Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

A Family of Vertebrate-Specific Polycombs Encoded by the LCOR/LCORL Genes Balance PRC2 Subtype Activities.

The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.

Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias.

AIM: Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human. METHODS AND RESULTS: Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations. CONCLUSION: IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.

Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes.

The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.

Loss of Pcgf5 Affects Global H2A Monoubiquitination but Not the Function of Hematopoietic Stem and Progenitor Cells.

Polycomb-group RING finger proteins (Pcgf1-Pcgf6) are components of Polycomb repressive complex 1 (PRC1)-related complexes that catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1), an epigenetic mark associated with repression of genes. Pcgf5 has been characterized as a component of PRC1.5, one of the non-canonical PRC1, consisting of Ring1a/b, Rybp/Yaf2 and Auts2. However, the biological functions of Pcgf5 have not yet been identified. Here we analyzed the impact of the deletion of Pcgf5 specifically in hematopoietic stem and progenitor cells (HSPCs). Pcgf5 is expressed preferentially in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) compared with committed myeloid progenitors and differentiated cells. We transplanted bone marrow (BM) cells from Rosa::Cre-ERT control and Cre-ERT;Pcgf5fl/fl mice into lethally irradiated recipient mice. At 4 weeks post-transplantation, we deleted Pcgf5 by injecting tamoxifen, however, no obvious changes in hematopoiesis were detected including the number of HSPCs during a long-term observation period following the deletion. Competitive BM repopulating assays revealed normal repopulating capacity of Pcgf5-deficient HSCs. Nevertheless, Pcgf5-deficient HSPCs showed a significant reduction in H2AK119ub1 levels compared with the control. ChIP-sequence analysis confirmed the reduction in H2AK119ub1 levels, but revealed no significant association of changes in H2AK119ub1 levels with gene expression levels. Our findings demonstrate that Pcgf5-containing PRC1 functions as a histone modifier in vivo, but its role in HSPCs is limited and can be compensated by other PRC1-related complexes in HSPCs.

Activation of Endogenous Retroviruses in Dnmt1(-/-) ESCs Involves Disruption of SETDB1-Mediated Repression by NP95 Binding to Hemimethylated DNA.

Repression of endogenous retroviruses (ERVs) in mammals involves several epigenetic mechanisms. Acute loss of the maintenance methyltransferase Dnmt1 induces widespread DNA demethylation and transcriptional activation of ERVs, including CpG-rich IAP (intracisternal A particle) proviruses. Here, we show that this effect is not due simply to a loss of DNA methylation. Conditional deletions reveal that both Dnmt1 and Np95 are essential for maintenance DNA methylation. However, while IAPs are derepressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these ERVs remain silenced when Np95 is deleted alone or in combination with Dnmt1. This paradoxical phenotype results from an ectopic interaction between NP95 and the H3K9 methyltransferase SETDB1. Normally, SETDB1 maintains silencing of IAPs, but in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA transiently disrupts SETDB1-dependent H3K9me3 deposition. Thus, our observations reveal an unexpected antagonistic interplay between two repressive pathways involved in retroviral silencing in mammalian cells.

Foxc2 in pharyngeal arch mesenchyme is important for aortic arch artery remodelling and ventricular septum formation.

The forkhead box C2 (Foxc2) protein is a member of the forkhead/winged helix transcription factor family and plays an essential role in cardiovascular development. Previous studies showed that Foxc2 null mouse embryos die during midgestation or just after birth with severe cardiovascular defects, including interruption, coarctation of the aortic arch and ventricular septal defects. These are also seen in human congenital heart disease. However, the tissue specific role of Foxc2 in aortic arch remodelling is not yet fully understood. Here we show that Foxc2 is expressed in a restricted pattern in several cell populations, including the mesenchyme and endothelium of pharyngeal arch arteries, which are important for cardiovascular development. In this study, we use a conditional knockout approach to examine the tissue specific role of Foxc2 in aortic arch remodelling. We demonstrate that mouse embryos lacking Foxc2 in Nkx2.5-expressing mesenchyme and endothelium of pharyngeal arch arteries display aortic arch interruption type B and ventricular septal defects. In contrast, conditional deletion of Foxc2 in Tie2-expressing endothelial cells does not result in aortic arch or ventricular septal defects, but does result in embryonic lethality due to peripheral oedema. Our data therefore provide for a detailed understanding of the role of mesenchymal Foxc2 in aortic arch remodelling and in the development of ventricular septum.

Histone acetylation mediated by Brd1 is crucial for Cd8 gene activation during early thymocyte development.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRß(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.

SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing.

The Polycomb-group (PcG) repressive complex-1 (PRC1) forms microscopically visible clusters in nuclei; however, the impact of this cluster formation on transcriptional regulation and the underlying mechanisms that regulate this process remain obscure. Here, we report that the sterile alpha motif (SAM) domain of a PRC1 core component Phc2 plays an essential role for PRC1 clustering through head-to-tail macromolecular polymerization, which is associated with stable target binding of PRC1/PRC2 and robust gene silencing activity. We propose a role for SAM domain polymerization in this repression by two distinct mechanisms: first, through capturing and/or retaining PRC1 at the PcG targets, and second, by strengthening the interactions between PRC1 and PRC2 to stabilize transcriptional repression. Our findings reveal a regulatory mechanism mediated by SAM domain polymerization for PcG-mediated repression of developmental loci that enables a robust yet reversible gene repression program during development.

Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.

Ticks are ectoparasitic arthropods that can transmit a variety of microorganisms to humans and animals during blood feeding, causing serious infectious disorders, including Lyme disease. Acaricides are pharmacologic agents that kill ticks. The emergence of acaricide-resistant ticks calls for alternative control strategies for ticks and tick-borne diseases. Many animals develop resistance to ticks after repeated infestations, but the nature of this acquired anti-tick immunity remains poorly understood. Here we investigated the cellular and molecular mechanisms underlying acquired resistance to Haemaphysalis longicornis ticks in mice and found that antibodies were required, as was IgFc receptor expression on basophils but not on mast cells. The infiltration of basophils at tick-feeding sites occurred during the second, but not the first, tick infestation. To assess the requirement for basophil infiltration to acquired tick resistance, mice expressing the human diphtheria toxin receptor under the control of the mast cell protease 8 (Mcpt8) promoter were generated. Diphtheria toxin administration to these mice selectively ablated basophils. Diphtheria toxin-mediated basophil depletion before the second tick infestation resulted in loss of acquired tick resistance. These data provide the first clear evidence, to our knowledge, that basophils play an essential and nonredundant role in antibody-mediated acquired immunity against ticks, which may suggest new strategies for controlling tick-borne diseases.