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Analysis of archaic human haplotypes suggests that 5hmC acts as an epigenetic guide for NCO recombination
Get PDFBACKGROUND: Non-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates. METHODS: NCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans. RESULTS: We detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5'- and 3'-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and 'poised' bivalent regions. CONCLUSION: Our results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci
Genomics reveals the history of a complex plant invasion and improves the management of a biological invasion from the South African-Australian biotic exchange
Get PDFMany plants exchanged in the global redistribution of species in the last 200 years, particularly between South Africa and Australia, have become threatening invasive species in their introduced range. Refining our understanding of the genetic diversity and population structure of native and alien populations, introduction pathways, propagule pressure, naturalization, and initial spread, can transform the effectiveness of management and prevention of further introductions. We used 20,221 single nucleotide polymorphisms to reconstruct the invasion of a coastal shrub, Chrysanthemoides monilifera ssp. rotundata (bitou bush) from South Africa, into eastern Australia (EAU), and Western Australia (WAU). We determined genetic diversity and population structure across the native and introduced ranges and compared hypothesized invasion scenarios using Bayesian modeling. We detected considerable genetic structure in the native range, as well as differentiation between populations in the native and introduced range. Phylogenetic analysis showed the introduced samples to be most closely related to the southern-most native populations, although Bayesian analysis inferred introduction from a ghost population. We detected strong genetic bottlenecks during the founding of both the EAU and WAU populations. It is likely that the WAU population was introduced from EAU, possibly involving an unsampled ghost population. The number of private alleles and polymorphic SNPs successively decreased from South Africa to EAU to WAU, although heterozygosity remained high. That bitou bush remains an invasion threat in EAU, despite reduced genetic diversity, provides a cautionary biosecurity message regarding the risk of introduction of potentially invasive species via shipping routes
The Bruce effect: Representational stability and memory formation in the accessory olfactory bulb of the female mouse
Get PDFIn the Bruce effect, a mated female mouse becomes resistant to the pregnancy-blocking effect of the stud. Various lines of evidence suggest that this form of behavioral imprinting results from reduced sensitivity of the female's accessory olfactory bulb (AOB) to the stud's chemosignals. However, the AOB's combinatorial code implies that diminishing responses to one individual will distort representations of other stimuli. Here, we record extracellular responses of AOB neurons in mated and unmated female mice while presenting urine stimuli from the stud and from other sources. We find that, while initial sensory responses in the AOB (within a timescale required to guide social interactions) remain stable, responses to extended stimulation (as required for eliciting the pregnancy block) display selective attenuation of stud-responsive neurons. Such temporal disassociation could allow attenuation of slow-acting endocrine processes in a stimulus-specific manner without compromising ongoing representations that guide behavior
Evaluating the differential expression of TAM family receptors and efferocytosis activities in differentiated and polarized THP-1 macrophage
No full textIn Vivo Imaging of Circadian NET Formation During Lung Injury by Four-Dimensional Intravital Microscopy
No full textNeutrophil extracellular traps (NETs) are toxic extracellular structures deployed by neutrophils in response to pathogens and sterile danger signals. NETs are circadian in nature as mouse and human neutrophils preferentially deploy them at night or early morning. Traditionally, NETs have been quantified using a plethora of methods including immunofluorescence and ELISA-based assays; however few options are available to visualize them in vivo. Here we describe a method to directly visualize and quantify NET formation and release in the microvasculature of the lung using intravital imaging in a model of acute lung injury. The method allows four-dimensional capture and quantification of NET formation dynamics over time and should be a useful resource for those interested in visualizing neutrophil responses in vivo
PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling
Get PDFFor RNA virus families except Picornaviridae, viral RNA sensing includes Toll-like receptors and/or RIG-I. Picornavirus RNAs, whose 5' termini are shielded by a genome-linked protein, are predominately recognized by MDA5. This has important ramifications for adaptive immunity, as MDA5-specific patterns of type-I interferon (IFN) release are optimal for CD4+T cell TH1 polarization and CD8+T cell priming. We are exploiting this principle for cancer immunotherapy with recombinant poliovirus (PV), PVSRIPO, the type 1 (Sabin) PV vaccine containing a rhinovirus type 2 internal ribosomal entry site (IRES). Here we show that PVSRIPO-elicited MDA5 signaling is preceded by early sensing of the IRES by the double-stranded (ds)RNA-activated protein kinase (PKR). PKR binding to IRES stem-loop domains 5-6 led to dimerization and autoactivation, displaced host translation initiation factors, and suppressed viral protein synthesis. Early PKR-mediated antiviral responses tempered incipient viral translation and the activity of cytopathogenic viral proteinases, setting up accentuated MDA5 innate inflammation in response to PVSRIPO infection. IMPORTANCE Among the RIG-I-like pattern recognition receptors, MDA5 stands out because it senses long dsRNA duplexes independent of their 5' features (RIG-I recognizes viral [v]RNA 5'-ppp blunt ends). Uniquely among RNA viruses, the innate defense against picornaviruses is controlled by MDA5. We show that prior to engaging MDA5, recombinant PV RNA is sensed upon PKR binding to the viral IRES at a site that overlaps with the footprint for host translation factors mediating 40S subunit recruitment. Our study demonstrates that innate antiviral type-I IFN responses orchestrated by MDA5 involve separate innate modules that recognize distinct vRNA features and interfere with viral functions at multiple levels
MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated
Get PDFMetastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients
Recruitment of an ancient branching program to suppress carpel development in maize flowers
Get PDFCarpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form
Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals
Get PDFRNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals
BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network
No full textInhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM