S1 Nuclease Protection Research Articles

LC-TOF-MS methods to quantify siRNAs and major metabolite in plasma, urine and tissues.

There are a few different bioanalytical approaches that have been used for the quantification of siRNA in biological matrices, such as S1 nuclease protection 'cutting ELISA', fluorescent probe hybridization HPLC, HPLC UV, LC-MS/high-resolution accurate-mass (HRAM) and LC-MS/MS. We have developed and validated plasma assays for several oligonucleotides such as GalNAc-conjugated siRNA, using uHPLC and high-resolution mass spectrometer by TOF detection. Although the molecular weights are in the range of 7000-9000, we were able to meet the same assay acceptance criteria as for the small molecules based on regulatory bioanalytical method validation guidance. The antisense strand and the sense strand can both be monitored. The method was also used in the tissue lysate matrices without a full validation.

Chicken GHR antisense transcript regulates its sense transcript in hepatocytes

An increasing number of evidences indicated that long noncoding RNAs (LncRNAs) regulate a variety of biological progresses via different mechanisms. Our previous study had identified a chicken growth hormone receptor (GHR) antisense transcript (GHR-AS) which regulated GHR sense transcript (GHR-S) in LMH cells. In the present study, roles of GHR-AS and its regulatory mechanism were analyzed in chicken hepatocytes. The expression patterns of liver GHR-S, GHR-AS and Let-7b ascended with the development of chicken. The hepatocytes proliferation was promoted and more cells entered into DNA synthesis (S) phase when GHR-AS was overexpressed while the cell proliferation was slowed and fewer cells were in S phase when GHR-AS was interfered. Meanwhile, the GHR-S increased when we overexpressed GHR-AS while it reduced when GHR-AS was inhibited. The S1 Nuclease protection assay indicated that GHR-S and GHR-AS formed RNA duplex via GHR-S 3′ untranslation regon (3′UTR). In hepatocytes or LMH cells, the half-time of GHR-S showed a delayed trend when GHR-AS or GHR-AS 5′ untranslation regon (5′UTR) was overexpressed. Furthermore, the level of GHR-S can be decreased by Let-7b mimics whereas it was partially rescued when co-transfected pGHR-AS or pGHR-AS 5′UTR with Let-7b mimics. Based on our findings, GHR-AS affected hepatocytes proliferation and improved GHR-S stability possibly by forming RNA duplex between GHR-S and GHR-AS, competing with Let-7b.

Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system.

Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.

LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

Identification of the in vitro target of an iron-responsive AraC-like protein from Neisseria meningitidis that is in a regulatory cascade with Fur

In this study we characterized a genetic locus that is predicted to encode one of the three AraC-like regulators of Neisseria meningitidis, a homologue of MpeR of Neisseria gonorrhoeae which is specific to the pathogenic Neisseria species. Previous microarray studies have suggested that this gene is a member of the Fur regulon. In strain MC58, it is a pseudogene (annotated as two ORFs, NMB1879 and NMB1878) containing a frameshift mutation which we show is common to all strains tested belonging to the ST-32 hypervirulent clonal complex. Using primer extension and S1 nuclease protection assays, we mapped two promoters in the upstream intergenic region: the mpeR promoter and the NMB1880 promoter. The latter promoter drives transcription of the divergent upstream locus, which is predicted to encode a high-affinity iron uptake system. We demonstrated that both promoters are induced during iron limitation and that this regulation is also mediated by the Fur regulator. DNA-binding studies with the purified MpeR protein revealed that it binds to a region directly upstream of the NMB1880 divergent promoter, suggesting a role in its regulation. Mutants of N. meningitidis strains lacking MpeR or overexpressing MpeR showed no significant differences in expression of the P(NMB1880) promoter, nor did global transcriptional profiling of an MpeR knockout identify any deregulated genes, suggesting that the MpeR protein is inactive under the conditions used in these experiments. The presence of MpeR in a regulatory cascade downstream of the Fur master iron regulator implicates it as being expressed in the iron-limiting environment of the host, where it may in turn regulate a group of genes, including the divergent iron transport locus, in response to signals important for infection.

Feed-Forward Regulation of Microbisporicin Biosynthesis in Microbispora corallina

Lantibiotics are ribosomally synthesized, posttranslationally modified peptide antibiotics. Microbisporicin is a potent lantibiotic produced by the actinomycete Microbispora corallina and contains unique chlorinated tryptophan and dihydroxyproline residues. The biosynthetic gene cluster for microbisporicin encodes several putative regulatory proteins, including, uniquely, an extracytoplasmic function (ECF) σ factor, σ(MibX), a likely cognate anti-σ factor, MibW, and a potential helix-turn-helix DNA binding protein, MibR. Here we examine the roles of these proteins in regulating microbisporicin biosynthesis. S1 nuclease protection assays were used to determine transcriptional start sites in the microbisporicin gene cluster and confirmed the presence of the likely ECF sigma factor -10 and -35 sequences in five out of six promoters. In contrast, the promoter of mibA, encoding the microbisporicin prepropeptide, has a typical Streptomyces vegetative sigma factor consensus sequence. The ECF sigma factor σ(MibX) was shown to interact with the putative anti-sigma factor MibW in Escherichia coli using bacterial two-hybrid analysis. σ(MibX) autoregulates its own expression but does not directly regulate expression of mibA. On the basis of quantitative reverse transcriptase PCR (qRT-PCR) data, we propose a model for the biosynthesis of microbisporicin in which MibR functions as an essential master regulator and the ECF sigma factor/anti-sigma factor pair, σ(MibX)/MibW, induces feed-forward biosynthesis of microbisporicin and producer immunity.

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

Autoregulation of hpdR and its effect on CDA biosynthesis in Streptomyces coelicolor

HpdR, an IclR-family regulator in Streptomyces coelicolor, is a substrate-dependent repressor for the tyrosine catabolic gene hppD. In this study, S1 nuclease protection assays revealed that hpdR is subject to a negative autoregulation. Purified HpdR showed specific DNA-binding activity for the promoter region of hpdR, indicating that the autoregulation of hpdR is performed directly. The disruption of hpdR led to reduced production of CDA by S. coelicolor J1501, suggesting a positive effect of hpdR on CDA biosynthesis. Electrophoretic mobility shift assays showed that HpdR specifically bound to the promoter region of hmaS (SCO3229 in the CDA gene cluster), encoding 4-hydroxymandelic acid synthase. Disruption of hmaS in J1501 abolished CDA production. It is possible that hpdR regulates CDA biosynthesis by controlling the transcription of hmaS.

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochon­drial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y) revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

The mitochondrial housekeeping gene 16S is inappropriate as an internal standard in comparative studies of rare mitochondrial transcripts using S1-nuclease protection assays

The analysis of rare mitochondrial transcripts derived from the L-strand of the mitochondrial genome requires a sensitive method such as the S1-nuclease protection assay. We examined whether the ribosomal mitochon­drial transcript 16S is suitable as an internal standard in a multiplex S1-nuclease protection assay for the measurement of different mitochondrial transcripts. For reliable quantification of rare mitochondrial transcripts with the RNase protection assay, a minimum of 2 μg of total RNA is necessary. Standard curves of 16S RNA produced with total RNA from human kidney, liver, brain, and a human neuroblastoma cell line (SH-SY5Y) revealed dose-response relationships that were saturated already at less than 0.5 μg of total RNA. Therefore, 16S is inappropriate as an internal standard for analyzing mitochondrial transcripts with RNase protection assays when more than 0.5 μg of total RNA have to be analyzed.

Desmin-regulated Lentiviral Vectors for Skeletal Muscle Gene Transfer

Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors.

Identification of sas, a conserved gene cluster involved in the regulation of aerial mycelium formation in Streptomyces griseus

We cloned a DNA fragment that suppressed the aerial-mycelium-deficient phenotype in an amfS mutant of Streptomyces griseus when it was introduced into the cells via a high-copy-number plasmid. The sasABCDR gene cluster was identified as being responsible for this suppressive activity. The proteins encoded by sasABCD were of unknown function, but the operon structure was found to be conserved in all the strains of Streptomyces spp. and related organisms whose genomes have been sequenced. sasR, the flanking opposite coding sequence, encoded a putative DNA-binding protein. Subcloning revealed that the presence of all five coding sequences was essential for complete suppression. Scanning electron microscopy of Streptomyces griseus strains carrying the sas gene cluster at a high copy-number revealed that bundle-like structures consisting of several aerial hyphae were often formed. S1 nuclease protection analyses were performed to identify the transcriptional start site in the promoters preceding sasA and sasR. The promoter preceding sasA was highly active during vegetative growth. Null mutants for sasABCD among the S. griseus and S. coelicolor A3(2) cells exhibited bald phenotypes; this suggested a positive regulatory role of this gene cluster in the onset of morphogenesis in these two phylogenetically distinct Streptomyces species.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

A signal transduction system in Streptomyces coelicolor that activates expression of a putative cell wall glycan operon in response to vancomycin and other cell wall‐specific antibiotics

This article was published in Mol Microbiol (2002) 44(5), 1199–1211. The authors wish to make the following correction. The panel labelled “ΔsigE” in Fig. 3 (B) was in error, and is in fact the data from a ΔcseA mutant, correctly published as such in Hutchings et al. (2006), and the ΔsigE panel has therefore never been published. To clarify, the complete data set, originally split between the two papers, is reproduced here. Figure. Induction of the sigE promoter (sigEp) in its native chromosomal context by vancomycin. S1 nuclease protection analysis of the sigE transcript in S. coelicolor M600 (wt), J2130 (ΔsigE), J2172 (ΔcseA), and J2142 (ΔcseB::hyg) grown to mid-late exponential phase in NMMP liquid medium containing 2 mM MgCl2 and exposed to 10 µg/ml vancomycin. RNA was extracted from samples taken immediately prior to the addition of vancomycin and at subsequent 30 min intervals. Relative signal strengths were quantified on a Phosphorimager; each signal is expressed as a percentage of the strongest signal.

Regulation of the Yeast Ace2 Transcription Factor during the Cell Cycle*

Previous studies have revealed many parallels in the cell cycle regulation of the Ace2 and Swi5 transcription factors. Although both proteins begin entry into the nucleus near the start of mitosis, here we show that Ace2 accumulates in the nucleus and binds DNA about 10 min later in the cell cycle than Swi5. We used chimeric fusions to identify the N-terminal region of Ace2 as responsible for the delay, and this same region of Ace2 was required for interaction with Cbk1, a kinase necessary for both transcriptional activation by Ace2 and asymmetric distribution of Ace2. Ace2 and Swi5 also showed differences in prevalence during the cell cycle. Swi5 is apparently degraded soon after nuclear entry, whereas constant Ace2 levels throughout the cell cycle suggest Ace2 is exported from the nucleus. Our work suggests that the precise timing of Ace2 accumulation in the nucleus involves both a nuclear export sequence and a nuclear localization signal, whose activities are regulated by phosphorylation.

Zinc deficiency response in Saccharomyces cerevisiae: Characterization of the Zap1‐responsive regulon

The yeast Zap1 transcription factor is a central player in zinc homeostasis. To identify genes important for low zinc growth, DNA microarrays were used to identify genes directly regulated by Zap1. A previous study indicated that Zap1 activates expression of 46 genes under moderate zinc deficiency. Our new experiments identified Zap1 targets that respond only to severe deficiency. We also applied motif identification tools to find candidate Zap1 binding sites (ZREs) in the promoters of these genes. Using these methods, we found 31 new Zap1 target genes. We confirmed the expression levels of several by S1 nuclease protection assay and verified Zap1 binding to putative ZREs by EMSA. We also identified 36 genes that are repressed in a Zap1‐responsive manner. Among these genes, MET3/14/16 encode enzymes of sulfur assimilation. It was known that these genes are regulated by the Met4 transcriptional activator via ubiquitination by Met30, a component of SCF ubiquitin ligase. We found that MET30 is a Zap1 target up‐regulated under severe zinc deficiency. We also showed that less Met4 protein accumulates in zinc‐limited cells and in cells expressing a constitutive Zap1 allele. These results suggest that MET3/14/16 genes repression is due, at least in part, to Met4 down‐regulation following MET30 induction by Zap1. Hence, Zap1‐mediated repression may control sulfur assimilation during the zinc deficiency.

Transcription of all amoC copies is associated with recovery of Nitrosomonas europaea from ammonia starvation.

The chemolithotrophic ammonia-oxidizing bacterium Nitrosomonas europaea is known to be highly resistant to starvation conditions. The transcriptional response of N. europaea to ammonia addition following short- and long-term starvation was examined by primer extension and S1 nuclease protection analyses of genes encoding enzymes for ammonia oxidation (amoCAB operons) and CO(2) fixation (cbbLS), a third, lone copy of amoC (amoC(3)), and two representative housekeeping genes (glyA and rpsJ). Primer extension analysis of RNA isolated from growing, starved, and recovering cells revealed two differentially regulated promoters upstream of the two amoCAB operons. The distal sigma(70) type amoCAB promoter was constitutively active in the presence of ammonia, but the proximal promoter was only active when cells were recovering from ammonia starvation. The lone, divergent copy of amoC (amoC(3)) was expressed only during recovery. Both the proximal amoC(1,2) promoter and the amoC(3) promoter are similar to gram-negative sigma(E) promoters, thus implicating sigma(E) in the regulation of the recovery response. Although modeling of subunit interactions suggested that a nonconservative proline substitution in AmoC(3) may modify the activity of the holoenzyme, characterization of a DeltaamoC(3) strain showed no significant difference in starvation recovery under conditions evaluated. In contrast to the amo transcripts, a delayed appearance of transcripts for a gene required for CO(2) fixation (cbbL) suggested that its transcription is retarded until sufficient energy is available. Overall, these data revealed a programmed exit from starvation likely involving regulation by sigma(E) and the coordinated regulation of catabolic and anabolic genes.

Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>or=10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP-1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>or=10 nm), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.

Persistent Human γ-Globin Expression in Adult Transgenic Mice Following Selective Deletion of Two Repressor Elements Located 3′ to the Aγ-Globin Gene.

Adult β-globin gene expression is tightly regulated during development and hematopoiesis. The human globin genes undergoing two developmental switches are regulated by a complex interplay between cis-acting elements and stage-specific trans-acting factors. Understanding the molecular basis of globin gene switching is of particular interest as persistent expression of the fetal γ-globin genes in the adult ameliorates the effects of hemoglobinopathies. Natural occurring deletions within the human β-globin gene cluster lead to specific clinical syndromes characterized by increased production of fetal hemoglobin (HbF) in adult life. These clinical syndromes provide an excellent model to reveal and delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. One major hypothesis, which accounts for these distinct phenotypic features, assumes that silencers located within the Aγ to δ gene region, are deleted in both HPFH and δβ-thalassemias leading to the failure of switching.Previous studies of our laboratory suggested that four elements (Enh, F, O and P) located within the Aγ toδ globin intergenic region, exhibited silencer activity in transient assays (Clin Res 41:308, 1993 and Blood 84:506, 1994) and that the Enh and F elements were capable of down-regulating transcription of the human β-globin locus in an embryonic-specific manner in transgenic mice (Exp Hematol 32:224, 2004). In the present study, we sought to further clarify the in vivo role of the Enh and F elements in the silencing of the fetal Aγ-gene. To this end, we have generated transgenic mice by using cosmid constructs containing the full length human globin LCR linked to the 3.3 kb Aγ gene, lacking both the Enh and F elements. As controls, we used transgenic lines containing the full length LCR linked to the 5.6 kb Aγ-gene construct, which includes both the Enh and F elements, previously shown by us (Blood 102:3412, 2003) and others (Nature 350:252, 1991) to be autonomously regulated during the perinatal period.Three transgenic lines for the LCR 3.3 kb Aγ-gene construct have been generated. Cosmid integrity and copy numbers (2, 3 and 4 copies respectively) were determined by Southern blot analysis. Expression analysis in adult blood RNA performed by S1 nuclease protection and real-time reverse transcriptase PCR, documented persistence of expression of Aγ-gene in adult life. To further investigate whether the persistence of Aγ-gene expression was not a non-specific effect of the multicopy integrants, we generated a new series of single copy mice by cross-breeding the three transgenic lines with a line expressing the Cre recombinase gene (CAG-Cre). As expected, in the control LCR-5.6 kb Aγ lines, containing the Enh and F elements, the Aγ-globin gene was silenced in all lines tested in the adult stage. In contrast, high levels of Aγ-globin gene expression, similar to those of multicopy integrants were documented in all three generated single copy LCR-3.3 kb Aγ lines, lacking the Enh and F elements.Thus, this study documents directly for the first time the in vivo role of of these two gene-proximal negative regulatory elements on the silencing of the Aγ-gene in the perinatal period and may permit the design of future therapeutic strategies for their exploitation in therapeutic approaches for thalassemias.