A gene-specific non-enhancer sequence is critical for expression from the promoter of the small heat shock protein gene αB-crystallin
© Jing et al.; licensee BioMed Central Ltd. 2014
Received: 14 December 2013
Accepted: 10 February 2014
Published: 3 March 2014
Deciphering of the information content of eukaryotic promoters has remained confined to universal landmarks and conserved sequence elements such as enhancers and transcription factor binding motifs, which are considered sufficient for gene activation and regulation. Gene-specific sequences, interspersed between the canonical transacting factor binding sites or adjoining them within a promoter, are generally taken to be devoid of any regulatory information and have therefore been largely ignored. An unanswered question therefore is, do gene-specific sequences within a eukaryotic promoter have a role in gene activation? Here, we present an exhaustive experimental analysis of a gene-specific sequence adjoining the heat shock element (HSE) in the proximal promoter of the small heat shock protein gene, αB-crystallin (cryab). These sequences are highly conserved between the rodents and the humans.
Using human retinal pigment epithelial cells in culture as the host, we have identified a 10-bp gene-specific promoter sequence (GPS), which, unlike an enhancer, controls expression from the promoter of this gene, only when in appropriate position and orientation. Notably, the data suggests that GPS in comparison with the HSE works in a context-independent fashion. Additionally, when moved upstream, about a nucleosome length of DNA (−154 bp) from the transcription start site (TSS), the activity of the promoter is markedly inhibited, suggesting its involvement in local promoter access. Importantly, we demonstrate that deletion of the GPS results in complete loss of cryab promoter activity in transgenic mice.
These data suggest that gene-specific sequences such as the GPS, identified here, may have critical roles in regulating gene-specific activity from eukaryotic promoters.
A eukaryotic promoter is heterogeneous in structure. It contains multiple transacting factor binding sites that are shared amongst multiple genes, yet it contains specific information for how and when a gene should be active. Investigations on eukaryotic promoters have sought a common mechanistic thread in cis-regulatory modules of enhancer sequences and transcription factor binding sites (both distant as well as proximal) for an understanding of the control of gene expression [1–3]. There is, however, a finite number of transcription factors that are shared among a large number of promoters  (at least 70,000 promoters and 1,800 transcription factors) ; thus, combinatorial schemes have been invoked to explain specific gene activation via a ‘regulatory grammar’ that remains to be deciphered [6–9]. Thus, there is no known concrete mechanistic detail that explains the control of specific gene activity .
Our understanding of the regulatory information in the eukaryotic promoters has largely come from functional understanding of the shared presence of universal or conserved sequence elements in different genes [1–3, 11, 12] and has established a major role for transcription factors (transacting factors, coactivators, and basal factors) and their binding sequences in the regulation of gene activity [4, 6, 13, 14]. The import of gene-specific sequences, if any, in the regulation has thus remained uninvestigated. While the commonality of the sequence elements in the promoters of various genes has contributed to the identification and validation of shared sequence motifs experimentally as well as computationally, these approaches, however, cannot be meaningfully applied for the elucidation of the role of gene-specific promoter sequences. At this time, the role of gene-specific sequences can only be determined experimentally, on a gene to gene basis. In this investigation, we have examined one such gene-specific sequence adjoining the heat shock element (HSE) in the proximal promoter of the small heat shock protein gene αB-crystallin (cryab) and found it to be essential for expression both in cultured cells as well as in transgenic mice.
Cryab is the archetypical, conserved, small heat shock protein gene expressed ubiquitously in multiple tissues in vertebrates in a developmentally dictated fashion. Its expression attends a host of pathologies ranging from cardiomyopathies and cataracts to oncogenesis and neurodegenerations such as Alzheimer's disease, multiple sclerosis, and age-related macular degeneration [15, 16]. In specific cell types, in culture, it is also expressed in response to heat and osmotic stress [17–19].
A 10-bp sequence in the cryabpromoter is required for expression in cultured ARPE cells
Much against this perception, however, the 10-bp sequence adjoining the HSE seems to work in a context-independent fashion. This is revealed by the observation that the same level of inhibition is obtained when this sequence is mutated within a complete promoter (Figure 2A, numbers 1–6) as well as when it is part of the truncated version of the promoter (Figure 2A, numbers 7–9). In comparison, when HSE (−54/−40) in the truncated promoter is mutated, the inhibition of the expression is not as pronounced as when the whole promoter is used (Figure 2A, compare numbers 1 and 3 and numbers 7 and 8) reiterating the known context-dependent  functioning of individual promoter motifs or a transcription factor binding site, in this case, the HSE. We conclude that the 5′ 10-bp sequence (−64/−55) adjoining the HSE contains information that is required for expression from the cryab promoter in human ARPE cells.
The 10-bp sequence functions in a position and orientation-specific fashion
Impact of GPS on promoter activity when moved to various distances from the transcription start site
The GPS, when moved to the 3′ of the HSE-αB inhibits expression (Figure 3A, number 5). The GPS was also moved to −71, −154, −267, −392, and −496 (with transcription start site (TSS) as +1) which corresponds to 7, 90, 203, 328 and 432 bp upstream from its original position (at −64/−55), respectively (Figure 3B). When moved upstream to −71 or −154 positions, the effect on expression is minimal (Figure 3B, numbers 6 and 7). However, when moved farther than −154 bp from the TSS, there is a precipitous loss of promoter activity (Figure 3B, numbers 3–5).
It is important to note that the movement of the GPS from its original site to a new site (Figure 3B) does not disrupt any essential sequences required for expression. GPS is part of one of the two consensus Pax6 binding sites (−160/−140 and −77/−55) in the cryab promoter (Figure 1A) [21–23, 28]. Note that moving the GPS from −64 to −71, which disrupts the proximal Pax6 site, hardly impacts the expression (Figure 3B, number 7) as does the placement of the GPS at −154, which disrupts the distal Pax6 site (see Figure 1A), reducing the expression only by about 15% (Figure 3B, number 6) suggesting that neither Pax6 site significantly contributes to the expression from the cryab promoter in ARPE cells in culture.
GPS is required for expression in transgenic mice
In a multicellular organism, differential gene activity is the outcome of the initial decision that a cell makes whether a particular gene should be active or inactive followed by the modulation of the gene activity by the tissue/organ function. The data presented here demonstrates an overriding requirement for a GPS in the functioning of the cryab promoter both in cells in culture as well as in the transgenic mice (Figures 2, 3, 4, 5, and 6). Significantly, the GPS, unlike an enhancer, is position and orientation-specific (Figure 3).
The proximity of the GPS to the TSS and its orientation and position-specific function in the cryab promoter may suggest a simpler and direct mechanism of gene-specific control through its involvement with the development of transcriptional competence (or opening up of a promoter) . The analysis presented here, however, does not preclude the existence of GPS-like elements at longer distances from the TSS in the eukaryotic promoters. It is interesting to note that sequences 100 bp upstream of the TSS have been previously suggested to control the lymphoid cell specificity of the expression from a κ-light chain immunoglobulin promoter .
The GPS identified here is either a binding site or a hub for transacting factor(s) or simply a landmark that dictates the physical state of the chromatin that allows gene activity [34–39]. The data presented in Figure 3 provides significant insight about the relationship between expression and the location of the GPS (distance of this sequence from the TSS). We know that changing the location of the GPS from the 5′ to the 3′ side of HSE-αB inhibits expression (Figure 3A, number 5). However, moving it more than 90 bp, 5′ upstream of the HSE (−64/−35), reduces the promoter activity marginally (Figure 3B, number 6). This tolerance to change in location, 5′ upstream of the HSE, becomes unacceptable when the GPS is moved more than 154 bp from the TSS, which results in drastic inhibition of the promoter activity (Figure 3B, numbers 2–5). A plot of the expression versus distance of the GPS from the TSS indicates a biphasic response, a slow less dramatic phase when at positions −71 and −154 and a fast declining component beyond −154 (Figure 3C). This data leads to two important inferences: (1) GPS must remain in proximity of the TSS, 5′ to the HSE to be functional, and (2) considering that 154 bp is roughly the size of DNA wrapped around a nucleosome bead, the GPS may have an influence on nucleosome spacing and/or the physical status of the nucleosomes in the vicinity of the TSS [35, 36]. It is known that HSF4 (that binds to the HSE) has been reported to recruit BRG1 (Brahma-related gene 1), a member of the chromatin remodeling complex to cryab promoter [38, 39] suggesting a possible function of the GPS via positioning of the nucleosomes in regulating access to the promoter.
While it remains to be established if trans-acting factor binding sites (including transcription factors) become functional only in the presence of a GPS, it is tempting to speculate that the apparent promiscuity in some DNA binding transcription factors, e.g., Pax6 [37, 40] and possibly HSF4, may be brought about by gene-specific sequences like the GPS.
We have demonstrated recently that HSF4 is detected on the cryab promoter in ARPE cells indicative of its involvement in the expression from this promoter . In light of this observation, the inhibition of the promoter activity upon deletion of GPS (Figure 3A, number 2) or upon change of its position (Figure 3A, number 5) suggests that HSF4 binding to HSE is not enough for eliciting gene activity but may also require a functional GPS. If this interpretation is extrapolated to the data obtained with transgenic mice, it is obvious that GPS may be essential for keeping the promoter open (active). This is borne out by the complete inhibition of cryab promoter activity in multiple tissues in transgenic mice made with constructs without the GPS (ΔGPS) in comparison with constructs that contained GPS (+GPS) (Figures 5 and 6). These data suggest that GPS may be obligatory for the activation of cryab transcription.
We have identified a non-enhancer gene-specific, position- and orientation-dictated 10-bp sequence (GPS) within the heat shock promoter of the αB-crystallin gene that is required for expression from this promoter, in cultured cells as well as in transgenic mice. The data presented here brings up three important corollaries: (1) Since GPS is essential for expression even before transcription factors and/or enhancer sequences get involved, the initial activation of a gene may be dictated by the gene-specific information in the promoter DNA. (2) Because GPS sequences do not represent universal motifs, they cannot be computed. Thus, they may have to be identified through labor-intensive experimentation as done here on a gene-to-gene basis. (3) GPS sequences could become targets for manipulation of a cell's phenotype.
Construction of recombinant plasmids
A 940-bp DNA fragment, −896/+44 (upstream of the ATG in the first exon of the αB gene) was amplified from the rat (Sprague Dawley) genomic DNA using primers: F (forward) 5′-ATAGTGCCGAGCCTCTTG-3′ and R (reverse) 5′-GGGAGTGGAAAGGAAAGAA-3′ and cloned into pTOPO4 vector (Invitrogen, Carlsbad, CA, USA). This promoter sequence in pTOPO4 was used as the template for all downstream manipulations. The −896/+44 sequences represent complete rat cryab promoter. Beyond −896, there is another gene (HspB2), which is transcribed in the opposite orientation .
Oligonucleotides used in site- directed mutagenesis
Primer set number
Figure 2A/number 2
Figure 2A/number 3
Figure 2A/number 5
Figure 2A/number 9
Figure 2B/number 2
Figure 2B/number 3
Figure 2B/number 4
Figure 3A/number 3
Figure 3A/number 4
Figure 3A/number 5
Figure 3B/number 2
Figure 3B/number 3
Figure 3B/number 4
Figure 3B/number 5
Figure 3B/number 6
Figure 3B/number 7
Figure 4/number 5
Figure 4/number 6
Cell culture and transfection experiments
ARPE-19 cells (ATCC, Manassas, VA, USA)  at 70% to 90% confluence were transfected with a mixture of experimental αB-tGFP plasmid DNA and pCMV-DsRed vector (Clontech, Mountain View, CA, USA) (50:1) using Lipofectamine2000 (Invitrogen). The pCMV-DsRed plasmid was used as an internal standard to normalize transfection efficiency. The experiments were done in triplicate and repeated three times.
Transgenic mice and genotyping
The animal care and use protocol were followed as per institutional guidelines of the Animal Research Committee, University of California, Los Angeles, CA, USA. The whole promoter αB-tGFP constructs with or without GPS (construct with GPS shown in Figure 5A) were double digested with Xho I and Afl II to obtain a 2-kb fragment containing polyA signal (polyA is from the backbone of pTurbo-GFP-pRL plasmid). The fragment (αB-tGFP-polyA) was purified from the vector backbone and used for the generation of transgenic mice  at the UCLA Transgenic/Knockout Injection Facility. We generated five founders for + GPS and nine founders for ΔGPS constructs. Three lines each for + GPS and ΔGPS were examined for expression of the tGFP.
Genotyping was performed using PureLink™ Genomic DNA Mini Kit (Invitrogen) employing two primer sets (1F 5′-GTGTCACCCTGCCAAATC-3′, 1R 5′-GCTCGAACTCCACGCCGTT-3′; 2F 5′-GCCACCATGGAGAGCGACGAGA-3′, 2R 5′-GATGCGGGTGTTGGTGTAG-3′). To determine the copy number of αB-tGFP inserts in different transgenic strains, absolute qPCR assays were performed with 10-ng genomic DNA using the LightCycler 480 SYBR Master Mix (Roche, Indianapolis, IN, USA) [43, 44]. The whole promoter αB-tGFP constructs were serial diluted as template, and four different amounts of DNA (1 ng, 100, 10, and 1 pg) were used in a 10-μl reaction for the generation of the standard curve. All reactions were done in triplicate. The qPCR thermal cycling conditions were as follows: 95°C for 5 min for hot start, followed by 45 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 30 s. Specific primers were used in SYBR Green qPCRs were as follows: tGFP: 2F 5′-GCCACCATGGAGAGCGACGAGA-3′, 2R 5′-GATGCGGGTGTTGGTGTAG-3′. The average copy number in + GPS and ΔGPS transgenic mice were determined to be 6.2 and 18.0, respectively.
Confocal microscopy and immunofluorescence
The whole embryos from + GPS (embryonic day 16, E16) and ΔGPS transgenic mice (embryonic day 15, E15) were fixed in 4% paraformaldehyde and processed as detailed previously  using anti-tGFP antibody (Axxora LLC., San Diego, CA, USA). Serial z-stack images were acquired from the whole eye, heart, and liver using a confocal microscope (FluoView 1000, Olympus, Tokyo, Japan) and processed using Adobe Photoshop Elements version 9.
Immunoblotting and RT-qPCR
Mouse tissue extracts (post-natal, day 10 pups) were prepared in T-PER Protein Extraction Reagent (Pierce, Rockford, IL, USA). About 30 μg of protein/lane was electrophoresed on 4% to 12% SDS-PAGE gradient gels (Invitrogen) and transferred to nitrocellulose membranes for immunoblotting . The reactive protein bands (anti-tGFP) were quantified using the LiCOR Odyssey dual wavelength IR system (LiCOR Biosciences, Lincoln, NE, USA). Gapdh was used as an internal control for all blots. Similar data was obtained with three lines of + GPS and ΔGPS transgenic lines.
Total RNAs were extracted 48 h after transfection of ARPE cells or from mouse tissues using TRIzol Plus RNA Purification System (Invitrogen, Carlsbad, CA, USA). RT-qPCR was conducted as described . RT-qPCRs were performed in triplicate for each RNA sample in the Lightcycler 480 (Roche) (95°C for 5 min, followed by 45 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 30 s). To calculate the relative change of tGFP expression, PCRs were normalized with reference to corresponding internal controls (DsRed RNA isolated for transiently transfected APRE-19 cells and Gapdh RNA for transgenic and wild-type mice tissues using the ΔΔCt method) and expressed as a percentage of the wild-type construct. Specific primers used were as follows: tGFP: F 5′-CTACCACTTCGGCACCTACC-3′, and R 5′-GATGCGGGTGTTGGTGTAG-3′; DsRed: F 5′-TACCTGGTGGAGTTCAAGTCC-3′ and R 5′-TCGTTGTGGGAGGTGATGT-3′. Gapdh: F 5′-GGTGAAGGTCGGTGTGAACG-3′ and R 5′-CTCGCTCCTGGAAGATGGTG-3′. We also assessed expression of tGFP in transfected ARPE cells (Figures 2, 3, and 4) with immunoblotting. This data mirrored the RT-qPCR data and is therefore not shown.
This work was supported by NIH grants to Suraj P Bhat. We thank Garen Polatoglu and Josh Lee for the technical help and for the help with purification of antibodies and immunoblotting. We thank UCLA Transgenic core facilities (Dr. Meisheng Jiang and Yoshiko Nagaoka) for their guidance in generating the transgenic mice.
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