Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model.

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.

Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair.

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

Meg3 Non-coding RNA Expression Controls Imprinting by Preventing Transcriptional Upregulation in cis.

Although many long non-coding RNAs (lncRNAs) are imprinted, their roles often remain unknown. The Dlk1-Dio3 domain expresses the lncRNA Meg3 and multiple microRNAs and small nucleolar RNAs (snoRNAs) on the maternal chromosome and constitutes an epigenetic model for development. The domain's Dlk1 (Delta-like-1) gene encodes a ligand that inhibits Notch1 signaling and regulates diverse developmental processes. Using a hybrid embryonic stem cell (ESC) system, we find that Dlk1 becomes imprinted during neural differentiation and that this involves transcriptional upregulation on the paternal chromosome. The maternal Dlk1 gene remains poised. Its protection against activation is controlled in cis by Meg3 expression and also requires the H3-Lys-27 methyltransferase Ezh2. Maternal Meg3 expression additionally protects against de novo DNA methylation at its promoter. We find that Meg3 lncRNA is partially retained in cis and overlaps the maternal Dlk1 in embryonic cells. Combined, our data evoke an imprinting model in which allelic lncRNA expression prevents gene activation in cis.

Chromatin mechanisms in the developmental control of imprinted gene expression.

Hundreds of protein-coding genes and regulatory non-coding RNAs (ncRNAs) are subject to genomic imprinting. The mono-allelic DNA methylation marks that control imprinted gene expression are somatically maintained throughout development, and this process is linked to specific chromatin features. Yet, at many imprinted genes, the mono-allelic expression is lineage or tissue-specific. Recent studies provide mechanistic insights into the developmentally-restricted action of the 'imprinting control regions' (ICRs). At several imprinted domains, the ICR expresses a long ncRNA that mediates chromatin repression in cis (and probably in trans as well). ICRs at other imprinted domains mediate higher-order chromatin structuration that enhances, or prevents, transcription of close-by genes. Here, we present how chromatin and ncRNAs contribute to developmental control of imprinted gene expression and discuss implications for disease. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

ICR noncoding RNA expression controls imprinting and DNA replication at the Dlk1-Dio3 domain.

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.