Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance.

In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability of heterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.

Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance.

Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.

Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation.

Heterochromatin assembly requires methylation of histone H3 lysine 9 (H3K9me) and serves as a paradigm for understanding the importance of histone modifications in epigenetic genome control. Heterochromatin is nucleated at specific genomic sites and spreads across extended chromosomal domains to promote gene silencing. Moreover, heterochromatic structures can be epigenetically inherited in a self-templating manner, which is critical for stable gene repression. The spreading and inheritance of heterochromatin are believed to be dependent on preexisting H3K9 tri-methylation (H3K9me3), which is recognized by the histone methyltransferase Clr4/Suv39h via its chromodomain, to promote further deposition of H3K9me. However, the process involving the coupling of the "read" and "write" capabilities of histone methyltransferases is poorly understood. From an unbiased genetic screen, we characterize a dominant-negative mutation in histone H3 (H3G13D) that impairs the propagation of endogenous and ectopic heterochromatin domains in the fission yeast genome. H3G13D blocks methylation of H3K9 by the Clr4/Suv39h methyltransferase and acts in a dosage-dependent manner to interfere with the spreading and maintenance of heterochromatin. Our analyses show that the incorporation of unmethylatable histone H3G13D into chromatin decreases H3K9me3 density and thereby compromises the read-write capability of Clr4/Suv39h. Consistently, enhancing the affinity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome the defects in heterochromatin assembly caused by H3G13D Our work directly implicates methylated histones in the transmission of epigenetic memory and shows that a critical density threshold of H3K9me3 is required to promote epigenetic inheritance of heterochromatin through the read-write mechanism.

MUSASHI-Mediated Expression of JMJD3, a H3K27me3 Demethylase, Is Involved in Foamy Macrophage Generation during Mycobacterial Infection.

Foamy macrophages (FM)s harbor lipid bodies that not only assist mycobacterial persistence within the granulomas but also are sites for intracellular signaling and inflammatory mediators which are essential for mycobacterial pathogenesis. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs during mycobacterial infection are not clear. Here, we report for the first time that jumonji domain containing protein (JMJD)3, a demethylase of the repressive H3K27me3 mark, orchestrates the expression of M. tuberculosis H37Rv-, MDR-JAL2287-, H37Ra- and M. bovis BCG-induced genes essential for FM generation in a TLR2-dependent manner. Further, NOTCH1-responsive RNA-binding protein MUSASHI (MSI), targets a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein, to positively regulate infection-induced JMJD3 expression, FM generation and M2 phenotype. Investigations in in vivo murine models further substantiated these observations. Together, our study has attributed novel roles for JMJD3 and its regulators during mycobacterial infection that assist FM generation and fine-tune associated host immunity.

Aspergillus fumigatus Cell Wall α-(1,3)-Glucan Stimulates Regulatory T-Cell Polarization by Inducing PD-L1 Expression on Human Dendritic Cells.

Background: Human dendritic cell (DC) response to α-(1,3)-glucan polysaccharide of Aspergillus fumigatus and ensuing CD4+ T-cell polarization are poorly characterized. Methods: α-(1,3)-Glucan was isolated from A. fumigatus conidia and mycelia cell wall. For the analysis of polarization, DCs and autologous naive CD4+ T cells were cocultured. Phenotype of immune cells was analyzed by flow cytometry, and cytokines by enzyme-linked immunosorbent assay (ELISA). Blocking antibodies were used to dissect the role of Toll-like receptor 2 (TLR2) and programmed death-ligand 1 (PD-L1) in regulating α-(1,3)-glucan-mediated DC activation and T-cell responses. DCs from TLR2-deficient mice were additionally used to consolidate the findings. Results: α-(1,3)-Glucan induced the maturation of DCs and was dependent in part on TLR2. "α-(1,3)-Glucan-educated" DCs stimulated the activation of naive T cells and polarized a subset of these cells into CD4+CD25+FoxP3+ regulatory T cells (Tregs). Mechanistically, Treg stimulation by α-(1,3)-glucan was dependent on the PD-L1 pathway that negatively regulated interferon-gamma (IFN-γ) secretion. Short α-(1,3)-oligosaccharides lacked the capacity to induce maturation of DCs but significantly blocked α-(1,3)-glucan-induced Treg polarization. Conclusions: PD-L1 dictates the balance between Treg and IFN-γ responses induced by α-(1,3)-glucan. Our data provide a rationale for the exploitation of immunotherapeutic approaches that target PD-1-PD-L1 to enhance protective immune responses to A. fumigatus infections.

WNT-inflammasome signaling mediates NOD2-induced development of acute arthritis in mice.

In addition to its role in innate immunity, the intracellular pathogen sensor nucleotide-binding oligomerization domain 2 (NOD2) has been implicated in various inflammatory disorders, including the development of acute arthritis. However, the molecular mechanisms involved in the development of NOD2-responsive acute arthritis are not clear. In this study, we demonstrate that NOD2 signals to a cellular protein, Ly6/PLAUR domain-containing protein 6, in a receptor-interacting protein kinase 2-TGF-ß-activated kinase 1-independent manner to activate the WNT signaling cascade. Gain- or loss-of-function of the WNT signaling pathway in an in vivo experimental mouse arthritis model or in vitro systems established the role for WNT-responsive X-linked inhibitor of apoptosis during the development of acute arthritis. Importantly, WNT-stimulated X-linked inhibitor of apoptosis mediates the activation of inflammasomes. The subsequent caspase-1 activation and IL-1ß secretion together contribute to the phenotypic character of the inflammatory condition of acute arthritis. Thus, identification of a role for WNT-mediated inflammasome activation during NOD2 stimulation serves as a paradigm to understand NOD2-associated inflammatory disorders and develop novel therapeutics.

Ac2PIM-responsive miR-150 and miR-143 target receptor-interacting protein kinase 2 and transforming growth factor beta-activated kinase 1 to suppress NOD2-induced immunomodulators.

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress ß-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

NOD2-nitric oxide-responsive microRNA-146a activates Sonic hedgehog signaling to orchestrate inflammatory responses in murine model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress major histocompatibility complex II expression during Mycobacterium bovis bacillus Calmette-Guerin infection.

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis.

BACKGROUND: Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. METHODS: Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment. RESULTS: Here, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. CONCLUSION: Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

Outcomes and quality of life of frail patients following elective ventral hernia repair: Retrospective review of a national hernia collaborative.

BACKGROUND: Ventral hernia repair (VHR) is one of the most common general surgery procedures among older adults but is often deferred due to a higher risk of complications. This study compares postoperative quality of life (QOL) and complications between frail and non-frail patients undergoing elective VHR. We hypothesized that frail patients would have higher complication rates and smaller gains in quality of life compared to non-frail patients. STUDY DESIGN: Patients 65 years of age and older, undergoing elective VHR between 2018 and 2022 were selected from the ACHQC (Abdominal Core Health Quality Collaborative) and grouped based on frailty scores obtained using the Modified Frailty Index (mFI-5). Logistic regression adjusting for hernia characteristics (size, recurrent, parastomal, incisional) were performed for 30-day outcomes including surgical site infections (SSI), surgical site occurrences (SSO), surgical site infections/occurrences requiring procedural intervention (SSOPI), and readmission. Multivariable analyses controlling for patient and procedure characteristics were performed comparing QOL scores (HerQLes scale, 0-100) at baseline, 30 days, 6 months and 1 year postoperatively. RESULTS: A total of 4888 patients were included, 29.17% non-frail, 47.87% frail, and 22.95% severely frail. On adjusted analysis, severely frail patients had higher odds of SSO (most commonly seroma formation) but no evidence of a difference in SSI, SSOPI, readmission or mortality. Severely frail patients had lower median QOL scores at baseline (48.3/100, IQR 26.1-71.7, p â€‹= â€‹0.001) but reported higher QOL scores at both 30-days (68.3/100, IQR 41.7-88.3, p â€‹= â€‹0.01) and 6-months (86.7/100, IQR 65.0-93.3, p â€‹= â€‹0.005). CONCLUSION: Severely frail patients reported similar increases in QOL and similar complications to their not frail counterparts. Our results demonstrate that appropriately selected older patients, even those who are severely frail, may benefit from elective VHR in the appropriate clinical circumstance.

Quality of life among older patients after elective ventral hernia repair: A retrospective review.

BACKGROUND: Ventral hernia repair is a common elective general surgery procedure among older patients, a population at greater risk of complications. Prior research has demonstrated improved quality of life in this population despite increased risk of complications. This study sought to assess the relationship between post-ventral hernia repair quality of life and patient frailty. We hypothesized that frail patients would report smaller gains in quality of life compared to the non-frail group. METHODS: The Abdominal Core Health Quality Collaborative was used to identify a cohort of patients 65 years of age or older undergoing elective ventral hernia repair from 2018 to 2022. Patients were categorized based on their modified frailty index scores as not frail/prefrail, frail, and severely frail. Quality of life was assessed using a patient-reported 12-item scale preoperatively, 30 days, 6 months, and 1 year postoperatively. RESULTS: A total of 3,479 patients were included: 30.93% non-frail, 47.17% frail, and 21.90% severely frail. Severely frail patients had lower quality of life scores at baseline (P = .001) but reported higher quality of life at both 30 days (1.24 points higher, 95% confidence interval (-1.51, 2.52), P = .010) and 6 months (0.92 points higher, 95% confidence interval (-2.29, 4.13), P = .005). Severely frail patients had higher rates of surgical site complications (P < .001) but no difference in 30-day readmissions. CONCLUSION: Our results found that frail patients reported the greatest increase in quality of life 1 year from baseline, showing that they, when selected appropriately, can gain equal benefits and have similar surgical outcomes as their non-frail counterparts.

Limited effects of dehydration on object discrimination in the novel object recognition paradigm in young and middle-aged male and female rats.

Dehydration is associated with impaired cognitive function in humans. Limited animal research also suggests that disruptions in fluid homeostasis impair performance in cognitive tasks. We previously demonstrated that extracellular dehydration impaired performance in the novel object recognition memory test in a sex and gonadal hormone specific manner. The experiments in this report were designed to further characterize the behavioral effects of dehydration on cognitive function in male and female rats. In Experiment 1, we tested whether dehydration during the training trial in the novel object recognition paradigm would impact performance, while euhydrated, in the test trial. Regardless of hydration status during training, all groups spent more time investigating the novel object during the test trial. In Experiment 2, we tested whether aging exacerbated dehydration-induced impairments on test trial performance. Although aged animals spent less time investigating the objects and had reduced activity levels, all groups spent more time investigating the novel object, compared to the original object, during the test trial. Aged animals also had reduced water intake after water deprivation and, unlike the young adult rats, there was no sex difference in water intake. Together these results, in combination with our previous findings, suggest that disruptions in fluid homeostasis have limited effects on performance in the novel object recognition test and may only impact performance after specific types of fluid manipulations.

Histone deacetylation primes self-propagation of heterochromatin domains to promote epigenetic inheritance.

Heterochromatin assembly, involving histone H3 lysine-9 methylation (H3K9me), is nucleated at specific genomic sites but can self-propagate across extended domains and, indeed, generations. Self-propagation requires Clr4/Suv39h methyltransferase recruitment by pre-existing H3K9 tri-methylation (H3K9me3) to perpetuate H3K9me deposition and is dramatically affected by chromatin context. However, the mechanism priming self-propagation of heterochromatin remains undefined. We show that robust chromatin association of fission yeast class II histone deacetylase Clr3 is necessary and sufficient to support heterochromatin propagation in different chromosomal contexts. Efficient targeting of Clr3, which suppresses histone turnover and maintains H3K9me3, enables self-propagation of an ectopic heterochromatin domain via the Clr4/Suv39h read-write mechanism requiring methylated histones. The deacetylase activity of Clr3 is necessary and, when inactivated, heterochromatin propagation can be recapitulated by removing two major histone acetyltransferases. Our results show that histone deacetylation, a conserved heterochromatin feature, preserves H3K9me3 that transmits epigenetic memory for stable propagation of silenced chromatin domains through multiple generations.

Conserved protein Pir2ARS2 mediates gene repression through cryptic introns in lncRNAs.

Long non-coding RNAs (lncRNAs) are components of epigenetic control mechanisms that ensure appropriate and timely gene expression. The functions of lncRNAs are often mediated through associated gene regulatory activities, but how lncRNAs are distinguished from other RNAs and recruit effector complexes is unclear. Here, we utilize the fission yeast Schizosaccharomyces pombe to investigate how lncRNAs engage silencing activities to regulate gene expression in cis. We find that invasion of lncRNA transcription into the downstream gene body incorporates a cryptic intron required for repression of that gene. Our analyses show that lncRNAs containing cryptic introns are targeted by the conserved Pir2ARS2 protein in association with splicing factors, which recruit RNA processing and chromatin-modifying activities involved in gene silencing. Pir2 and splicing machinery are broadly required for gene repression. Our finding that human ARS2 also interacts with splicing factors suggests a conserved mechanism mediates gene repression through cryptic introns within lncRNAs.

A conserved dimer interface connects ERH and YTH family proteins to promote gene silencing.

Gene regulatory mechanisms rely on a complex network of RNA processing factors to prevent untimely gene expression. In fission yeast, the highly conserved ortholog of human ERH, called Erh1, interacts with the YTH family RNA binding protein Mmi1 to form the Erh1-Mmi1 complex (EMC) implicated in gametogenic gene silencing. However, the structural basis of EMC assembly and its functions are poorly understood. Here, we present the co-crystal structure of the EMC that consists of Erh1 homodimers interacting with Mmi1 in a 2:2 stoichiometry via a conserved molecular interface. Structure-guided mutation of the Mmi1Trp112 residue, which is required for Erh1 binding, causes defects in facultative heterochromatin assembly and gene silencing while leaving Mmi1-mediated transcription termination intact. Indeed, EMC targets masked in mmi1∆ due to termination defects are revealed in mmi1W112A. Our study delineates EMC requirements in gene silencing and identifies an ERH interface required for interaction with an RNA binding protein.

CPF Recruitment to Non-canonical Transcription Termination Sites Triggers Heterochromatin Assembly and Gene Silencing.

In eukaryotic genomes, heterochromatin is targeted by RNAi machinery and/or by pathways requiring RNA elimination and transcription termination factors. However, a direct connection between termination machinery and RNA polymerase II (RNAPII) transcriptional activity at heterochromatic loci has remained elusive. Here, we show that, in fission yeast, the conserved cleavage and polyadenylation factor (CPF) is a key component involved in RNAi-independent assembly of constitutive and facultative heterochromatin domains and that CPF is broadly required to silence genes regulated by Clr4SUV39H. Remarkably, CPF is recruited to non-canonical termination sites within the body of genes by the YTH family RNA-binding protein Mmi1 and is required for RNAPII transcription termination and facultative heterochromatin assembly. CPF loading by Mmi1 also promotes the selective termination of long non-coding RNAs that regulate gene expression in cis. These analyses delineate a mechanism in which CPF loaded onto non-canonical termination sites specifies targets of heterochromatin assembly and gene silencing.