Targeting a SWI/SNF-related chromatin remodeling complex to the β-globin promoter in erythroid cells

Chromatin remodeling complexes such as the SWI/SNF complex make DNA accessible to transcription factors by disrupting nucleosomes. However, it is not known how such complexes are targeted to the promoter. For example, a SWI/SNF1-like chromatin remodeling complex erythroid Krüppel-like factor (EKLF) coactivator-remodeling complex 1 (E-RC1) disrupts the nucleosomes over the human β-globin promoter in an EKLF-dependent manner. However, it is not known whether E-RC1 is targeted specifically to the β-globin promoter or whether E-RC1 is randomly targeted, but its activity is evident only at the β-globin promoter. Because E-RC1 cannot remodel chromatin over the β-globin promoter without EKLF in vitro, it has been proposed that SWI/SNF1-like complexes such as E-RC1 are targeted specifically to the promoter by selectively interacting with promoter-associated transcription factors such as EKLF. In this report, we test this hypothesis in the cellular context by using the ProteIN POsition Identification with Nuclease Tail (PIN*POINT) assay. We find that the Brahma-related gene (BRG) 1 and BRG1-associated factor (BAF) 170 subunits of E-RC1 are both recruited near the transcription initiation site of the β-globin promoter. On transiently transfected templates, both the locus control region and the EKLF-binding site are important for their recruitment to the β-globin promoter in mouse erythroleukemia cells. When the β-globin promoter was linked to the cytomegalovirus enhancer, the E-RC1 complex was not recruited, suggesting that recruitment of the E-RC1 complex is not a general property of enhancers.

RAG2 is regulated differentially in B and T cells by elements 5′ of the promoter

To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5′ of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5′ region starting just upstream of the RAG2 promoter, as well as the region from 2–7 kb 5′, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5′ region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5′ elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5′ region.

Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at −30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30–31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

Cloning and characterization of the mouse vitamin D receptor promoter

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5′-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5′-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5′-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

The 3′-untranslated region of CaMKIIα is a cis-acting signal for the localization and translation of mRNA in dendrites

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The α subunit of Ca2+-calmodulin-dependent protein kinase II (CaMKIIα) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKIIα provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKIIα promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3′-untranslated region of the CaMKIIα gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKIIα gene, only the lacZ transcripts bearing the 3′-untranslated region are localized to dendrites. The β-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

The structural basis for the oriented assembly of a TBP/TFB/promoter complex

Recently the definition of the metazoan RNA polymerase II and archaeal core promoters has been expanded to include a region immediately upstream of the TATA box called the B recognition element (BRE), so named because eukaryal transcription factor TFIIB and its archaeal orthologue TFB interact with the element in a sequence-specific manner. Here we present the 2.4-Å crystal structure of archaeal TBP and the C-terminal core of TFB (TFBc) in a complex with an extended TATA-box-containing promoter that provides a detailed picture of the stereospecific interactions between the BRE and a helix–turn–helix motif in the C-terminal cyclin repeat of TFBc. This interaction is important in determining the level of basal transcription and explicitly defines the direction of transcription.

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor β (TGF-β)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4−/− breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-β response element in the region −496/−444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1–5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-β-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

A σ32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity

σ32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of σ32 with other sigma factors in the σ70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant σ32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of σ70, which competes with σ32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σD restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σA also has a dramatic effect on start-site selection even though the σA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σA. Thus, σA appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.

A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo

Polymorphic regions consisting of a variable number of tandem repeats within intron 2 of the gene coding for the serotonin transporter protein 5-HTT have been associated with susceptibility to affective disorders. We have cloned two of these intronic polymorphisms, Stin2.10 and Stin2.12, into an expression vector containing a heterologous minimal promoter and the bacterial LacZ reporter gene. These constructs were then used to produce transgenic mice. In embryonic day 10.5 embryos, both Stin2.10 and Stin2.12 produced consistent β-galactosidase expression in the embryonic midbrain, hindbrain, and spinal cord floor plate. However, we observed that the levels of β-galactosidase expression produced by both the Stin2.10 and Stin2.12 within the rostral hindbrain differed significantly at embryonic day 10.5. Our data suggest that these polymorphic variable number of tandem repeats regions act as transcriptional regulators and have allele-dependent differential enhancer-like properties within an area of the hindbrain where the 5-HTT gene is known to be transcribed at this stage of development.

Silencer elements controlling the B29 (Igβ) promoter are neither promoter- nor cell-type-specific

The murine B29 (Igβ) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5′ DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5′ DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5′ segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5′ segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5′ of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5′ DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Activation of Utrophin Promoter by Heregulin via the ets-related Transcription Factor Complex GA-binding Protein α/β

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein α/β transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein α/β complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal–regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne’s muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

Lipophosphoglycan from Leishmania suppresses agonist-induced interleukin 1β gene expression in human monocytes via a unique promoter sequence

Leishmania are parasites that survive within macrophages by mechanism(s) not entirely known. Depression of cellular immunity and diminished production of interleukin 1β (IL-1β) and tumor necrosis factor α are potential ways by which the parasite survives within macrophages. We examined the mechanism(s) by which lipophosphoglycan (LPG), a major glycolipid of Leishmania, perturbs cytokine gene expression. LPG treatment of THP-1 monocytes suppressed endotoxin induction of IL-1β steady-state mRNA by greater than 90%, while having no effect on the expression of a control gene. The addition of LPG 2 h before or 2 h after endotoxin challenge significantly suppressed steady-state IL-1β mRNA by 90% and 70%, respectively. LPG also inhibited tumor necrosis factor α and Staphylococcus induction of IL-1β gene expression. The inhibitory effect of LPG is agonist-specific because LPG did not suppress the induction of IL-1β mRNA by phorbol 12-myristate 13-acetate. A unique DNA sequence located within the −310 to −57 nucleotide region of the IL-1β promoter was found to mediate LPG’s inhibitory activity. The requirement for the −310 to −57 promoter gene sequence for LPG’s effect is demonstrated by the abrogation of LPG’s inhibitory activity by truncation or deletion of the −310 to −57 promoter gene sequence. Furthermore, the minimal IL-1β promoter (positions −310 to +15) mediated LPG’s inhibitory activity with dose and kinetic profiles that were similar to LPG’s suppression of steady-state IL-1β mRNA. These findings delineated a promoter gene sequence that responds to LPG to act as a “gene silencer,” a function, to our knowledge, not previously described. LPG’s inhibitory activity for several mediators of inflammation and the persistence of significant inhibitory activity 2 h after endotoxin challenge suggest that LPG has therapeutic potential and may be exploited for therapy of sepsis, acute respiratory distress syndrome, and autoimmune diseases.