimeric construct containing CPRE-1 in the distal promoter (-2521/-890 T{alpha}CP) significantly increases expression from the minimal zUV opsin heterologous promoter. Deletion of CPRE-1 from (-2501/-890 T{alpha}CP) results in significant loss of EGFP expression. Multiple copies of the 20-bp CPRE-1 are not sufficient to enhance expression of the heterologous promoter. Chimeric constructs containing the distal zebrafish T{alpha}CP (zT{alpha}CP) fragment with CPRE-1 upstream of the human T{alpha}CP (hTaCP) or the minimal SV 40 promoter are inactive. B-F, confocal micrographs of retinal sections from 5-dpf embryos injected with (-2521/-890 bp T{alpha}CP)-(-800 bp zUVOP)-EGFP-1. Single optical section (B) and stacked z-series (C and E), Z-series projection (D) demonstrating the EGFP-expressing cells co-localize with UV cones (white arrow) and not with double cones (Zpr1), blue cones (blue opsin), or rods (rhodopsin). All EGFP cells are situated in the outer nuclear layer. ONL, outer nuclear layer; INL, inner nuclear layer; and GCL, ganglion cell layer.

 
Thus, CPRE-1 is necessary, in the context of the -2521/-890 distal zebrafish T{alpha}CP-UV opsin chimeric construct, to enhance transgene expression from a heterologous promoter. However, it is not sufficient to enhance transcription from the heterologous promoter nor to overwrite the UV cone specificity of the minimal promoter by itself.

CPRE-1 Is Conserved in T{alpha}CP Orthologues—Alignment of the CPRE-1 sequence with orthologous T{alpha}C promoters reveals a conserved copy within divergent vertebrate species (Fig. 6A). The location of the CPRE-1 site is also conserved among more closely related species, e.g. primates and rodents. Bioinformatics reveal a CPRE-1 consensus sequence of 5'-CTGGAGTG(A/T)TGGA(G/A)GCAGGG(G/C)T-3' (Fig. 6B), which does not match any known transcription factor site in the Transfac data base (37). We searched for CPRE-1 consensus sites in the promoter regions of other zebrafish cone-specific (arr3, cnga3, gngt2, pde6C, and pde6h), rod-specific (rho, pde6a, and gnat1), and housekeeping genes (tubulin and β-actin). A significantly matching consensus site was identified in the cone-specific arrestin (arr3) and cone cyclicgated nucleotide channel {alpha}3 subunit (cnga3) genes. Thus, CPRE-1 is an evolutionarily conserved enhancer element in a cohort of cone-specific genes.


Figure 6
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 6.
CPRE-1 is evolutionarily conserved in the promoter regions of T{alpha}C orthologues. A, conserved CPRE-1-like sequences in the 5' regulatory regions of T{alpha}C genes of various vertebrates. ClustalW pairwise alignments of consensus CPRE-1 sites in the 5' regulatory regions of T{alpha}C promoters are to the right. Location of the consensus enhancer element in the 10-kb T{alpha}C promoters of a variety of vertebrate species is shown on the left. The position of the consensus CPRE-1 sequence is indicated in brackets and by the red boxes. B, consensus sequence of highest scoring motif from alignment of CPRE-1 sites from the promoter regions of GNAT2 orthologues. The size of each letter is proportional to the percentage of sites containing the respective nucleotide. Red line under sequences, indicate the nine central nucleotides essential for binding eye nuclear protein to Probe 1. The position of the four nucleotides that regulate efficiency of binding to Probe 1 are underlined in green.

 


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7.
Proposed model of transcriptional regulation of the zebrafish T{alpha}C gene.A, linear model of regulatory regions on -3173 T{alpha}CP. A cone photoreceptor-specific promoter is illustrated in green, enhancer-dependent region in yellow, CPRE-1 site in blue, and enhancer regions in black. B, molecular apparatus controlling transcription of T{alpha}C in zebrafish cone photoreceptors. Tissue specificity is regulated by activators or repressors that interact with the basal transcription machinery (labeledA,B,F,E,andH) on the-0.7-kbproximalpromoter. L and R activators bound to the CPRE-1 site regulate transcription of T{alpha}C by interacting with unknown activators bound to the enhancer-dependent region.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cone photoreceptors are highly specialized sensory neurons that enable color vision and visual acuity. Severe forms of human blindness associated with cone defects include rare genetic diseases, e.g. achromatopsia, and prevalent complex diseases, e.g. age-related macular degeneration. To develop our understanding of the molecular genetics of cones we focused on identifying cis-elements in the zebrafish T{alpha}C promoter region.

This study identifies and characterizes cone photoreceptor regulatory element-1 (CPRE-1) a 20-bp enhancer of cone-specific expression. CPRE-1 is located in the distal zebrafish T{alpha}C promoter region, ~2.5 kb upstream of the coding sequence. CPRE-1 is necessary for robust, cone-specific expression, and regulates expression in all cone photoreceptor types. CPRE-1 comprises a modular arrangement of two 10-bp cis-elements. These L and R elements have separate but co-dependent transcriptional activities that are required to drive high levels of cone-specific expression. The activity of CPRE-1 appears orientation-dependent as inversion and reversion of LR result in significantly less activity than the wild-type orientation. CPRE-1 is not sufficient to drive expression from heterologous promoters. However, a larger distal fragment of the zebrafish T{alpha}CP is sufficient to enhance expression from a heterologous UV cone-specific promoter, and CPRE-1 is required for this enhanced activity. Notably, CPRE-1 does not override the UV specificity of the heterologous promoter. Thus, CPRE-1 is an enhancer module that increases the rate of transcriptional activity, but not the tissue specificity, of cone-specific promoters in vivo.

Enhancement of transcriptional activity by CPRE-1 may result from direct binding of transcriptional activators on site or by modification of the chromatin architecture to enable access of transcription factors to the promoter region. However, in the transient transgenesis assay, the reporter constructs are microinjected as closed-circular DNA, which does not integrate into the zebrafish genome, but remains as episomal DNA (38). Chromatin assembly on episomal DNA is a "time-dependent" process with the majority of episomal DNA not bound by chromatin until ~5 weeks after DNA administration (39). At 1 and 7 days post DNA administration there is ~5- to 10-fold less episomal DNA bound by chromatin than at 5 weeks (39). Because our reporter constructs are assayed for activity at 5 days post microinjection, it is unlikely that regulation of chromatin accessibility is a significant determinant of reporter activity. Thus, we conclude that CPRE-1 most likely enhances transcriptional activity by assembly of transcriptional activators.

Bioinformatic analyses suggest that CPRE-1 sequences are evolutionarily conserved in the promoter regions of T{alpha}C orthologues from zebrafish to humans and identify a novel consensus sequence of 5'-CTGGAGTG(A/T)TGGA(G/A)GCAGGG(G/C)T-3'. In vitro experiments confirm that DNA probes encompassing the CPRE-1 element preferentially bind eye nuclear protein in a dose-dependent manner, and CPRE-1 is required for these interactions. Within the CPRE-1 of zebrafish T{alpha}CP we identify nine central nucleotides essential for binding eye nuclear factors (-2518/-2512 and -2507/-2504 bp), and four nucleotides regulating the efficacy of binding (-2511 and -2501/-2499 bp). These sites include four of the eight most conserved bases identified in the CPRE-1 consensus sequence.

This study advances our understanding of the transcriptional regulation of the cone-specific GNAT2 gene family. Previous studies of the human promoter reported several silencer sequences that bind nuclear protein in vitro, but their functional importance in vivo has not been reported (40). A transgenic line consisting of a 277-bp proximal region of the human T{alpha}C promoter directs expression in murine cones when a heterologous enhancer from the human interphotoreceptor retinoid-binding protein gene is placed upstream (18), but a native human T{alpha}C enhancer has not been identified. Chimeric reporter constructs with a distal fragment of the zebrafish T{alpha}C promoter region containing CPRE-1 cannot regulate a proximal human T{alpha}C promoter, and vice versa, suggesting species-specific differences in promoter organization or transcription factor binding.

Our proposed model of transcriptional regulation of the zebrafish T{alpha}C gene is depicted in Fig. 7. The proximal 0.7 kb of promoter has weak transcriptional activity but controls the "all" cone specificity of expression (19). In the distal promoter, enhancer activities are located between -3.2/-2.8 kb (enhancer region 2) and -2.8/-2.5 kb (enhancer region 1). A novel 20-bp enhancer, CPRE-1 is located within enhancer region 1. CPRE-1 comprises of separate but co-dependent L and R modules necessary for robust, cone-specific expression. CPRE-1 enhances the rate of transcriptional activity, but not the tissue specificity. The region from -2.5/-0.9 kb, bridging the tissue specificity and enhancer domains, is essential for the enhancer activity of CPRE-1. This dependence is not a trivial spatial dependence, because neither a generic spacer nor an equivalent region of the human T{alpha}C distal promoter can substitute in chimeric reporter assays. We speculate that transcription factors binding to these regulatory DNA regions are brought into contact in a complex resulting in high level cone-specific expression (Fig. 7B). Our future studies are focused on identifying these factors and characterizing their expression and function.


   FOOTNOTES
 
* This work was supported in part by Science Foundation Ireland Research Frontiers Program 2005-2008 (Grant 05/RFP/Gen0027), Science Foundation Ireland Investigator Grant Award (Grant 04/IN3/B559), and University College Dublin Presidents Award 2003-2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 Supported by under the Science Foundation Ireland UREKA (Undergraduate Research Experience & Knowledge Award) program. Back

2 To whom correspondence should be addressed: Tel.: 353-1-716-6740; Fax: 353-1-283-7211; E-mail: brendan.kennedy{at}ucd.ie.

3 The abbreviations used are: T{alpha}C, cone transducin {alpha} subunit; T{alpha}CP, cone transducin {alpha} subunit 5' promoter; dpf, days post fertilization; CPRE-1, cone photoreceptor regulatory element 1; TBS, Tris-buffered saline; zUVOP, UV zebrafish opsin promoter; EGFP, enhanced green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Profs. Tom Vihtelic and David Hyde for the generous gift of zebrafish opsin antibodies; Prof. Finian Martin, Dr. Yolanda Alvarez, Dr. Victor Vendrell, Maria Morrissey, Sarah Mc Loughlin, and Theresa Heffernan for helpful technical assistance and comments on the manuscript; and Beata Sapetto-Rebow for management of our zebrafish facility.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Forrester, J. V., Dick, A. D., McMenamin, P. G., and Lee, W. R. (2002) The Eye: Basic Sciences in Practice, Elsevier Health Sciences, New York
  2. Kimble, T. D., and Williams, R. W. (2000) Anat. Embryol. (Berl.) 201, 305-316[CrossRef][Medline] [Order article via Infotrieve]
  3. Silva, E., Yang, J. M., Li, Y., Dharmaraj, S., Sundin, O. H., and Maumenee, I. H. (2000) Invest. Ophthalmol. Vis. Sci. 41, 2076-2079[Abstract/Free Full Text]
  4. Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531-541[CrossRef][Medline] [Order article via Infotrieve]
  5. Cheng, H., Khanna, H., Oh, E. C., Hicks, D., Mitton, K. P., and Swaroop, A. (2004) Hum. Mol. Genet 13, 1563-1575[Abstract/Free Full Text]
  6. Chen, J., Rattner, A., and Nathans, J. (2005) J. Neurosci. 25, 118-129[Abstract/Free Full Text]
  7. Peng, G. H., Ahmad, O., Ahmad, F., Liu, J., and Chen, S. (2005) Hum. Mol. Genet. 14, 747-764[Abstract/Free Full Text]
  8. Mears, A. J., Kondo, M., Swain, P. K., Takada, Y., Bush, R. A., Saunders, T. L., Sieving, P. A., and Swaroop, A. (2001) Nat. Genet. 29, 447-452[CrossRef][Medline] [Order article via Infotrieve]
  9. Nikonov, S. S., Daniele, L. L., Zhu, X., Craft, C. M., Swaroop, A., and Pugh, E. N., Jr. (2005) J. Gen. Physiol. 125, 287-304[Abstract/Free Full Text]
  10. Oh, E. C., Khan, N., Novelli, E., Khanna, H., Strettoi, E., and Swaroop, A. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1679-1684[Abstract/Free Full Text]
  11. Haider, N. B., Jacobson, S. G., Cideciyan, A. V., Swiderski, R., Streb, L. M., Searby, C., Beck, G., Hockey, R., Hanna, D. B., Gorman, S., Duhl, D., Carmi, R., Bennett, J., Weleber, R. G., Fishman, G. A., Wright, A. F., Stone, E. M., and Sheffield, V. C. (2000) Nat. Genet. 24, 127-131[CrossRef][Medline] [Order article via Infotrieve]
  12. Haider, N. B., Demarco, P., Nystuen, A. M., Huang, X., Smith, R. S., Mc-Call, M. A., Naggert, J. K., and Nishina, P. M. (2006) Vis. Neurosci. 23, 917-929[CrossRef][Medline] [Order article via Infotrieve]
  13. Ng, L., Hurley, J. B., Dierks, B., Srinivas, M., Salto, C., Vennstrom, B., Reh, T. A., and Forrest, D. (2001) Nat. Genet. 27, 94-98[Medline] [Order article via Infotrieve]
  14. Roberts, M. R., Srinivas, M., Forrest, D., Morreale de Escobar, G., and Reh, T. A. (2006) in Proc. Natl. Acad. Sci. U. S. A. 103, 6218-6223[Abstract/Free Full Text]
  15. Luo, W., Williams, J., Smallwood, P. M., Touchman, J. W., Roman, L. M., and Nathans, J. (2004) J. Biol. Chem. 279, 19286-19293[Abstract/Free Full Text]
  16. Pickrell, S. W., Zhu, X., Wang, X., and Craft, C. M. (2004) Invest. Ophthalmol. Vis. Sci. 45, 3877-3884[Abstract/Free Full Text]
  17. Glushakova, L. G., Timmers, A. M., Pang, J., Teusner, J. T., and Hauswirth, W. W. (2006) Invest. Ophthalmol. Vis. Sci. 47, 3505-3513[Abstract/Free Full Text]
  18. Ying, S., Fong, S. L., Fong, W. B., Kao, C. W., Converse, R. L., and Kao, W. W. (1998) Curr. Eye Res. 17, 777-782[CrossRef][Medline] [Order article via Infotrieve]
  19. Kennedy, B. N., Alvarez, Y., Brockerhoff, S. E., Stearns, G. W., Sapetto-Rebow, B., Taylor, M. R., and Hurley, J. B. (2007) Invest. Ophthalmol. Vis. Sci. 48, 522-529[Abstract/Free Full Text]
  20. Gouras, P., Kjeldbye, H., and Zack, D. J. (1994) Vis. Neurosci. 11, 1227-1231[Medline] [Order article via Infotrieve]
  21. Chen, J., Tucker, C. L., Woodford, B., Szel, A., Lem, J., Gianella-Borradori, A., Simon, M. I., and Bogenmann, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2611-2615[Abstract/Free Full Text]
  22. Boatright, J. H., Borst, D. E., Peoples, J. W., Bruno, J., Edwards, C. L., Si, J. S., and Nickerson, J. M. (1997) Mol. Vis. 3, 15[Medline] [Order article via Infotrieve]
  23. Babu, S., McIlvain, V., Whitaker, S. L., and Knox, B. E. (2006) FEBS Lett. 580, 1479-1484[CrossRef][Medline] [Order article via Infotrieve]
  24. Kohl, S., Baumann, B., Rosenberg, T., Kellner, U., Lorenz, B., Vadala, M., Jacobson, S. G., and Wissinger, B. (2002) Am. J. Hum. Genet. 71, 422-425[CrossRef][Medline] [Order article via Infotrieve]
  25. Weinstein, L. S., Chen, M., Xie, T., and Liu, J. (2006) Trends Pharmacol. Sci. 27, 260-266[CrossRef][Medline] [Order article via Infotrieve]
  26. Michaelides, M., Aligianis, I. A., Holder, G. E., Simunovic, M., Mollon, J. D., Maher, E. R., Hunt, D. M., and Moore, A. T. (2003) Br. J. Ophthalmol. 87, 1317-1320[Abstract/Free Full Text]
  27. Brockerhoff, S. E., Rieke, F., Matthews, H. R., Taylor, M. R., Kennedy, B., Ankoudinova, I., Niemi, G. A., Tucker, C. L., Xiao, M., Cilluffo, M. C., Fain, G. L., and Hurley, J. B. (2003) J. Neurosci. 23, 470-480[Abstract/Free Full Text]
  28. Goldsmith, P., and Harris, W. A. (2003) Semin. Cell Dev. Biol. 14, 11-18[CrossRef][Medline] [Order article via Infotrieve]
  29. Collery, R. F., Cederlund, M. L., Smyth, V. A., and Kennedy, B. N. (2006) Adv. Exp. Med. Biol. 572, 201-207[Medline] [Order article via Infotrieve]
  30. Raymond, P. A., and Barthel, L. K. (2004) Int. J. Dev. Biol. 48, 935-945[CrossRef][Medline] [Order article via Infotrieve]
  31. Branchek, T., and Bremiller, R. (1984) J. Comp. Neurol. 224, 107-115[CrossRef][Medline] [Order article via Infotrieve]
  32. Tsujimura, T., Chinen, A., and Kawamura, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12813-12818[Abstract/Free Full Text]
  33. Barthel, L. K., and Raymond, P. A. (1990) J. Histochem. Cytochem. 38, 1383-1388[Abstract]
  34. Vihtelic, T. S., Doro, C. J., and Hyde, D. R. (1999) Vis. Neurosci. 16, 571-585[CrossRef][Medline] [Order article via Infotrieve]
  35. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics 23, 2947-2948[Abstract/Free Full Text]
  36. Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004) Genome Res. 14, 1188-1190[Abstract/Free Full Text]
  37. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A., Podkolodny, N. L., and Kolchanov, N. A. (1998) Nucleic Acids Res. 26, 362-367[Abstract/Free Full Text]
  38. Chen, Z. Y., Yant, S. R., He, C. Y., Meuse, L., Shen, S., and Kay, M. A. (2001) Mol. Ther. 3, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  39. Riu, E., Chen, Z. Y., Xu, H., He, C. Y., and Kay, M. A. (2007) Mol. Ther. 15, 1348-1355[CrossRef][Medline] [Order article via Infotrieve]
  40. Morris, T. A., Fong, W. B., Ward, M. J., Hu, H., and Fong, S. L. (1997) Invest. Ophthalmol. Vis. Sci. 38, 196-206[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/16/10881    most recent
M710454200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smyth, V. A.
Right arrow Articles by Kennedy, B. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smyth, V. A.
Right arrow Articles by Kennedy, B. N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement