scholarly journals Molecular Mechanisms ofhsdSInversions in thecodLocus ofStreptococcus pneumoniae

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Jing-Wen Li ◽  
Jing Li ◽  
Juanjuan Wang ◽  
Chunhao Li ◽  
Jing-Ren Zhang
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Phenotypic Diversity ◽  
Transcriptional Analysis ◽  
Inverted Repeats ◽  
Type I ◽  
Content Type ◽  
Dna Inversion ◽  
Methylation Patterns

ABSTRACTStreptococcus pneumoniae(pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferasehsdSgenes (hsdSA,hsdSB, andhsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all threehsdSgenes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression ofhsdSAbut nothsdSBandhsdSC, indicating thathsdSBandhsdSCdo not produce functional proteins and instead act as sources for altering the sequence ofhsdSAby DNA inversions. Consistent with our previous finding that thehsdSinversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion betweenhsdSAandhsdSB. Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in thepsrAmutant. Surprisingly, PsrA-independent inversions were also detected in thehsdSsequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed thatS. pneumoniaevaries the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in thecodlocus.IMPORTANCEStreptococcus pneumoniaeis a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferasehsdSgenes of thecodlocus. This work revealed that only thehsdSgene that is in the same orientation ashsdMis actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of thehsdSinversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed thatS. pneumoniaeswitches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

scholarly journals Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Leonardo Furi ◽  
Liam A. Crawford ◽  
Guillermo Rangel-Pineros ◽  
Ana S. Manso ◽  
Megan De Ste Croix ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lytic Replication ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Type I ◽  
Phase Variable ◽  
Abortive Infection ◽  
Content Type ◽  
Variable Type ◽  
Restriction Modification

ABSTRACTVirus-host interactions are regulated by complex coevolutionary dynamics. InStreptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of thenrdRnucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogenS. pneumoniae.IMPORTANCEWith antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogenStreptococcus pneumoniaeand explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

scholarly journals Type I Interferon Signaling Is a Common Factor Driving Streptococcus pneumoniae and Influenza A Virus Shedding and Transmission

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tonia Zangari ◽  
Mila B. Ortigoza ◽  
Kristen L. Lokken-Toyli ◽  
Jeffrey N. Weiser
Keyword(s):  
Streptococcus Pneumoniae ◽  
Respiratory Tract ◽  
Influenza A Virus ◽  
Influenza A ◽  
Common Factor ◽  
Type I ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Infant Mouse ◽  
Upper Respiratory

ABSTRACT The dynamics underlying respiratory contagion (the transmission of infectious agents from the airways) are poorly understood. We investigated host factors involved in the transmission of the leading respiratory pathogen Streptococcus pneumoniae. Using an infant mouse model, we examined whether S. pneumoniae triggers inflammatory pathways shared by influenza A virus (IAV) to promote nasal secretions and shedding from the upper respiratory tract to facilitate transit to new hosts. Here, we show that amplification of the type I interferon (IFN-I) response is a critical host factor in this process, as shedding and transmission by both IAV and S. pneumoniae were decreased in pups lacking the common IFN-I receptor (Ifnar1−/− mice). Additionally, providing exogenous recombinant IFN-I to S. pneumoniae-infected pups was sufficient to increase bacterial shedding. The expression of IFN-stimulated genes (ISGs) was upregulated in S. pneumoniae-infected wild-type (WT) but not Ifnar1−/− mice, including genes involved in mucin type O-glycan biosynthesis; this correlated with an increase in secretions in S. pneumoniae- and IAV-infected WT compared to Ifnar1−/− pups. S. pneumoniae stimulation of ISGs was largely dependent on its pore-forming toxin, pneumolysin, and coinfection with IAV and S. pneumoniae resulted in synergistic increases in ISG expression. We conclude that the induction of IFN-I signaling appears to be a common factor driving viral and bacterial respiratory contagion. IMPORTANCE Respiratory tract infections are a leading cause of childhood mortality and, globally, Streptococcus pneumoniae is the leading cause of mortality due to pneumonia. Transmission of S. pneumoniae primarily occurs through direct contact with respiratory secretions, although the host and bacterial factors underlying transmission are poorly understood. We examined transmission dynamics of S. pneumoniae in an infant mouse model and here show that S. pneumoniae colonization of the upper respiratory tract stimulates host inflammatory pathways commonly associated with viral infections. Amplification of this response was shown to be a critical host factor driving shedding and transmission of both S. pneumoniae and influenza A virus, with infection stimulating expression of a wide variety of genes, including those involved in the biosynthesis of mucin, a major component of respiratory secretions. Our findings suggest a mechanism facilitating S. pneumoniae contagion that is shared by viral infection.

scholarly journals Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Neil D. Ritchie ◽  
Tom J. Evans
Keyword(s):  
Streptococcus Pneumoniae ◽  
Small Rnas ◽  
Pneumococcal Infection ◽  
Pleural Space ◽  
Type I Interferons ◽  
Type I ◽  
Bacterial Cells ◽  
Rna Seq ◽  
Bacterial Killing ◽  
Content Type

ABSTRACT Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease. IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.

scholarly journals Systematic Analysis of REBASE Identifies Numerous Type I Restriction-Modification Systems with Duplicated, Distinct hsdS Specificity Genes That Can Switch System Specificity by Recombination

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
John M. Atack ◽  
Chengying Guo ◽  
Thomas Litfin ◽  
Long Yang ◽  
Patrick J. Blackall ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Phase Variation ◽  
Dna Methyltransferases ◽  
Inverted Repeats ◽  
Global Changes ◽  
Type I ◽  
Phase Variable ◽  
Global Methylation ◽  
Content Type ◽  
Restriction Modification

ABSTRACT N6-Adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to undergo phase variation, randomly switching expression ON or OFF by varying the length of locus-encoded simple sequence repeats (SSRs). This variation of methyltransferase expression results in genome-wide methylation differences and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 3.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which could have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. IMPORTANCE Many bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems, called phasevarions (phase-variable regulons), control the expression of multiple genes by global methylation changes. In every previously characterized phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. Our systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems will provide valuable information for understanding how bacteria regulate genes and is key to the study of physiology, virulence, and vaccine development; therefore, it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

scholarly journals Human mitochondrial DNA is extensively methylated in a non-CpG context

2019 ◽  
Vol 47 (19) ◽  
pp. 10072-10085 ◽  
Author(s):  
Vibha Patil ◽  
Cyrille Cuenin ◽  
Felicia Chung ◽  
Jesus R Rodriguez Aguilera ◽  
Nora Fernandez-Jimenez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mitochondrial Dna ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Dna Methyltransferases ◽  
Functional Link ◽  
Functional Consequences ◽  
Single Base Pair ◽  
Methylation Patterns ◽  
Global Reduction

Abstract Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.

scholarly journals Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Dane Parker ◽  
Francis J. Martin ◽  
Grace Soong ◽  
Bryan S. Harfenist ◽  
Jorge L. Aguilar ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type I Interferon ◽  
Bacterial Pathogen ◽  
Central Component ◽  
Type I ◽  
Respiratory Pathogens ◽  
Airway Epithelial ◽  
Type I Ifn ◽  
Mucosal Epithelium ◽  
Content Type

ABSTRACTThe mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogenStreptococcus pneumoniaeactivates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade.Tlr4−/−,Myd88−/−,Trif−/−, andNod2−/−mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization withS. pneumoniaecompared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.IMPORTANCEThe bacteriumStreptococcus pneumoniaeis a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clearS. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

scholarly journals Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

2011 ◽  
Vol 80 (3) ◽  
pp. 1166-1180 ◽  
Author(s):  
Constantine Bitsaktsis ◽  
Bibiana V. Iglesias ◽  
Ying Li ◽  
Jesus Colino ◽  
Clifford M. Snapper ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Bactericidal Activity ◽  
Protein A ◽  
Pneumococcal Infection ◽  
Lavage Fluid ◽  
Complement C3 ◽  
Type I ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Content Type

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen,Francisella tularensis. To determine if a similar strategy could be applied to the important pathogenStreptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevatedS. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethalS. pneumoniaechallenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed againstS. pneumoniaethat appears to be lactoferrin mediated.

scholarly journals The cGAS/STING Pathway Detects Streptococcus pneumoniae but Appears Dispensable for Antipneumococcal Defense in Mice and Humans

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Juan Sebastian Ruiz-Moreno ◽  
Lutz Hamann ◽  
Lei Jin ◽  
Leif E. Sander ◽  
Monika Puzianowska-Kuznicka ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type I ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Type I Ifns ◽  
Mouse Macrophages ◽  
Upper Respiratory ◽  
Sting Pathway ◽  
And Control ◽  
Wall Components

ABSTRACT Streptococcus pneumoniae is a frequent colonizer of the upper respiratory tract and a leading cause of bacterial pneumonia. The innate immune system senses pneumococcal cell wall components, toxin, and nucleic acids, which leads to production of inflammatory mediators to initiate and control antibacterial defense. Here, we show that the cGAS (cyclic GMP-AMP [cGAMP] synthase)-STING pathway mediates detection of pneumococcal DNA in mouse macrophages to primarily stimulate type I interferon (IFN) responses. Cells of human individuals carrying HAQ TMEM173 , which encodes a common hypomorphic variant of STING, were largely or partly defective in inducing type I IFNs and proinflammatory cytokines upon infection. Subsequent analyses, however, revealed that STING was dispensable for restricting S. pneumoniae during acute pneumonia in mice. Moreover, explorative analyses did not find differences in the allele frequency of HAQ TMEM173 in nonvaccinated pneumococcal pneumonia patients and healthy controls or an association of HAQ TMEM173 carriage with disease severity. Together, our results indicate that the cGAS/STING pathway senses S. pneumoniae but plays no major role in antipneumococcal immunity in mice and humans.

scholarly journals Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Xue Liu ◽  
Jing-Wen Li ◽  
Zhixing Feng ◽  
Youfu Luo ◽  
Jan-Willem Veening ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Treatment Failure ◽  
Molecular Mechanisms ◽  
Transcriptional Repressor ◽  
Drug Tolerance ◽  
Microbial Populations ◽  
Luciferase Reporter ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA sequencing, quantiative reverse transcriptase PCR, and transcriptional luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB, and ptvC) is induced by exposure to vancomycin. Further investigation showed that transcription of the ptv operon is repressed by PtvR, a PadR family repressor. Transcriptional induction of the ptv operon by vancomycin was achieved by transcriptional derepression of this locus, which was mediated by PtvR. Importantly, fully derepressing ptvABC by deleting ptvR or overexpressing the ptv operon with an exogenous promoter significantly enhanced vancomycin tolerance. Gene deletion analysis revealed that PtvA, PtvB, and PtvC are all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, the results of an electrophoretic mobility shift assay with recombinant PtvR showed that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences within the ptv promoter. Together, the ptv locus represents an inducible system in S. pneumoniae in response to stressful conditions, including those caused by antibiotics. IMPORTANCE Reversible or phenotypic tolerance to antibiotics within microbial populations is associated with treatment failure of bacterial diseases, but the underlying mechanisms regulating phenotypic drug tolerance remain obscure. This study reports our finding of a multigene locus that contributes to inducible tolerance to vancomycin in Streptococcus pneumoniae, an important opportunistic human pathogen. The vancomycin tolerance phenotype depends on the PtvR transcriptional repressor and three predicted membrane-associated proteins encoded by the ptv locus. This represents the first example of a gene locus in S. pneumoniae that is responsible for antibiotic tolerance and has important implications for further understanding bacterial responses and phenotypic tolerance to antibiotic treatment in this and other pathogens.

scholarly journals Selective Anchoring of DNA Methyltransferases 3A and 3B to Nucleosomes Containing Methylated DNA

2009 ◽  
Vol 29 (19) ◽  
pp. 5366-5376 ◽  
Author(s):  
Shinwu Jeong ◽  
Gangning Liang ◽  
Shikhar Sharma ◽  
Joy C. Lin ◽  
Si Ho Choi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Dna Methyltransferases ◽  
Micrococcal Nuclease ◽  
Nuclear Antigen ◽  
Heterochromatin Protein 1 ◽  
Histone Deacetylase 1 ◽  
Methylation Patterns

ABSTRACT Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.

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