Airway epithelium structure/function can be altered by local inflammatory/immune signals, and this process is called epithelial remodeling. The mechanism by which this innate response is regulated, ...which causes mucin/mucus overproduction, is largely unknown. Exosomes are nanovesicles that can be secreted and internalized by cells to transport cellular cargo, such as proteins, lipids, and miRNA. The objective of this study was to understand the role exosomes play in airway remodeling through cell-cell communication. We used two different human airway cell cultures: primary human tracheobronchial (HTBE) cells, and a cultured airway epithelial cell line (Calu-3). After intercellular exosomal transfer, comprehensive proteomic and genomic characterization of cell secretions and exosomes was performed. Quantitative proteomics and exosomal miRNA analysis profiles indicated that the two cell types are fundamentally distinct. HTBE cell secretions were typically dominated by fundamental innate/protective proteins, including mucin MUC5B, and Calu-3 cell secretions were dominated by pathology-associated proteins, including mucin MUC5AC. After exosomal transfer/intake, approximately 20% of proteins, including MUC5AC and MUC5B, were significantly altered in HTBE secretions. After exosome transfer, approximately 90 miRNAs (∼4%) were upregulated in HTBE exosomes, whereas Calu-3 exosomes exhibited a preserved miRNA profile. Together, our data suggest that the transfer of exosomal cargo between airway epithelial cells significantly alters the qualitative and quantitative profiles of airway secretions, including mucin hypersecretion, and the miRNA cargo of exosomes in target cells. This finding indicates that cellular information can be carried between airway epithelial cells via exosomes, which may play an important role in airway biology and epithelial remodeling.
We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, ...Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs-a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.
The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSβ-dependent MMR pathways ...correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by ∼17-fold and loss of MutSβ-dependent MMR elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSβ-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSβ-dependent MMR for protection from 1-bp insertions, while MutSβ-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5′-NCN-3′ trinucleotides, 5′-GCA-3′ trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the −1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSβ-dependent MMR pathways.
In eukaryotes, meiotic recombination events are distributed nonrandomly in the genome, with certain regions having high levels of recombination (hotspots) and others having low levels (coldspots). ...Species with similar DNA sequences (for example, chimpanzees and humans) can have strikingly different patterns of hotspots and coldspots. Below, by using a microarray analysis that allows us to measure the frequency of the meiosis-specific double-strand DNA breaks (DSBs) of all 6,000 yeast genes, we show that mutation of a single gene (SIR2), which encodes a histone deacetylase, significantly changes DSB frequencies of 12% of yeast genes, elevating DSBs of 5%, and reducing DSBs of 7%. Many of the genes with repressed recombination are located in large (50-100 kb) regions located near, but not at, the telomeres. Some of the genes with altered frequencies of DSBs (including the ribosomal RNA gene cluster) are known targets of Sir2p deacetylation in the wild-type strain.
DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. ...Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers.
Abstract
Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is ...driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.
Colonization of the lung by
Yersinia pestis
is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by ...which
Y. pestis
adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify
Y. pestis
genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple
Y. pestis
adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing
Y. pestis
adherence and the subsequent development of pneumonic plague.
ABSTRACT
Following inhalation,
Yersinia pestis
rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in
Yersinia
spp., the factors mediating early
Y. pestis
adherence in the lung remain unknown. To identify genes important for
Y. pestis
adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δ
caf1
)
Y. pestis
transposon mutant libraries were serially passaged
in vivo
to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δ
caf1
Y. pestis
backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for
YPO3903
in
Y. pestis
adherence in the lung and showed that YPO3903 regulated transcript levels of
psaA,
which encodes a fimbria previously implicated in
Y. pestis
adherence
in vitro
. Deletion of
psaA
had a minor effect on
Y. pestis
adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to
psaA
. By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early
Y. pestis
adherence during primary pneumonic plague.
IMPORTANCE
Colonization of the lung by
Yersinia pestis
is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which
Y. pestis
adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify
Y. pestis
genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple
Y. pestis
adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing
Y. pestis
adherence and the subsequent development of pneumonic plague.
DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates ...of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G₂ phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.
Genome instability continuously presents perils of cancer, genetic disease and death of a cell or an organism. At the same time, it provides for genome plasticity that is essential for development ...and evolution. We address here the genome instability confined to a small fraction of DNA adjacent to free DNA ends at uncapped telomeres and double-strand breaks. We found that budding yeast cells can tolerate nearly 20 kilobase regions of subtelomeric single-strand DNA that contain multiple UV-damaged nucleotides. During restoration to the double-strand state, multiple mutations are generated by error-prone translesion synthesis. Genome-wide sequencing demonstrated that multiple regions of damage-induced localized hypermutability can be tolerated, which leads to the simultaneous appearance of multiple mutation clusters in the genomes of UV- irradiated cells. High multiplicity and density of mutations suggest that this novel form of genome instability may play significant roles in generating new alleles for evolutionary selection as well as in the incidence of cancer and genetic disease.
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