Suppression of HBV reverse transcription with nucleos t ide analogs NA reduces viral load and liver damage, but has little effect on the nuclear cccDNA pool.
Sas Jean Marc Brocard. Direction Commerciale JM Brocard. Get a mail digest of vacancies that match your profile! Thousand of vacancies are available for you on Wizbii: Setup my job digest. Membership Representative - New Mexico. Find your next job on Wizbii,. By continuing to browse this website, you accept the terms of our cookie use policy. D Sucrose gradient centrifugation of MNase-digested chromatin. An aliquot of each fraction bottom was reverse cross-linked, separated by agarose gel electrophoresis, and visualized by ethidium bromide staining.
S1 A and B. F Mononucleosomes were isolated from cells infected and cultured in the presence of ETV. For sufficient sequencing coverage 1, paired mapped reads would be required SI Materials and Methods. The difference in the total number of reads is indicated in the upper left corner of each sequencing track. PTMs associated with both active and repressive transcriptional states in cellular chromatin were chosen for cccDNA evaluation Fig.
H3K4me3 is found at the transcription start site TSS of actively transcribed genes 22 , 23 , whereas histone acetylation marks H3K27ac and H3Kac indicate active TSS and also active gene enhancers 24 — H3K9me3 can be found predominantly in transcriptionally silent heterochromatin and H3K27me3 at transcriptionally repressed genes A Histone posttranslational modifications PTMs used in this study and their primary location in cellular chromatin.
PTM associated with active and repressed transcription are highlighted in green and red, respectively. B The fraction of all mapped paired reads in each sequencing library that align to HBV. PTMs associated with active and repressed transcription are indicated by the green and red bar, respectively. Read density for each track is represented by height on the y axis scaled to a minimum of reads. HBV transcripts are shown in blue, Enhancer elements in green and the basal core promoter BCP in red and represented on the x axis.
Transcription occurs from left to right. Because PTMs appear in specific patterns, e. A nucleosome-depleted region followed by a strong signal of active PTMs resembles the pattern that can be observed at the TSS of many transcribed genes in human chromatin 25 , Although HepG2 cells are a valuable in vitro model system for HBV infection, they differ in their transcriptional profile 32 and other aspects such as innate immune signaling 33 , 34 from primary liver cells.
We next compared the genomic distribution of the highly enriched PTMs between all three samples Fig. A feature that is shared between all three sources of cccDNA is a distinct peak of active PTMs upstream of Enhancer I, followed by a nucleosome-depleted region.
This comparative analysis of PTMs suggests that the distribution of PTMs in cccDNA chromatin can vary substantially between cell culture models and infected tissue, and that cccDNA chromatin is therefore not exclusively determined by viral components, but can adapt to different cellular environments. Consistent with this observation, Northern blot analysis Fig. The bottom row shows 18S rRNA loading control visualized by methylene blue staining.
These observations are summarized in Fig. A Localization of amplicons 1—4 across the HBV genome. ChIP signal for each amplicon was normalized to the mononucleosome input material. Enrichment for different histone modifications at different human loci in HepG2-NTCP1 cells is shown relative to mononucleosome input material.
However, this hypothesis has not been tested for specific PTMs in the context of de novo infected cells. C treatment reduced HBV transcription in a dose-dependent manner Fig. S6 D are functionally linked. Level of enrichment relative to nucleosomes was normalized to a cellular control locus as described for Fig. C p inhibition by C reduces HBV transcription.
Global effect of C treatment on PTM levels. The level of enrichment relative to nucleosomes was normalized to a cellular control locus as described for Fig. A tremendous amount of research in the past years has been devoted to the genomewide mapping of PTMs in cellular chromatin of numerous cells types and tissues. From this body of work, we have learned that PTMs are distributed in specific patterns, e. Although HBV cccDNA is assembled into chromatin as well, its circular conformation, small genome size, and compact coding and transcript organization are remarkably different from the cellular genome.
It is therefore open to question whether within this context the typical PTM patterns and regulatory mechanisms that apply to cellular chromatin are maintained. Our analysis reveals several key features common to all of the infected cells that we examined. In cellular chromatin, H3K4me3 and H3K27ac enrichment at promoters is known to stimulate transcription by recruiting components of the preinitiation complex and other transcriptional activators 41 — Separate from the mechanisms of transcriptional activation achieved by H3K4me3 and H3K27ac, H3Kac can activate transcription by directly increasing nucleosome mobility This data could indicate that the small circular cccDNA tolerates only lower levels of H3Kac, maybe because the unmodified state of H3K is important for transcriptional regulation in the context of overlapping transcription units 44 that are present in the HBV genome.
Surprisingly, we detected relatively low levels of H3K36me3, a PTM that is usually enriched in the gene body of transcribed genes to suppress aberrant transcription initiation in the wake of Pol2 transcription The low levels of H3K36me3 are possibly due to the negative regulation by the highly enriched mark H3K4me3 46 present throughout cccDNA chromatin.
It remains to be determined whether higher levels of H3K36me3 would interfere with the regulation of cccDNA transcription. In sharp contrast to most activating marks, the repressive mark H3K27me3 is strikingly underrepresented in HBV chromatin, and this feature too was preserved in all three contexts of HBV infection. Interestingly, Riviere et al. Although cccDNA chromatin appears to be generally in an open, transcriptionally permissive conformation throughout the HBV genome, the relative transcription of the four viral promoters differed substantially between the different samples analyzed.
HBV transcription can be modulated by different cellular transcription factors 48 , and different expression levels especially of liver-specific transcription factors among HepG2, PHH, and liver tissue 49 might likely contribute to the selective promoter activation or silencing. However, because we do not detect significant levels of repressive H3K27me3 even at silent HBV promoters e.
Therefore, it remains to be determined exactly how selected HBV promoters are silenced. Hence, an extension of our study to additional individuals and different phases of the chronic HBV infection will be required to get a more complete picture of cccDNA chromatin regulation in vivo.
Interestingly, even near-complete transcriptional silencing of cccDNA seems to occur in vivo, because it has been observed that patients who overcame HBV infection can still carry intact, but apparently transcriptionally largely silent, cccDNA molecules in their liver One intracellular pathway that has been shown to control HBV transcription and replication is the type I IFN response pathway 7 , 55 , High levels of H3K27ac, possibly in concert with other active marks, have been shown to antagonize H3K27me3 deposition in cellular chromatin 58 ; therefore, removal of active PTMs seems a logic first step toward the transcriptional silencing of cccDNA.
Our finding that the epigenetic inhibitor C inhibits HBV transcription in a dose-dependent manner in the absence of measurable toxicity provides a proof of concept that epigenetic manipulation of cccDNA can lead to substantial reduction of HBV transcription and, hence, could present a novel approach to the therapy of chronic HBV infection.
Realizing such an approach is likely to require considerable additional effort, including the analysis of additional PTMs, additional viral strains, clinical samples, and signaling pathways to further elucidate the role of modulation of HBV chromatin structure in the regulation of viral infection.
Our data and cccDNA ChIP-Seq assay described here should facilitate additional investigations aimed at exploring the therapeutic potential of this approach. Such an approach will further reduce viral particle and antigen production and could facilitate alone or in combination with other manipulations the ability of the immune system to reestablish control over or even clear chronic HBV infection Fixation was quenched by adding mM glycine final concentration for 2 min.
Nuclei were pelleted and resuspended in Mnase digestion buffer [50 mM Tris pH 7. Cross-linked liver tissue was washed in ice cold nuclei isolation buffer once, resuspended in the same buffer, and homogenized with 10 strokes of a dounce homogenizer. After 10 min on ice, the homogenized sample was passed through a cell strainer to remove debris, the released nuclei pelleted and resuspended in Mnase digestion buffer. The antibodies used were as follows: For the target enrichment reaction, equal amounts of barcoded sequencing libraries were pooled —1, ng of DNA total amount and enriched for HBV DNA according to the manufacturer's xGen rapid capture protocol v2 with slight modifications: Raw and processed sequencing data were deposited in Gene Expression Omnibus under accession no.
Shown is the total number of paired sequencing reads and the number of paired reads aligning concordantly to the human genome hg19 or hepatitis B virus genome HBV. Several clones were evaluated in HBV infection assays, and the clone with the highest infection rate based on eAg secretion at 7 dpi was used throughout this study.
After 16 h, virus containing media was removed, the cells washed four times, and further incubated in HMM. Media was changed every other day. In some experiments, cells were infected and cultured in the presence of nM Entecavir. Primary human hepatocytes PHH were obtained from Gibco. HBV viral particles were concentrated from cell culture supernatant of a HepG2 cell line with a stably integrated 1. On the next day, the cells were washed four times with incubation buffer and stained with DAPI Calbiochem and secondary antibody goat anti-rabbit IgG conjugated with Alexa Fluor Life Technologies 1: After washing three times in PBS with 0.
For Southern blot analysis, 0. After electrophoresis the DNA was depurinated, denatured, and neutralized exactly as described by Cai et al. Primers used throughout this study are listed in Table S1.
The cccDNA copies per cell in Fig. Paired-end sequencing reads in fastq format were mapped to the HBV or human genome with Bowtie2 61 in no-discordant and no-mixed mode.
Andrea de Bartolomeis 2 Andrea de Bartolomeis. April 1, Received: March 09, ; Revisions received: June 22, ; Accepted: Pansini 5, Naples, Italy E-mail: Abstract Full Text References Abstract.
Remember me Forgotten your password? Subscribe to this journal. Vol 26, Issue 4, Tips on citation download. Brain Res Brain Res Protocols Ann N Y Acad Sci Brain Res Mol Brain Res J Cereb Blood Flow Metab Hossmann, KA Periinfarct depolarizations. Cerebrovasc Brain Metab Rev 8: Kiedrowski, L N -methyl-d-aspartate excitotoxicity: Kiessling, M, Gass, P Stimulus-transcription coupling in focal cerebral ischemia.
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