Acid-rich Domain Research Articles

Nuclear Magnetic Resonance Structure of the N-Terminal Domain of Nonstructural Protein 3 from the Severe Acute Respiratory Syndrome Coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

Phosphorylation-dependent Regulation of Stability and Transforming Potential of ETS Transcriptional Factor ESE-1 by p21-activated Kinase 1

Differential phosphorylation of transcription factors by signal transduction pathways play an important role in regulation of gene expression and functions. ESE-1 is an epithelium-specific ETS transcription factor that transforms human breast epithelial cells through a serine- and aspartic acid-rich domain (SAR) by an unknown cytoplasmic mechanism. Here we found that a signaling kinase, p21-activated kinase-1 (Pak1), interacts with and phosphorylates ESE-1. Interestingly, Pak1 selectively phosphorylates ESE-1 at Ser(207), which is located within the SAR domain. A S207A substitution in ESE-1 reduced its ability to transform breast cancer cells. We also found that ESE-1 is a labile protein and by interacting with F-box-binding protein beta-TrCP, undergoes ubiquitin-dependent proteolysis. Intriguingly, Pak1 phosphorylation inactive mutant ESE1-S207A is more unstable than either wild-type ESE-1 or its Pak1 phosphorylation mimetic mutant, i.e. ESE1-S207E. These findings provide novel insights into the mechanism of transformation potential of ESE-1 and discovered that ESE-1 functions are coordinately regulated by Pak1 phosphorylation and beta-TrCP-dependent ubiquitin-proteasome pathways.

Histidine-rich Ca-binding protein interacts with sarcoplasmic reticulum Ca-ATPase

Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.

Localization of glucocorticoid-induced leucine zipper (GILZ) expressing neurons in the central nervous system and its relationship to the stress response

The glucocorticoid-induced leucine zipper (GILZ) is a 137 amino acid protein, which was originally identified as a dexamethasone-inducible gene, and is characterized by a leucine zipper domain, an N-terminal domain and a C-terminal proline and glutamic acid rich domain. In this study, we performed in situ hybridization analyses and found that many neurons from the olfactory bulb to the spinal cord express GILZ mRNA, suggesting that GILZ is involved in fundamental and common cellular events of neurons. In addition, the predominant expression of GILZ mRNA in the motor nuclei indicated a strong relationship between GILZ and the motor nervous system. On the other hand, real-time RT-PCR analyses of brains of animals exposed to water-immersion restraint stress (WRS) showed that WRS significantly up-regulated GILZ expression in the medial prefrontal cortex (mPFC) and hippocampus, which are known to be mental stress-related areas, suggesting the multiple roles of GILZ in the brain. This up-regulation was not observed in adrenalectomized mice, confirming its dependency on hypothalamic–pituitary–adrenal axis activation.

A novel gene with antisalt and anticadmium stress activities from a halotolerant marine green alga Chlamydomonas sp. W80

A novel gene with antistress activities against both salt (NaCl) and cadmium stresses was isolated from the cDNA library of halotolerant green alga Chlamydomonas sp. strain W80 by a functional expression screening with Escherichia coli. The C-terminal region of this protein is responsible for the antistress activity, because N-terminal truncated clone of this gene retains the antistress activity, and the C-terminal truncated clone loses the activity. In the C-terminal region, there is a histidine and aspartic acid-rich domain (HD-rich domain).

Characterization of Compositional Heterogeneity in Chemically Amplified Photoresist Polymer Thin Films with Infrared Spectroscopy

We demonstrate a general approach to characterize compositional heterogeneity in polymer thin films using Fourier transform infrared (FTIR) spectroscopy. Polymer films with varying degrees of heterogeneity were prepared using a model chemically amplified photoresist where a photoacid catalyzed reaction-diffusion process results in the formation of methacrylic acid (MAA) rich domains. Within these domains, the carboxylic acid groups dimerize through hydrogen bonding. FTIR measurements of the relative fraction of hydrogen-bonded vs free carboxylic groups are used to quantify the degree of compositional heterogeneity. The FTIR data from compositionally homogeneous systems, statistical copolymers, and polymer blends follow the expected linear dependence of the relative hydrogen-bonded fraction with composition. For the chemically amplified photoresist system, the FTIR data deviate substantially from that of the homogeneous distribution. Furthermore, parameters describing the size of the heterogeneity can be determined from a solid sphere model of the deprotected regions catalyzed by the diffusing photoacid. The degree of the spatial heterogeneity varies with changes in the photoacid concentration, reaction conditions, and initial copolymer composition. Increased nonreactive comonomer content decreases the degree of heterogeneity by reducing the hydrogen-bonding efficiency, which is consistent with the dilution of MAA groups.

Different Domains of the Transcription Factor ELF3 Are Required in a Promoter-specific Manner and Multiple Domains Control Its Binding to DNA

Elf3 is an epithelially restricted member of the ETS transcription factor family, which is involved in a wide range of normal cellular processes. Elf3 is also aberrantly expressed in several cancers, including breast cancer. To better understand the molecular mechanisms by which Elf3 regulates these processes, we created a large series of Elf3 mutant proteins with specific domains deleted or targeted by point mutations. The modified forms of Elf3 were used to analyze the contribution of each domain to DNA binding and the activation of gene expression. Our work demonstrates that three regions of Elf3, in addition to its DNA binding domain (ETS domain), influence Elf3 binding to DNA, including the transactivation domain that behaves as an autoinhibitory domain. Interestingly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA. On the basis of these studies, we suggest a model for autoinhibition of Elf3 involving intramolecular interactions. Importantly, this model is consistent with our finding that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C terminus, which contains the ETS domain. In parallel studies, we demonstrate that residues flanking the N- and C-terminal sides of the ETS domain of Elf3 are crucial for its binding to DNA. Our studies also show that an AT-hook domain, as well as the serine- and aspartic acid-rich domain but not the pointed domain, is necessary for Elf3 activation of promoter activity. Unexpectedly, we determined that one of the AT-hook domains is required in a promoter-specific manner.

Monitoring release of ketoprofen enantiomers from biodegradable poly( d, l-lactide-co-glycolide) injectable implants

A stereoselective reversed-phase HPLC assay was developed that could simultaneously quantify S-(+) and R-(−) enantiomers of ketoprofen in release samples. Racemic ketoprofen (rac-KET) and its S-(+) enantiomer ( S-(+)-KET) were dissolved in an injectable viscous polymer solution consisting of the biodegradable poly( d, l-lactide-co-glycolide, 70:30) ( d, l-PLG) and a solvent, N-methyl-2-pyrrolidone (NMP). Once injected into an aqueous environment, the polymeric mixture solidified into a solid implant due to the leaching of NMP. In vitro release studies show that such implants with ketoprofen can provide sustained release of the drug lasting about three months in a pH 7.4 release medium. Moreover, a preferential faster S-(+)-KET release over R-(−)-KET was observed for the implants containing 4%, 7%, and 10% of racemic ketoprofen in the neutral pH 7.4 release medium. Stereoselective release was minimal in the first 42 days in vitro but became very pronounced at later time points. When S-(+)-KET was incorporated into the polymeric mixture, its release was also faster than that of the racemic ketoprofen, confirming the stereoselective release of ketoprofen from the d, l-PLG implants. The observed stereoselective release of KET at pH 7.4 was most likely produced by chiral interactions between KET enantiomers and transiently produced d-lactic acid or l-lactic acid rich domains within the implants during d, l-PLG degradation. However, such stereoselective release was not observed in pH 10.0 release medium, probably due to a much faster and homogeneous polymer degradation. The study suggests possible stereoselective release of racemic drugs from d, l-PLG microspheres and implants in vivo.

RPGRmutation analysis and disease: an update

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the most common single cause of retinitis pigmentosa, accounting for up to 15 to 20% of cases in Caucasians. A total of 240 different RPGR mutations have been reported, including 24 novel ones in this work, which are associated with X-linked retinitis pigmentosa (XLRP) (95%), cone, cone-rod dystrophy, or atrophic macular atrophy (3%), and syndromal retinal dystrophies with ciliary dyskinesia and hearing loss (2%). All disease-causing mutations occur in one or more RPGR isoforms containing the carboxyl-terminal exon open reading frame 15 (ORF15), which are widely expressed but show their highest expression in the connecting cilia of rod and cone photoreceptors. Of reported RPGR mutations, 55% occur in a glutamic acid-rich domain within exon ORF15, which accounts for only 31% of the protein. RPGR forms complexes with a variety of other proteins and appears to have a role in microtubular organization and transport between photoreceptor inner and outer segments.

The C-Terminal Portion of the Hrs Protein Interacts with Tsg101 and Interferes with Human Immunodeficiency Virus Type 1 Gag Particle Production

The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in "lump-like" structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.

Computational study of coagulation factor VIIa’s affinity for phospholipid membranes

The interaction between the gamma-carboxyglutamic acid-rich domain of coagulation factor VIIa (FVIIa), a vitamin-K-dependent enzyme, and phospholipid membranes plays a major role in initiation of blood coagulation. However, despite a high sequence and structural similarity to the Gla domain of other vitamin-K-dependent enzymes with a high membrane affinity, its affinity for negatively charged phospholipids is poor. A few amino acid differences are responsible for this observation. Based on the X-ray structure of lysophosphatidylserine (lysoPS) bound to the Gla domain of bovine prothrombin (Prth), models of the Gla domain of wildtype FVIIa and mutated FVIIa Gla domains in complex with lysoPS were built. Molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on the complexes were applied to investigate the significant difference in the binding affinity. The MD simulation approach provides a structural and dynamic support to the role of P10Q and K32E mutations in the improvement of the membrane contact. Hence, rotation of the Gly11 main chain generated during the MD simulation results in a hydrogen bond with Q10 side chain as well as the appearance of a hydrogen bond between E32 and Q10 forcing the loop harbouring Arg9 and Arg15 to shrink and thereby enhances the accessibility of the phospholipids to the calcium ions. Furthermore, the application of the SMD simulation method to dissociate C6-lysoPS from a series of Gla domain models exhibits a ranking of the rupture force that can be useful in the interpretation of the PS interaction with Gla domains. Finally, adiabatic mapping of Gla6 residue in FVIIa with or without insertion of Tyr4 confirms the critical role of the insertion on the conformation of the side chain Gla6 in FVIIa and the corresponding Gla7 in Prth.

Transmembrane Gla Protein 4 as a Novel Modulator of ERK2.

Transmembrane Gla protein 4 (TMG4) is a single pass, type I transmembrane protein with a vitamin-K dependent γ-carboxy-glutamic acid-rich domain (Kulman et al PNAS,2001). Bioinformatic analysis of the primary sequence indicates a very high degree of conservation (ca. 80%) among mammals and to-date no known functional roles have been assigned to this protein. In our efforts to elucidate molecular functions of TMG4, we first analyzed sub cellular localization of this protein. Microscopic analysis of HEK293T cells expressing a TMG4-GFP fusion construct showed juxtanuclear localization of the tagged protein at 24h post transfection. Staining with organelle specific fluorescent dyes revealed that the TMG4-GFP primarily localizes to the trans Golgi network in the cell. Recently, there have been increasing number of reports of golgi-localized proteins participating in compartmentalized signal transduction pathways and affecting cell differentiation and proliferation by activating major signaling cascades like the Ras-Raf/MAPK signaling cascades. Moreover, the transmembrane localization and the current lack of evidence for a direct role for TMG4 in the coagulation pathways encouraged us to hypothesize that TMG4 may play a role in signal transduction. To test this we generated a TMG4 over-expressing cell line (high-TMG4) by stably transfecting HEK293T cells with a CMV-promoter driven TMG4 plasmid , and a TMG4 knock-down cell line (low-TMG4) by stable transfections with a TMG4 short-hairpin RNA (shRNA) expression vector. The levels of TMG4 gene expression were confirmed in both the cell lines by RNA and protein expression analysis. The low-TMG cells showed >90% knockdown of TMG4 in comparison to untransfected control cells. The cells were grown under standard tissue culture conditions for 48h after which they were harvested. Total cell lysates from the cell lines were analyzed by western blotting using primary antibodies against a panel of signal transduction proteins representing major signaling pathways including threonine kinase, tyrosine kinase and ERK2. The results showed no significant changes in activation of signal transduction proteins tested except for extracellular-signal-regulated kinase (ERK) -2. The high-TMG cells showed a down modulation of phosphorylated ERK2 while the low-TMG4 cell lysates showed a 500-fold up regulation of phosphorylated ERK2 when compared to the levels in the low-TMG cells. Our observations were confirmed visually by assaying the cells for intracellular localization of ERK2. We used immuno-fluorescent staining to show that TMG4 levels regulate ERK2 activation based on TMG4 dependent translocation of ERK2 using anti phospho-ERK2 and anti-ERK2 antibodies. More than 85% of high-TMG4 cells showed extra nuclear localization of ERK2 while phosphorylated ERK2 was localized in the nucleus in the >80% of low-TMG4 cells. Thus there is an inverse correlation between TMG4 levels and ERK2 phosphorylation. We conclude that TMG4 is a molecular modulator of ERK2. Whether TMG4 has a direct interaction or affects ERK2 indirectly is under investigation. These studies suggest novel roles for vitamin-K dependent Gla-proteins in regulating thrombopoietin dependent megakaryocytic differentiation, platelet activation and myelopoiesis and myeloproliferation.

Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ–carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

Mouse Cristin/R-spondin Family Proteins Are Novel Ligands for the Frizzled 8 and LRP6 Receptors and Activate β-Catenin-dependent Gene Expression

Wnt signaling plays critical biological roles during normal embryonic development and homeostasis in adults. In the canonical pathway, binding of Wnt ligands to the Frizzled (Fzd) receptor and the low density lipoprotein-related receptor (LRP) 5 or LRP6 coreceptor initiates downstream signaling events leading to gene activation by beta-catenin and the T-cell factor (TCF)-lymphoid enhancer factor (LEF) family transcription factor complex. In this study, we provide several lines of evidence that the mouse Cristin/R-spondin family proteins function as Fzd8 and LRP6 receptor ligands and induce the canonical Wnt/beta-catenin signaling pathway, leading to TCF-dependent gene activation. First, conditioned medium containing Cristin/R-spondin proteins effectively induced reporter activity in a TCF-binding site-dependent manner. Second, stimulation of cells with Cristin/R-spondin was accompanied by stabilization of endogenous beta-catenin proteins and induction of canonical Wnt target genes. Third, Cristin/R-spondin proteins physically interacted with the extracellular domains of the LRP6 and Fzd8 receptors in vivo and in vitro. Interestingly, unlike canonical Wnt ligands, Cristin/R-spondin failed to form a ternary complex with both LRP6 and Fzd8 receptors, suggesting that R-spondin may activate the canonical Wnt signaling pathway by different mechanisms. Furthermore, Cristin/R-spondin proteins possess an intriguing positive modulatory activity on Wnt ligands, possibly through a direct interaction. Our findings expand the repertoire of ligands that induce beta-catenin/TCF-dependent gene activation and implicate the presence of active beta-catenin-dependent gene activation in a Wnt-free biological context.

Characterization of the RNA-binding domain in the Dendrolimus punctatus cytoplasmic polyhedrosis virus nonstructural protein p44

Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV-1) belongs to the Cypovirus genus in the Reoviridae family. The ORF of genome segment 8 (S8) of DpCPV-1 was cloned into vector pMAL-c2X and used to express a 44 kDa protein (p44) in E. coli, which was detected by Western blotting. The gel mobility shift assays showed that p44 had ssRNA-binding activity. Competitive assay indicated that this protein only bind to ssRNA and could not interact with DNA and dsRNA. The binding of p44 to ssRNA is sequence non-specific. To identify the domain(s) important for RNA binding of the protein, a number of deletions were made. These truncated proteins were expressed in E. coli and purified. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays and northwestern blot. The results indicated that glutamic acid-rich domain in the central region of p44 from residues 104 to 201 was the ssRNA-binding domain.

Crystallization of an anti-factor IX antibody and its complex

Initiation, propagation and regulatory processes of blood coagulation occur at the cell surface. During blood coagulation, many coagulation factors are anchored onto cell-surface membranes through their N-terminal carboxyglutamic acid-rich (Gla) domains found on such vitamin K-dependent blood coagulation factors as factors VII, IX, X, prothrombin, factors C and S. 10C12 is a conformation-specific calcium-dependent anti-factor IX antibody, which is directed at the calcium-stabilized Gla domain and interferes with factor IX-membrane interaction. In a variety of animal models, 10C12 has been demonstrated to be effective anticoagulant in attenuating thrombosis without severe bleeding. The crystallization of the Fab fragment of 10C12 and its complex with the Gla domain of human factor IX was not trivial. Here, the crystallization conditions and unusual aspects of this crystallization process are reported.

Osteopontin Expression Correlates with Melanoma Invasion

Osteopontin Expression Correlates with Melanoma Invasion

Zinc finger domain of Snail functions as a nuclear localization signal for importin β‐mediated nuclear import pathway

Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin) beta in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin beta in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal and Snail can be transported into the nucleus in an importin beta-mediated manner.

Cloning and Identification of a New Antimicrobial Protein, Salvic, from Human Salivary Gland

From a subtracted cDNA library of human salivary gland a C77-91 gene totally unknown to date was named as Salvic that expresses 46 amino acids peptide (pI=9.45) possessing an antimicrobial activity on E. coli. Salvic is consisted of a typical hydrophobic amino acid rich domain in the N-terminus, a cluster of basic amino acids, carbohydrate attachment sites, a possible transglutaminase catalyzed crosslinking site, and multiple consensus sequences of phosphorylation site in the C-terminus. Western blot analysis using the monospecific antibody to the synthetic Salvic peptide showed strong interacting proteins in the extracts from submandibular gland and parotid saliva, and the immunohistochemical staining detected a strong positive reaction in the cytoplasmic secretory granules of interlobular ductal cells of salivary gland. The Salvic was also distributed in the human sebaceous gland and prostate. These data suggest that the identification of Salvic may add further understanding of greater role of salivary proteins providing innate immunity by protecting and stabilizing the mucosal epithelium in the maintaining homeostasis of oral mucosa.

Multiple interactions between BRG1 and MHC class II promoter binding proteins

The class II transactivator (CIITA) interacts with the chromatin remodeling factor brahma related gene 1 (BRG1) as a necessary component of the transcriptional activation of human major histocompatibility complex (MHC) class II genes. We report here that RFXAP, a subunit of the DNA-binding RFX complex, also binds BRG1 and therefore provides a mechanism by which MHC class II gene chromatin can be remodeled in the absence of CIITA. RFXAP and CIITA bind to different regions of BRG1. The region of the RFXAP protein that binds BRG1 coincides with the minimally required fragment of RFXAP previously reported to be necessary to mediate MHC class II gene transcription. For CIITA, we found that BRG1 interacts with both the N-terminal acidic amino acid rich transactivation domain and a central region of CIITA that contains GTP-binding motifs. Analysis of chromatin structure of MHC class II genes in cell lines lacking CIITA or RFXAP, suggests that the RFXAP–BRG1 interaction may, in some cell types, produce chromatin remodeling.