Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response.

The hepatic acute-phase response is characterized by a massive upregulation of serum proteins, such as haptoglobin and serum amyloid A, at the expense of liver homeostatic functions. Although the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has a well-established role in safeguarding liver function and its cistrome spans around 50% of liver-specific genes, its role in the acute-phase response has received little attention so far. We demonstrate that HNF4A binds to and represses acute-phase genes under basal conditions. The reprogramming of hepatic transcription during inflammation necessitates loss of HNF4A function to allow expression of acute-phase genes while liver homeostatic genes are repressed. In a pre-clinical liver organoid model overexpression of HNF4A maintained liver functionality in spite of inflammation-induced cell damage. Conversely, HNF4A overexpression potently impaired the acute-phase response by retaining chromatin at regulatory regions of acute-phase genes inaccessible to transcription. Taken together, our data extend the understanding of dual HNF4A action as transcriptional activator and repressor, establishing HNF4A as gatekeeper for the hepatic acute-phase response.

Dually Modified Cellulose as a Non-Viral Vector for the Delivery and Uptake of HDAC3 siRNA.

RNA interference can be applied to different target genes for treating a variety of diseases, but an appropriate delivery system is necessary to ensure the transport of intact siRNAs to the site of action. In this study, cellulose was dually modified to create a non-viral vector for HDAC3 short interfering RNA (siRNA) transfer into cells. A guanidinium group introduced positive charges into the cellulose to allow complexation of negatively charged genetic material. Furthermore, a biotin group fixed by a polyethylene glycol (PEG) spacer was attached to the polymer to allow, if required, the binding of targeting ligands. The resulting polyplexes with HDAC3 siRNA had a size below 200 nm and a positive zeta potential of up to 15 mV. For N/P ratio 2 and higher, the polymer could efficiently complex siRNA. Nanoparticles, based on this dually modified derivative, revealed a low cytotoxicity. Only minor effects on the endothelial barrier integrity and a transfection efficiency in HEK293 cells higher than Lipofectamine 2000TM were found. The uptake and release of the polyplexes were confirmed by immunofluorescence imaging. This study indicates that the modified biopolymer is an auspicious biocompatible non-viral vector with biotin as a promising moiety.

Analysis of HDACi-Coupled Nanoparticles: Opportunities and Challenges.

Systemic administration of histone deacetylase inhibitors (HDACi), like valproic acid (VPA), is often associated with rapid drug metabolization and untargeted tissue distribution. This requires high-dose application that can lead to unintended side effects. Hence, drug carrier systems such as nanoparticles (NPs) are developed to circumvent these disadvantages by enhancing serum half-life as well as organ specificity.This chapter gives a summary of the biological characterization of HDACi-coupled NPs in vitro, including investigation of cellular uptake, biocompatibility, as well as intracellular drug release and activity. Suitable methods, opportunities, and challenges will be discussed to provide general guidelines for the analysis of HDACi drug carrier systems with a special focus on recently developed cellulose-based VPA-coupled NPs.

HDACi Delivery Systems Based on Cellulose Valproate Nanoparticles.

The ability of histone deacetylase inhibitors (HDACi) like valproic acid (VPA) as a therapeutic for inflammatory diseases or cancer has increased the interest in HDACi and their targeted transport to diseased tissues. Administration of VPA immobilized on polymeric carriers was found to be a suitable approach to circumvent drawbacks such as rapid metabolization, short serum half-life, or side effects. Polysaccharides are convenient biopolymeric carriers due to their biocompatibility and biodegradability. Furthermore, the hydroxy-, amino-, or carboxylic groups are predestinated for functionalization. The esterification of three hydroxy groups of cellulose with VPA leads to products having a high amount of VPA loading. Subsequent shaping yielded uniform nanoparticles (NPs) of around 150 nm in size capable of releasing VPA in a controlled way under physiological conditions.

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme.

Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2-6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular "labile" heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94-100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Biocompatible valproic acid-coupled nanoparticles attenuate lipopolysaccharide-induced inflammation.

Inflammatory diseases like sepsis are associated with dysregulated gene expression, often caused by an imbalance of epigenetic regulators, such as histone acetyltransferases (HATs) and histone deacetylases (HDACs), and consequently, altered epigenetic chromatin signatures or aberrant posttranslational modifications of signalling proteins and transcription factors. Thus, HDAC inhibitors (HDACi) are a promising class of anti-inflammatory drugs. Recently, an efficient drug delivery system carrying the class I/IIa selective HDACi valproic acid (VPA) was developed to circumvent common disadvantages of free drug administration, e.g. short half-life and side effects. The cellulose-based sulphated VPA-coupled (CV-S) nanoparticles (NPs) are rapidly taken up by cells, do not cause any toxic effects and are fully biocompatible. Importantly, VPA is intracellularly cleaved from the NPs and HDACi activity could be proven. Here, we demonstrate that CV-S NPs exhibit overall anti-inflammatory effects in primary human macrophages and are able to attenuate the lipopolysaccharide-induced inflammatory response. CV-S NPs show superior potential to free VPA to suppress the TLR-MyD88-NF-κB signalling axis, leading to decreased TNF-α expression and secretion.

The N-terminal BRCT domain determines MCPH1 function in brain development and fertility.

MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N'-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.

Biocompatible sulfated valproic acid-coupled polysaccharide-based nanocarriers with HDAC inhibitory activity.

The development of bio-based nanoparticles (NPs) as drug containers is of increasing interest to circumvent several obstacles in drug therapy such as rapid drug metabolization, short serum half-life, and unspecific side effects. The histone deacetylase inhibitor valproic acid (VPA) is known for its anti-inflammatory as well as for its anti-cancer activity. Here, recently developed VPA-loaded NPs based on cellulose- and dextran VPA esters were modified with sulfuric acid half ester moieties to improve intracellular drug release. The NPs show rapid cellular uptake, are non-toxic in vitro and in vivo, and able to induce histone H3 hyperacetylation. Thus, they represent a potent drug delivery system for the application in a variety of treatment settings, such as inflammation, sepsis and defined cancer types. In addition, the flexible NP-system offers a broad range of further options for modification, e.g. for targeting strategies and multi-drug approaches.

Polysaccharide Nanoparticles Bearing HDAC Inhibitor as Nontoxic Nanocarrier for Drug Delivery.

The histone deacetylase inhibitors (HDACi) are potent drugs in the treatment of inflammatory diseases and defined cancer types. However, major drawbacks of HDACi, such as valproic acid (VPA), are limited serum half-life, side effects and the short circulation time. Thus, the immobilization of VPA in a polysaccharide matrix is used to circumvent these problems and to design a suitable nanocarrier system. Therefore, VPA is covalently attached to cellulose and dextran via esterification with degree of substitution (DS) values of up to 2.20. The resulting hydrophobic polymers are shaped to spherical nanoparticles (NPs) with hydrodynamic diameter between 138 to 221 nm and polydispersity indices from 0.064 to 0.094 by nanoprecipitation and emulsification technique. Lipase treatment of the NPs leads to in vitro release of VPA and hence to an inhibition of HDAC2 activity in a HDAC2 assay. NPs are rapidly taken up by HeLa cells and mainly localize in the cytoplasm. The NPs are hemocompatible and nontoxic as revealed by the shell-less hen's egg model.

Epigenetic Erosion in Adult Stem Cells: Drivers and Passengers of Aging.

In complex organisms, stem cells are key for tissue maintenance and regeneration. Adult stem cells replenish continuously dividing tissues of the epithelial and connective types, whereas in non-growing muscle and nervous tissues, they are mainly activated upon injury or stress. In addition to replacing deteriorated cells, adult stem cells have to prevent their exhaustion by self-renewal. There is mounting evidence that both differentiation and self-renewal are impaired upon aging, leading to tissue degeneration and functional decline. Understanding the molecular pathways that become deregulate in old stem cells is crucial to counteract aging-associated tissue impairment. In this review, we focus on the epigenetic mechanisms governing the transition between quiescent and active states, as well as the decision between self-renewal and differentiation in three different stem cell types, i.e., spermatogonial stem cells, hematopoietic stem cells, and muscle stem cells. We discuss the epigenetic events that channel stem cell fate decisions, how this epigenetic regulation is altered with age, and how this can lead to tissue dysfunction and disease. Finally, we provide short prospects of strategies to preserve stem cell function and thus promote healthy aging.

HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3.

Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT1 and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90α/ß). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT1 and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis.

Analysis of Histone Deacetylases Sumoylation by Immunoprecipitation Techniques.

Histone deacetylases (HDACs) are controlling dynamic protein acetylation by removing acetyl moieties from lysine. Histone deacetylases themselves are regulated on the posttranslational level, including modifications with small ubiquitin-like modifier (SUMO) proteins. Detecting SUMO modifications of deacetylases by immunoblotting is technically challenging due to the typically low ratio of the modified compared to the unmodified species. Here, we describe a set of methods for the detection of endogenous sumoylated HDACs by immunoprecipitation and immunoblotting techniques.

Genetic and Epigenetic Mechanisms That Maintain Hematopoietic Stem Cell Function.

All hematopoiesis cells develop from multipotent progenitor cells. Hematopoietic stem cells (HSC) have the ability to develop into all blood lineages but also maintain their stemness. Different molecular mechanisms have been identified that are crucial for regulating quiescence and self-renewal to maintain the stem cell pool and for inducing proliferation and lineage differentiation. The stem cell niche provides the microenvironment to keep HSC in a quiescent state. Furthermore, several transcription factors and epigenetic modifiers are involved in this process. These create modifications that regulate the cell fate in a more or less reversible and dynamic way and contribute to HSC homeostasis. In addition, HSC respond in a unique way to DNA damage. These mechanisms also contribute to the regulation of HSC function and are essential to ensure viability after DNA damage. How HSC maintain their quiescent stage during the entire life is still matter of ongoing research. Here we will focus on the molecular mechanisms that regulate HSC function.

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally.

A father's lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

Transcription factor miz-1 is required to regulate interleukin-7 receptor signaling at early commitment stages of B cell differentiation.

B cell development requires the coordinated action of transcription factors and cytokines, in particular interleukin-7 (IL-7). We report that mice lacking the POZ (Poxvirus and zinc finger) domain of the transcription factor Miz-1 (Zbtb17(ΔPOZ/ΔPOZ)) almost entirely lacked follicular B cells, as shown by the fact that their progenitors failed to activate the Jak-Stat5 pathway and to upregulate the antiapoptotic gene Bcl2 upon IL-7 stimulation. We show that Miz-1 exerted a dual role in the interleukin-7 receptor (IL-7R) pathway by directly repressing the Janus kinase (Jak) inhibitor suppressor of cytokine signaling 1 (Socs1) and by activating Bcl2 expression. Zbtb17(ΔPOZ/ΔPOZ) (Miz-1-deficient) B cell progenitors had low expression of early B cell genes as transcription factor 3 (Tcf3) and early B cell factor 1 (Ebf1) and showed a propensity for apoptosis. Only the combined re-expression of Bcl2 and Ebf1 could reconstitute the ability of Miz-1-deficient precursors to develop into CD19(+) B cells.

Epigenetic mechanisms regulate stem cell expressed genes Pou5f1 and Gfra1 in a male germ cell line.

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia--here, predominantly mediated by HDAC-containing protein complexes.

Krüppel-like factor 4, a "pluripotency transcription factor" highly expressed in male postmeiotic germ cells, is dispensable for spermatogenesis in the mouse.

Krüppel-like factor 4 (Klf4, GKLF) was originally characterized as a zinc finger transcription factor essential for terminal differentiation and cell lineage allocation of several cell types in the mouse. Mice lacking Klf4 die postnatally within hours due to impaired skin barrier function and subsequent dehydration. Recently, KLF4 was also used in cooperation with other transcription factors to reprogram differentiated cells to pluripotent embryonic stem cell-like cells. Moreover, involvement in oncogenesis was also ascribed to KLF4, which is aberrantly expressed in some types of tumors such as breast, gastric and colon cancer. We previously have shown that Klf4 is strongly expressed in postmeiotic germ cells of mouse and human testes suggesting a role for Klf4 also during spermiogenesis. In order to analyze its function we deleted Klf4 in germ cells using the Cre-loxP system. Homologous recombination of the Klf4 locus has been confirmed by genomic southern blotting and the absence of the protein in germ cells was demonstrated by Western blotting and immunofluorescence. Despite its important roles in several significant biological settings, deletion of Klf4 in germ cells did not impair spermiogenesis. Histologically, the mutant testes appeared normal and the mice were fertile. In order to identify genes that were regulated by KLF4 in male germ cells we performed microarray analyses using a whole genome array. We identified many genes exhibiting changed expression in mutants even including the telomerase reverse transcriptase mRNA, which is a stem cell marker. However, in summary, the lack of KLF4 alone does not prevent complete spermatogenesis.

The dynamic epigenetic program in male germ cells: Its role in spermatogenesis, testis cancer, and its response to the environment.

Spermatogenesis is a truly remarkable process that requires exquisite control and synchronization of germ cell development. It is prone to frequent error, as paternal infertility contributes to 30-50% of all infertility cases; yet, in many cases, the mechanisms underlying its causes are unknown. Strikingly, aberrant epigenetic profiles, in the form of anomalous DNA and histone modifications, are characteristic of cancerous testis cells. Germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves stage- and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition. All are postulated to engender unique epigenetic controls. In support of this idea are the findings that mouse models with gene deletions for epigenetic modifiers have severely compromised fertility. Underscoring the importance of understanding how epigenetic marks are set and interpreted is evidence that abnormal epigenetic programming of gametes and embryos contributes to heritable instabilities in subsequent generations. Numerous studies have documented the existence of transgenerational consequences of maternal nutrition, or other environmental exposures, but it is only now recognized that there are sex-specific male-line transgenerational responses in humans and other species. Epigenetic events in the testis have just begun to be studied. New work on the function of specific histone modifications, chromatin modifiers, DNA methylation, and the impact of the environment on developing sperm suggests that the correct setting of the epigenome is required for male reproductive health and the prevention of paternal disease transmission.

Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary.

Follicular growth and oogenesis involve highly dynamic changes in morphogenesis, chromatin structure, and gene transcription. The tight coordination of these events leads to ovulation of a mature oocyte and formation of the luteal tissue necessary to regulate embryo implantation and development. This entire process is regulated by numerous endocrine and in situ mechanisms. The role of epigenetic mechanisms in folliculogenesis, such as the biochemical modification of the DNA packaging proteins, the histones, is not well understood. Our objective was to determine the cellular and follicular stage-specific patterns of histone H3 methylation at lysine 4 (K4) in porcine preovulatory follicles and during luteinization in pig ovaries. Ovary tissues were collected from slaughtered prepubertal and cyclic gilts at various stages of the estrous cycle, pregnancy, and from ovaries recovered from gonatropin-treated gilts at 0, 24, and 38 h post human chorionic gonadotropin (hCG) injection. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin and sectioned using standard histological protocols. Immunofluorescent staining was performed on 3 microm thick sections. The immunostaining pattern of mono-, di-, and tri-methylated histone H3-K4 and lysine-specific demethylase 1 (LSD1, also known as KDM1 or AOF1) was assessed. Interestingly, H3-K4 mono-, di-, and tri-methylation in follicles of prepubertal gilts was specifically distributed and developmentally regulated. While granulosa cells of primary, secondary, and early antral follicles were negative for H3-K4 methylation those from large antral follicles showed a striking upregulation in the cells located in the proximity to the oocyte. Specifically, the cumulus oophorus displayed intense staining for H3-K4 methylation and signals were strongest in the granulosa cells in the inner two cell layers of the follicular wall. Although all oocytes from primary to large antral stage follicles were positive for H3-K4 mono-, di-, and tri-methylation, the patterns of distribution were altered through oocyte follicle development. H3-K4 methylation in granulosa cells was dramatically reduced as time to ovulation approached and was low to undetected at 38 h post hCG treatment. H3-K4 mono-, di-, and tri-methylation in large luteal cells increased as differentiation evolved but remained low in small luteal cells. Strikingly, LSD1 (KDM1) expression was found to be restricted to the corpus luteum. In summary, this study provides new information on histone H3-K4 methylation patterns in the oocyte and follicle during folliculogenesis, which suggests that these epigenetic markers serve an essential regulatory role during folliculogenesis.