Double-stranded Oligonucleotide Probe Research Articles

Recognition of mixed-sequence double-stranded DNA regions using chimeric Invader/LNA probes.

Development of robust oligonucleotide-based probe technologies, capable of recognizing specific regions of double-stranded DNA (dsDNA) targets, continues to attract considerable attention due to the promise of tools for modulation of gene expression, diagnostic agents, and new modalities against genetic diseases. Our laboratory pursues the development of various strand-invading probes. These include Invader probes, i.e., double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, i.e., heteroduplex probes between individual Invader strands and complementary γPNA strands. Here we report on the biophysical properties and dsDNA-recognition characteristics of a new class of chimeric probes-chimeric Invader/LNA probes-which are comprised of densely modified Invader strands and fully modified complementary LNA strands. The chimeric Invader/LNA probes form labile and distorted heteroduplexes, due to an apparent incompatibility between intercalating pyrene moieties and LNA strands. In contrast, the individual Invader and LNA strands form very stable duplexes with complementary DNA, which provides the driving force for near-stoichiometric recognition of model double-stranded DNA targets with single base-pair accuracy. The distinctive properties of chimeric Invader/LNA probes unlock exciting possibilities in molecular biology, and diagnostic and therapeutic fields.

Tuning assembled DNA nanoarchitecture into disassembled state to enhance accurate and ultrasensitive detection of KRAS G12D mutation

Cancer remains a formidable global health challenge, necessitating precise diagnostic tools. The KRAS gene, particularly the G12D mutation, holds pivotal clinical significance across various cancer subtypes. Existing DNA sensing techniques using single-stranded and double-stranded oligonucleotide probes are susceptible for enzymatic degradation, especially in complex biological samples like blood or serum. To address these challenges, we present an innovative approach that involves tuning assembled DNA nanoarchitecture into a disassembled state to accurate and ultrasensitive detection of the KRAS G12D mutation. Our method utilized a pair of multifunctional assembly hairpin probes to construct a DNA wire with both of a fluorophore labeled probe and a quencher labeled probe attached. Upon binding with the target DNA, the DNA wire undergoes gradual disassembly, separating all fluorophores from quenchers to significantly amplify fluorescence signals. This approach offers exceptional sensitivity and reaction kinetics, capable of detecting the KRAS G12D at femtomolar levels, and demonstrates outstanding specificity in discriminating single-base mutations. Furthermore, the DNA nanoarchitecture with well-demonstrated ability to nuclease resistance makes our method exhibits robustness in detecting KRAS G12D mutation in human serum samples. This innovative approach represents an advancement in cancer diagnostics, providing a promising tool for early disease diagnosis and biomedical study.

Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

DNA-Protein-Interaction (DPI)-ELISA Assay for PPAR-γ Receptor Binding.

Dysregulation of peroxisome proliferator-activated receptor (PPAR)-γ has been described in a plethora of pathological conditions, such as diabetes, obesity, inflammatory-related diseases, and cancer. Therefore, identifying novel drugs that are able to restore PPAR-γ activity is a current challenge, which is however slowed down by the lack of a rapid and reproducible activity assay. To date, only a few methods are able to characterize PPAR-γ activity and most of them are expensive, time-consuming, and not always quantitative.Herein, we presented a sensitive multi-well colorimetric assay, termed DNA-Protein-Interaction enzyme-linked immunosorbent assay (DPI-ELISA). This method is based on the ELISA principle, except that it allows to detect only activated PPAR-γ because, unlike classical ELISA, PPAR-γ is not captured by an antibody but by a double-stranded oligonucleotide probe containing its peroxisome proliferator response elements (PPRE) consensus sequence. Thus, DPI-ELISA represents a useful assay for PPAR-γ studies, as well as for the identification of novel PPAR-γ ligands for the development of innovative therapeutic approaches to human diseases where PPAR-γ signaling is dysregulated.

Molecular pathogenesis of Waardenburg syndrome type II resulting from SOX10 gene mutation

To explore the molecular mechanism of Waardenburg syndrome type II (WS2) resulting from SOX10 gene mutation E248fs through in vitro experiment. 293T cells were transiently transfected with wild type (WT) SOX10 and mutant type (MT) E248fs plasmids. The regulatory effect of WT/MT SOX10 on the transcriptional activity of MITF gene and influence of E248fs on WT SOX10 function were determined with a luciferase activity assay. The DNA binding capacity of the WT/MT SOX10 with the promoter of the MITF gene was determined with a biotinylated double-stranded oligonucleotide probe containing the SOX10 binding sequence cattgtc to precipitate MITF and E248fs, respectively. The stability of SOX10 and E248fs were also analyzed. As a loss-of-function mutation, the E248fs mutant failed to transactivate the MITF promoter as compared with the WT SOX10 (P<0.01), which also showed a dominant-negative effect on WT SOX10. The WT SOX10 and E248fs mutant were also able to bind specifically to the cattgtc motif in the MITF promoter, whereas E248fs had degraded faster than WT SOX10. Despite the fact that the E248fs has a dominant-negative effect on SOX10, its reduced stability may down-regulate the transcription of MITF and decrease the synthesis of melanin, which may result in haploinsufficiency of SOX10 protein and cause the milder WS2 phenotype.

Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′-O-(Pyren-1-yl)methyl-RNA Monomers

The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2'-O-(pyren-1-yl)methyl-RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2'-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

A Novel Single Fluorophore-Labeled Double-Stranded Oligonucleotide Probe for Fluorescence-Enhanced Nucleic Acid Detection Based on the Inherent Quenching Ability of Deoxyguanosine Bases and Competitive Strand-Displacement Reaction

We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

Fluorescence-Enhanced Potassium Ions Detection Based on Inherent Quenching Ability of Deoxyguanosines and K+-Induced Conformational Transition of G-Rich ssDNA from Duplex to G-Quadruplex Structures

Herein, we develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid fluorescence-enhanced K(+) detection, based on an inherent quenching ability of guanine bases and G-rich OND conformation transition from duplex to G-quadruplex. This probe presents high sensitivity and good selectivity for the detection of K(+), and the assay process is simple and fast.

PU.1 Directly Regulates cdk6 Gene Expression, Linking the Cell Proliferation and Differentiation Programs in Erythroid Cells

Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G(1) phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G(1) phase of the cell cycle, the period during which the decision to differentiate is made.

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr(-/-) mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4alpha or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4alpha, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Lack of DREAM Protein Enhances Learning and Memory and Slows Brain Aging

Memory deficits in aging affect millions of people and are often disturbing to those concerned. Dissection of the molecular control of learning and memory is paramount to understand and possibly enhance cognitive functions. Old-age memory loss also has been recently linked to altered Ca(2+) homeostasis. We have previously identified DREAM (downstream regulatory element antagonistic modulator), a member of the neuronal Ca(2+) sensor superfamily of EF-hand proteins, with specific roles in different cell compartments. In the nucleus, DREAM is a Ca(2+)-dependent transcriptional repressor, binding to specific DNA signatures, or interacting with nucleoproteins regulating their transcriptional properties. Also, we and others have shown that dream mutant (dream(-/-)) mice exhibit marked analgesia. Here we report that dream(-/-) mice exhibit markedly enhanced learning and synaptic plasticity related to improved cognition. Mechanistically, DREAM functions as a negative regulator of the key memory factor CREB in a Ca(2+)-dependent manner, and loss of DREAM facilitates CREB-dependent transcription during learning. Intriguingly, 18-month-old dream(-/-) mice display learning and memory capacities similar to young mice. Moreover, loss of DREAM protects from brain degeneration in aging. These data identify the Ca(2+)-regulated "pain gene" DREAM as a novel key regulator of memory and brain aging.

Yin Yang 1 Is a Critical Repressor of Matrix Metalloproteinase-9 Expression in Brain Neurons

Membrane depolarization controls long lasting adaptive neuronal changes in brain physiology and pathology. Such responses are believed to be gene expression-dependent. Notably, however, only a couple of gene repressors active in nondepolarized neurons have been described. In this study, we show that in the unstimulated rat hippocampus in vivo, as well as in the nondepolarized brain neurons in primary culture, the transcriptional regulator Yin Yang 1 (YY1) is bound to the proximal Mmp-9 promoter and strongly represses Mmp-9 transcription. Furthermore, we demonstrate that monoubiquitinated and CtBP1 (C-terminal binding protein 1)-bound YY1 regulates Mmp-9 mRNA synthesis in rat brain neurons controlling its transcription apparently via HDAC3-dependent histone deacetylation. In conclusion, our data suggest that YY1 exerts, via epigenetic mechanisms, a control over neuronal expression of MMP-9. Because MMP-9 has recently been shown to play a pivotal role in physiological and pathological neuronal plasticity, YY1 may be implicated in these phenomena as well.

The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.

Enhanced Deacetylation of p53 by the Anti-apoptotic Protein HSCO in Association with Histone Deacetylase 1

HSCO (hepatoma subtracted-cDNA library clone one, also called ETHE1) was originally identified by its frequent overexpression in hepatocellular carcinomas. HSCO inhibits function of NF-kappaB by binding to RelA and accelerating its export from the nucleus. We show here that HSCO exhibits anti-apoptotic activity in cells exposed to DNA-damaging agents by suppressing transcriptional activity of p53. Induction of pro-apoptotic genes, Noxa, Perp, PIG3, and Bax were suppressed in cells over-expressing HSCO. By increasing ubiquitylation and degradation of p53, HSCO reduces p53 protein levels. HSCO specifically associates with histone deacetylase 1 (HDAC1) independently of Mdm2 and facilitates deacetylation of p53 at Lys-373/382 by HDAC1. The metallo-beta-lactamase family consensus sequence in HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies suggested that HSCO, HDAC1, and p53 form a complex in the nucleus. Thus, HSCO is a cofactor that increases the deacetylase activity of HDAC1 toward p53, leading to suppression of apoptosis. Treatment of hepatocellular carcinomas that retain wild-type p53 and overexpress HSCO with anti-HSCO agents might re-establish the p53 response and revert chemoresistance.

Regulation of Pancreas Duodenum Homeobox-1 Expression by Early Growth Response-1

The homeodomain transcription factor pancreas duodenum homeobox-1 (PDX-1) is a key regulator of pancreatic beta-cell development, function, and survival. Deficits in PDX-1 expression result in insulin deficiency and hyperglycemia. We previously found that the glucose-responsive transcription factor early growth response-1 (Egr-1) activates the insulin promoter in part by increasing expression levels of PDX-1. We now report that Egr-1 binds and activates multiple regulatory sites within the pdx-1 promoter. We identified consensus Egr-1 recognition sequences within proximal and distal regions of the mouse pdx-1 promoter and demonstrated specific binding of Egr-1 by chromatin immunoprecipitation and electrophoretic mobility shift assays. Overexpression of Egr-1 increased transcriptional activation of the -4500 proximal pdx-1 promoter and of the highly conserved regulatory Areas I, II, and III. Mutagenesis of a specific Egr-1 binding site within Area III substantially decreased Egr-1-mediated activation. Egr-1 increased the transcriptional activation of Areas I and II, despite the absence of Egr-1 recognition sequences within this promoter segment, suggesting that Egr-1 also can regulate the pdx-1 promoter indirectly. Egr-1 increased, and a dominant-negative Egr-1 mutant repressed, the transcriptional activation of distal pdx-1 promoter sequences. Mutagenesis of a specific Egr-1 binding site within regulatory Area IV reduced basal and Egr-1-mediated transcriptional activation. Our data indicate that Egr-1 regulates expression of PDX-1 in pancreatic beta-cells by both direct and indirect activation of the pdx-1 promoter. We propose that Egr-1 expression levels may act as a sensor in pancreatic beta-cells to translate extracellular signals into changes in PDX-1 expression levels and pancreatic beta-cell function.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.