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PubMed1998

In vivo identification of nuclear factors interacting with the conserved elements of box C/D small nucleolar RNAs.

The U16 small nucleolar RNA (snoRNA) is encoded by the third intron of the L1 (L4, according to the novel nomenclature) ribosomal protein gene of Xenopus laevis and originates from processing of the pre-mRNA in which it resides. The U16 snoRNA belongs to the box C/D snoRNA family, whose members are known to assemble in ribonucleoprotein particles (snoRNPs) containing the protein fibrillarin. We have utilized U16 snoRNA in order to characterize the factors that interact with the conserved elements common to the other members of the box C/D class. In this study, we have analyzed the in vivo assembly of U16 snoRNP particles in X. laevis oocytes and identified the proteins which interact with the RNA by label transfer after UV cross-linking. This analysis revealed two proteins, of 40- and 68-kDa apparent molecular size, which require intact boxes C and D together with the conserved 5',3'-terminal stem for binding. Immunoprecipitation experiments showed that the p40 protein corresponds to fibrillarin, indicating that this protein is intimately associated with the RNA. We propose that fibrillarin and p68 represent the RNA-binding factors common to box C/D snoRNPs and that both proteins are essential for the assembly of snoRNP particles and the stabilization of the snoRNA.

Animals↗

PubMed1998

Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: pivotal role of M boxes (GTCATGTGCT) and of microphthalmia.

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.

Animals↗

PubMed2000

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

Amino Acid Motifs↗

PubMed2000

Identification of a novel element required for processing of intron-encoded box C/D small nucleolar RNAs in Saccharomyces cerevisiae.

Processing of intron-encoded box C/D small nucleolar RNAs (snoRNAs) in metazoans through both the splicing-dependent and -independent pathways requires the conserved core motif formed by boxes C and D and the adjoining 5'-3'-terminal stem. By comparative analysis, we found that five out of six intron-encoded box C/D snoRNAs in yeast do not possess a canonical terminal stem. Instead, complementary regions within the flanking host intron sequences have been identified in all these cases. Here we show that these sequences are essential for processing of U18 and snR38 snoRNAs and that they compensate for the lack of a canonical terminal stem. We also show that the Rnt1p endonuclease, previously shown to be required for the processing of many snoRNAs encoded by monocistronic or polycistronic transcriptional units, is not required for U18 processing. Our results suggest a role of the complementary sequences in the early recognition of intronic snoRNA substrates and point out the importance of base pairing in favoring the communication between boxes C and D at the level of pre-snoRNA molecules for efficient assembly with snoRNP-specific factors.

Animals↗

PubMed2001

Wild-type circadian rhythmicity is dependent on closely spaced E boxes in the Drosophila timeless promoter.

Transcriptional regulation plays an important role in Drosophila melanogaster circadian rhythms. The period promoter has been well studied, but the timeless promoter has not been analyzed in detail. Mutagenesis of the canonical E box in the timeless promoter reduces but does not eliminate timeless mRNA cycling or locomotor activity rhythms. This is because there are at least two other cis-acting elements close to the canonical E box, which can also be transactivated by the circadian transcription factor dCLOCK. These E-box-like sequences cooperate with the canonical E-box element to promote high-amplitude transcription, which is necessary for wild-type rhythmicity.

Animals↗

PubMed2002

Targeted histone acetylation at the yeast CUP1 promoter requires the transcriptional activator, the TATA boxes, and the putative histone acetylase encoded by SPT10.

The relationship between chromatin remodeling and histone acetylation at the yeast CUP1 gene was addressed. CUP1 encodes a metallothionein required for cell growth at high copper concentrations. Induction of CUP1 with copper resulted in targeted acetylation of both H3 and H4 at the CUP1 promoter. Nucleosomes containing upstream activating sequences and sequences farther upstream were the targets for H3 acetylation. Targeted acetylation of H3 and H4 required the transcriptional activator (Ace1p) and the TATA boxes, suggesting that targeted acetylation occurs when TATA-binding protein binds to the TATA box or at a later stage in initiation. We have shown previously that induction results in nucleosome repositioning over the entire CUP1 gene, which requires Ace1p but not the TATA boxes. Therefore, the movement of nucleosomes occurring on CUP1 induction is independent of targeted acetylation. Targeted acetylation of both H3 and H4 also required the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 was lethal at high copper concentrations and correlated with slower induction and reduced maximum levels of CUP1 mRNA. These observations constitute evidence for a novel mechanism of chromatin activation at CUP1, with a major role for the TATA box.

Acetylation↗

PubMed2003

Occupancy and function of the -150 sterol regulatory element and -65 E-box in nutritional regulation of the fatty acid synthase gene in living animals.

Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter.

Animals↗

PubMed2006

Cotranscriptional recognition of human intronic box H/ACA snoRNAs occurs in a splicing-independent manner.

Processing from pre-mRNA introns is a widespread mechanism to generate human box C/D and H/ACA snoRNAs. Recent studies revealed that an optimal position relative to the 3' splice site is important for efficient processing of most box C/D snoRNAs and that assembly of box C/D snoRNPs is stimulated by splicing factors likely bound to the branch point region. Here we have investigated the processing of another major class of human intron-encoded RNAs, the box H/ACA snoRNAs. Analysis of 80 H/ACA RNA genes revealed that human H/ACA RNAs possess no preferential localization close to the 3' or 5' splice site. In vivo processing experiments confirmed that H/ACA intronic snoRNAs are processed in a position-independent manner, indicating that there is no synergy between H/ACA RNA processing and splicing. We also showed that recognition of intronic H/ACA snoRNAs and assembly of pre-snoRNPs is an early event that occurs during transcription elongation parallel with pre-mRNA splice site selection. Finally, we found that efficient processing and correct nucleolar localization of the human U64 H/ACA snoRNA requires RNA polymerase II-mediated synthesis of the U64 precursor. This suggests that polymerase II-associated factors direct the efficient assembly and determine the correct subnuclear trafficking of human H/ACA snoRNPs.

Cell Cycle Proteins↗

PubMed1987

Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter.

We characterized the activity of a human hsp70 gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp70 promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp70 gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.

Base Sequence↗

PubMed1987

Duplicated CArG box domains have positive and mutually dependent regulatory roles in expression of the human alpha-cardiac actin gene.

An upstream region from the transcription initiation site to -177 base pairs (bp) of the human alpha-cardiac actin gene directs the transient expression of a bacterial chloramphenicol acetyltransferase (CAT) gene only in muscle cells (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986). We modified this promoter region by additional 5' deletions, linker-scanning mutations, and insertion-deletion mutations and demonstrated that the asymmetrical sequences in and adjacent to two CArG [for CC(A + T rich)6GG] motifs, located at -140 and -100 bp, play an important positive role in transcription. The significant impairment of transcriptional activity that accompanies the disruption of one CArG box region can be restored by either. This demonstrated that these two elements interact in a mutually dependent and similar manner. Furthermore, a DNA fragment that includes the CArG boxes had significant competitive activity for transcription directed by the alpha-cardiac actin promoter in an in vivo competition assay. We conclude that the two sequences around each CArG box may interact with the same class of trans-acting positive factor(s) and that these interactions may mediate muscle-specific expression. Each of the two CArG regions appears to be bound independently by such a positive factor(s), and the regions support high-level transcription in a synergistic manner. The transcriptional activity of this regulatory region is proportional to its distance from a TATA box (at -30 bp) and is strictly orientation dependent relative to the direction of transcription. Therefore this upstream region is not an enhancer but is a tissue-specific regulatory upstream element.

Actins↗

PubMed1988

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1988

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗

PubMed2006

Preliminary assessment of cognitive function in older adults by clock drawing, box copying and narrative writing.

OBJECTIVE: To examine 3 cognitive screening tasks in older adults and determine which demographic, medical and lifestyle variables obtained 13-17 years previously predicted cognitive performance. DESIGN: Population-based longitudinal cohort study begun in 1981. SETTING: Leisure World Laguna Hills, a California retirement community. PARTICIPANTS: The cohort is predominantly white, well educated, upper-middle class; two thirds are women. Data from 1,744 participants (mean age 83 years, range 57-103) were analyzed. MEASUREMENTS: The baseline postal survey (1981-1985) asked demographic information, medical history, selected drug use and personal habits. The 1998 follow-up included 3 tasks to assess cognitive function: (1) clock drawing, (2) copying a box drawing and (3) narrative writing to describe a pictured scene. RESULTS: Within age groups, women tended to have lower scores than men on box copying, similar scores on clock drawing, but higher informational and complexity scores on narrative writing. Performance decreased with increasing age and was poorest in individuals > or = 90 years old. In addition to age, sex and education, significant predictors of performance were: exercise, body mass index and cataract surgery (clock); vitamin E supplements, daily aspirin use and gallbladder surgery (box); vitamin A supplements (narrative writing). Mean scores on all tasks were lower in those later identified as demented compared with nondemented but significantly different only for clock and box drawings. CONCLUSION: Performance on 3 cognitive tests showed significant effects of age, sex and education and was related to later dementia. Better performance by users of antioxidant supplements and aspirin suggests that these drugs may maintain cognitive function in later life.

Aged↗

PubMed2003

Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.

Changes in the differentiated state of smooth muscle cells (SMCs) play a key role in vascular diseases, yet the mechanisms controlling SMC differentiation are still largely undefined. We addressed the role of basic helix-loop-helix (bHLH) proteins in SMC differentiation by first determining the role of two E-box (CAnnTG) motifs, binding sites for bHLH proteins, in the transcriptional regulation of the SMC differentiation marker gene, smooth muscle alpha-actin (SM alpha-actin), in vivo. Mutation of one or both E-boxes significantly reduced the expression of a -2560- to 2784-bp SM alpha-actin promoter/LacZ reporter gene in vivo in transgenic mice. We then determined the potential role of class I bHLH proteins, E12, E47, HEB, and E2-2, in SM alpha-actin regulation. In cotransfection experiments, E12, HEB, and E2-2 activated the SM alpha-actin promoter. Activation by HEB and E2-2 was synergistic with serum response factor. Additionally, the dominant-negative/inhibitory HLH proteins, Id2, Id3, and Twist, inhibited both the E12 and serum response factor-induced activations of the SM alpha-actin promoter. Finally, we demonstrated that E2A proteins (E12/E47) specifically bound the E-box-containing region of the SM alpha-actin promoter in vivo in the context of intact chromatin in SMCs. Taken together, these results provide the first evidence of E-box-dependent regulation of a SMC differentiation marker gene in vivo in transgenic mice. Moreover, they demonstrate a potential role for class I bHLH factors and their inhibitors, Id and Twist, in SM alpha-actin regulation and suggest that these factors may play an important role in control of SMC differentiation and phenotypic modulation.

3T3 Cells↗

PubMed2000

Specific sequences outside the E-box are required for proper per expression and behavioral rescue.

A 69 bp circadian regulatory sequence (CRS) upstream of the per gene is sufficient to drive circadian transcription, mediate proper spatial expression, and rescue behavioral rhythmicity in per01 flies. Within the CRS, an E-box is required for transcriptional activation by two basic-helix-loop-helix (bHLH) PERARNT-SIM (PAS) transcription factors, dCLOCK (dCLK) and CYCLE (CYC). To define sequences within the CRS that are required for spatial expression, circadian expression, and behavioral rhythmicity, a series of mutants that alter blocks of 3 to 12 nucleotides across the entire CRS were used to drive lacZ or per expression in vivo. As expected, the E-box within the CRS is necessary for high-level expression and behavioral rhythmicity, but sequences outside the E-box are also required for transcriptional activation, proper spatial expression, and behavioral rhythmicity. These results indicate that the dCLK-CYC target site extends beyond the E-box and that factors other than dCLK and CYC modulate per transcription.

ARNTL Transcription Factors↗

PubMed1996

Photic entrainment in hamsters: effects of simulated twilights and nest box availability.

Entrainment of wheel-running activity rhythms was compared in hamsters exposed to daily light-dark (LD) cycles with abrupt transitions between 0 and 10 lux or with artificial twilights simulating summer solstice conditions at 41 degrees N latitude but truncated at 10 lux. The photoperiod in LD-rectangular was set at 16.24 h, equating the total light (in lux.min) emitted under the two schedules. The LD cycles were maintained for 35 days and were followed by 14 days of constant darkness (DD). Half the animals in each condition had access to a dark nest box connected to the outer compartment by a tunnel, the remaining animals being confined to a single compartment. Body temperature and locomotor activity inside the nest boxes were recorded by telemetry. Movements between the nest box and the outer compartment were monitored and the data were used to calculate light exposure at different times of the day. In all groups, the phase angle difference between wheel-running onset and dusk was more positive than that between activity offset and dawn. Hamsters with access to nest boxes, however, had later onsets, earlier offsets, and shorter activity durations (alpha s) than those without. These effects could be accounted for by the difference in light exposure between the nest and no-nest animals, particularly light exposure in the morning. The inclusion of twilights also resulted in later onsets and shorter alpha s, but the differences were relatively small and were only observed in the nest animals. The day-to-day variability in activity onset was negatively correlated with onset time and was smaller in the twilight/nest animals than in the other three groups. Most animals showed an expansion of alpha during the first few days of DD, resulting from a rapid advance of activity onsets relative to offsets. The period of the rhythms, determined from the first five activity onsets in DD, was negatively correlated with the balance of evening and morning light exposure. These results are discussed in the context of nonparametric entrainment of compound pacemakers.

Animals↗

PubMed2004

Distinct activities of suppressor of cytokine signaling (SOCS) proteins and involvement of the SOCS box in controlling G-CSF signaling.

Granulocyte-colony stimulating factor (G-CSF) induces proliferation of myeloid progenitor cells and controls their differentiation into mature neutrophils. Signal transducer and activator of transcription (STAT) proteins STAT3 and STAT5 are activated by G-CSF and play distinct roles in neutrophil development. Suppressor of cytokine signaling (SOCS) proteins are induced by STATs and inhibit signaling through various negative-feedback mechanisms. SOCS proteins can compete with docking of signaling substrates to receptors, interfere with Janus tyrosine kinase activity, and target proteins for proteasomal degradation. The latter process is mediated through the conserved C-terminal SOCS box. We determined the role of various SOCS proteins in controlling G-CSF responses and investigated the involvement of the SOCS box therein. We show that SOCS1 and SOCS3, but not CIS and SOCS2, inhibited G-CSF-induced STAT activation in human embryo kidney 293 cells. In myeloid 32D cells, SOCS1 and SOCS3 are induced by G-CSF. However, relative to interleukin-3-containing cultures, during G-CSF-induced neutrophilic differentiation, SOCS3 expression was further elevated, while SOCS1 levels remained constant. SOCS box deletion mutants of SOCS1 and SOCS3 were severely hampered in their abilities to inhibit STAT activation and to efficiently suppress colony formation by primary myeloid progenitors in response to G-CSF. These data demonstrate the importance of the SOCS box for the inhibitory effects of SOCS proteins on G-CSF signaling and show that among the different SOCS family members, SOCS3 is the major negative regulator of G-CSF responses during neutrophilic differentiation.

Blotting, Western↗

PubMed2003

Two basic amino acids C-terminal of the proximal box specify functional binding of the vitamin D receptor to its rat osteocalcin deoxyribonucleic acid-responsive element.

Nuclear hormone receptor-responsive element binding specificity has been reported to reside predominantly in the proximal box (P-box), three amino acids located in a DNA-recognition alpha-helix situated on the C-terminal side of the first zinc finger. To further define the residues in the vitamin D receptor (VDR) DNA binding domain (DBD) that mediate its interaction as a retinoid X receptor (RXR) heterodimer with the rat osteocalcin vitamin D-responsive element (VDRE), chimeric receptors were created in which the core DBD of VDR was replaced with that of the homodimerizing glucocorticoid receptor (GR). Systematic alteration of GR DBD amino acids in these chimeras to VDR DBD residues identified arg-49 and lys-53, just C-terminal of the P-box within the base recognition alpha-helix of human VDR (hVDR), as the only two amino acids among 36 differences required to convert the GR core zinc finger domain to that of the VDR. Gel mobility shift and 1,25-dihydroxyvitamin D3-stimulated transcription assays verified that an hVDR-GR DBD chimera is functional on the rat osteocalcin VDRE with only the conservative change of lys-49 to arg, and of the negatively charged glu-53 to a basic amino acid (lys or arg). Thus, for RXR heterodimerizing receptors like VDR, the P-box requires redefinition and expansion to include a DNA specificity element corresponding to arg-49 and lys-53 of hVDR. Examination of DNA specificity element amino acids in other nuclear receptors in terms of conservation and base contact in cocrystal structures supports the conclusion that these residues are crucial for selective DNA recognition.

Amino Acid Sequence↗