Formation Of DNA-protein Complexes Research Articles

Prenatal caloric restriction enhances DNA methylation and MeCP2 recruitment with reduced murine placental glucose transporter isoform 3 expression

Diminished transplacental glucose transport plays an important role in prenatal calorie restriction (CR) induced reduction in fetal growth. Fetal growth restriction (FGR) has an impact in shaping the adult phenotype with transgenerational implications. To understand the mechanisms underlying prenatal CR-induced transplacental glucose transport, we examined the epigenetic regulation of placental glucose transporter (Glut1 and Glut3) expression. We restricted calories by 50% in C57BL6 pregnant mice from gestational days 10 to 19 (CR; n=8) vs. controls (CON; n=8) and observed a 50% diminution in placental Glut3 expression (P<.05) with no effect on Glut1 expression by reverse transcription and quantitative real-time polymerase chain reaction (PCR). CR enhanced DNA methylation of a CpG island situated ~1000 bp upstream from the transcriptional start site of the glut3 gene, with no such effect on the glut1 gene as assessed by methylation-sensitive PCR and bisulfite sequencing. Chromatin immunoprecipitation (ChIP) assays demonstrated enhanced MeCP2 binding to the CpG island of the glut3 gene in response to CR vs. CON (P<.05). Sequential ChIP demonstrated that enhanced MeCP2 binding of the glut3-mCpG island enhanced histone deacetylase 2 recruitment (P<.05) but interfered with Sp1 binding (P<.001), although it did not affect Sp3 or Creb/pCreb interaction. We conclude that late-gestation CR enhanced DNA methylation of the placental glut3 gene. This epigenetic change augmented specific nuclear protein–DNA complex formation that was associated with prenatal CR-induced reduction of placental glut3 expression and thereby transplacental glucose transport. This molecular complex provides novel targets for developing therapeutic interventions aimed at reversing FGR.

Cooperative Binding of PhoBDBD to Its Cognate DNA Sequence—A Combined Application of Single-Molecule and Ensemble Methods

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process.

Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement ⟨R2⟩ exhibits a scaling with time as ⟨R2⟩ ∼ tα, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ∼ tβ/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein–DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis.

Transrepression of the estrogen receptor promoter by calcitriol in human breast cancer cells via two negative vitamin D response elements

Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at -2488 to -2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at -94 to -70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter-reporter constructs revealed that both nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5' hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein-DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful in the treatment of ER+ BCa.

Analysis of DNA interactions using single-molecule force spectroscopy

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy

In this protocol, we describe a procedure to generate 'DNA dumbbells'-single molecules of DNA with a microscopic bead attached at each end-and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

BackgroundStreptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described.ResultsWe report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex.ConclusionsThis work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

MP 3 The basal ganglia in perceptual timing: timing performance in multiple system atrophy and Huntington's disease

Transcription machinery depends on the temporal formation of protein-DNA complexes. Recent experiments demonstrated that lifetime of the complex can also affect transcription. In parallel, <i>in vitro</i> single-molecule studies showed that nucleoid-associated proteins (NAPs) leave the DNA rapidly as the bulk concentration of the protein increases via facilitated dissociation (FD). Never-theless, whether such concentration-dependent mechanism is functional in a bacterial cell, in which NAP levels and the 3D chromosomal structure are often coupled, is not clear <i>a priori</i>. Here, by using extensive coarse-grained molecular simulations, we model the unbinding of specific and nonspecific dimeric NAPs from a high-molecular-weight circular DNA molecule in a cylindrical structure mimicking the cellular confinement of a bacterial chromosome. Our simulations show that physiologically relevant peak protein levels (tens of micromolar) lead to highly compact chromosomal structures. This compaction results in rapid off rates (shorter DNA-residence times) but only for specifically DNA-binding NAPs such as the factor for inversion stimulation (Fis). Contrarily, for nonspecific NAPs, the off rates decrease as the protein levels increase, suggesting an inverse FD pattern. The simulations with restrained chromosome models reveal that this inverse response is due to DNA-segmental fluctuations, and that chromosomal compaction is in favor of faster protein dissociation. Overall, our results indicate that cellular-concentration level of a structural DNA-binding protein can be highly intermingled with its DNA-residence time.

Direct Evidence of Allele-Specific Binding of CTCF and MeCP2 to Tsix in a HPRT-Deficient Female F1 Hybrid Mouse Cell Line

Mammalian dosage compensation requires silencing of one of the two X chromosomes in females and is controlled by the X inactivation center (Xic). Xic contains many of the regulatory elements for the mutual interplay of X-inactive specific transcript (Xist) and Tsix, the antisense counterpart of Xist. The regulatory elements control X chromosome inactivation (XCI) via the formation of DNA-DNA and DNA-protein complexes with cis- and trans-acting factors. However, the process-dependent regulation of Xist/Tsix by these elements in each XCI process remains largely unknown. In this study, a 6-thioguanine-resistant female F₁ hybrid mouse cell line (designated HOBMSKI2) was constructed from a cross between a female HPRT-deficient transgenic mouse (designated BM3) and a male wild type Mus spretus mouse (designated MS), which enabled the direct discrimination of both allele-specific expression of X-linked genes and allele-specific binding of proteins associated with XCI due to DNA polymorphisms between BM3 and MS. Using this cell line, we found that Tsix on the active X chromosome (Xa) was not expressed in somatic cells despite the fact that CTCF, which ensures Tsix expression in embryonic stem cells, was still bound to the 5′ end of Tsix on Xa, implying that CTCF may function differently during each XCI process and its trans-activating activity for Tsix expression may be lost in the maintenance process. In addition, the monoallelic expression of Tsix on Xa was inhibited by epigenetic modification of the chromatin in the maintenance process, which was mediated by protein complexes recruited by MeCP2. The results indicate the value of HOBMSKI2 in directly detecting the allele-specific binding of CTCF and MeCP2 to the 5′ end of Tsix. The HOBMSKI2 mouse line is a versatile and useful resource for studying the molecular mechanism of the XCI process.

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.

Functional analysis of promoter variants in KU70 and their role in cancer susceptibility

KU70 is involved in the DNA double-strand break repair pathway, which plays a critical role in maintaining genomic stability and preventing cancer. Genetic variation within the KU70 gene has been shown to be associated with increased risk of several types of cancers including breast cancer. Here, we used gene reporter and gel shift assays combined with site-directed mutagenesis to characterize genetic variation within the KU70 proximal promoter region and investigate the putative functional role of regulatory variation and altered KU70 expression in modulating an individual's susceptibility to disease. We show that the variant rs2267437C>G significantly influences KU70 transcriptional activity in an allele- specific manner and alters DNA-protein complex formation in breast cancer cell lines. Our data provide a possible molecular explanation for the associations observed between the KU70 regulatory variant rs2267437 and breast cancer risk.

Influence of centrin 2 on the interaction of nucleotide excision repair factors with damaged DNA

We have examined the influence of centrin 2 (Cen2) on the interaction of nucleotide excision repair factors (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes bearing the dUMP derivative 5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate. The fluorescein residue linked to the nucleotide base imitates a bulky lesion of DNA. Cen2 stimulated the binding and increased the yield of DNA adducts with XPC-HR23b, a protein recognizing bulky damages in DNA. Stimulation of the binding was most pronounced in the presence of Mg(2+) and demonstrated a bell-shaped dependence on Cen2 concentration. The addition of Cen2 changed the stoichiometry of RPA-DNA complexes and diminished the yield of RPA-DNA covalent crosslinks. We have shown that Cen2 influences the binding of RPA and XPA with DNA, which results in formation of additional DNA-protein complexes possibly including Cen2. We have also found some evidence of direct contacts between Cen2 and DNA. These results in concert with the literature data suggest that Cen2 can be a regulatory element in the nucleotide excision repair system.

Inhibition of Apolipoprotein A‐I Gene Expression by Obesity‐Associated Endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.

Folding Versus Charge: Understanding Selective Target Recognition by the Thrombin Aptamers

The use of nucleic acids as drugs represents a consistently growing approach. Different therapeutical strategies take advantage of the biological and biophysical properties of DNA and RNA to properly modulate activity of selected targets. A peculiar characteristic of these molecules is their structural flexibility which allows them to assume distinct foldings depending upon their sequence and/or environment. During the last twenty years this has led to the theoretical and experimental development of oligonucleotide aptamers, short sequences which can recognize a target with specificity and affinity comparable to antibodies. A leading example is represented by the Thrombin aptamer (15fTBA), a 15-mer DNA selected by its high affinity for the exosite I (fibrinogen binding site) of the coagulation factor. The very stable protein-DNA complex formation is the result of complementarities between the two macromolecules promoted by the aptamer sequence and folding as well as of electrostatic interactions generated by the charge balance at the binding site/s. Here, we investigated the relative role of these contributions and their involvement in defining the biological properties of the resulting complex. Thus we compared the Thrombin binding and inhibition properties of TBA to those of unrelated single stranded oligonucleotides. Additionally, the differences between the two protein exosites were assessed by using 29hTBA, a longer (29-mer) aptamer known to bind exosite II (heparin binding site). A subtle balance of aptamer folding and sequence is shown to cooperate with charge density for effective and selective recognition of exosite I or exosite II by TBAs.

Sequence-Dependent Mechanics of DNA

Double-stranded DNA is a semiflexible polymer that can naturally bend on length scales comparable to the size of large DNA-protein complexes like nucleosomes or protein-mediated DNA loops. The sequence of the substrate DNA does not only provide biochemical binding sites for the proteins, but also affects the local mechanical properties of the DNA. Notably, sequence can affect the intrinsic curvature of the DNA, as well as its bendability, or elasticity. While intrinsic bends in DNA and their role in protein-DNA complex formation are well studied, sequence-dependent elasticity still remains only vaguely explored. In order to separate sequence effects on elasticity from those on intrinsic curvature, we have designed sequences of DNA which have nearly identical curvatures but varied AT content and directly measured their mechanical elasticity using constant force axial optical tweezers. We found the persistence length to be highly dependent on the AT content of the DNA, differing almost thirty percent between sequences with nearly identical curvature but different sequence composition. This is a departure from conventional dinucleotide and trinucleotide models, which predict a much smaller difference between the two sequences, but consistent with estimates obtained from the crystallographic structures of protein-DNA complexes.

Comparative study of the different mechanisms for zinc ion stress sensing in two cyanobacterial strains, Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.

In response to an increased level of Zn2+, Synechococcus sp. PCC 7942 expresses SmtA, a metallothionein-like metal-chelating protein, while Synechocystis sp. PCC 6803 expresses ZiaA, a transporter of Zn2+. The gene expression of these proteins is regulated by repressor protein, SmtB and ZiaR, respectively. In spite of contributing to different response systems, both repressor proteins belong to the ArsR family and are highly homologous to each other. To understand the different systems responsible for dealing with excess Zn2+, we examined the cis-elements in the promoter regions of smtA and ziaA, as well as the binding affinities of recombinant SmtB and ZiaR proteins. The operator/promoter region of smtA included two palindromic sequences and that of ziaA included one. Electrophoretic mobility shift assay revealed that SmtB formed four different complexes with the operator/promoter region of smtA, whereas it formed only two different complexes with the corresponding region of ziaA. For ZiaR, the corresponding results were quite the same as those for SmtB. Furthermore, the complex formation between SmtB and operator/promoter regions is inhibited in the presence of Zn2+ at higher concentrations than 16 μM. On the other hand, the corresponding Zn2+ concentration is 128 μM. These results demonstrate that the degrees of protein-DNA complex formation between repressor proteins and the operator/promoter regions of regulated genes depend on the structures of the operator/promoter regions, and the effects of Zn2+ on the dissociation of these complexes are mainly associated with the structures of the repressors.

MiR-125b, a Target of CDX2, Regulates Cell Differentiation through Repression of the Core Binding Factor in Hematopoietic Malignancies

MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor β (CBFβ) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFβ was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFβ in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress-related response in meniscus cells remains unclear. We investigated mechanical stretch-regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry-type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real-time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch-related protein-DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA-binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch-induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression.

Structural Basis for Catalytic Activation of a Serine Recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.