Zoltan Simandi

A Versatile Method to Design Stem-Loop Primer-Based Quantitative PCR Assays for Detecting Small Regulatory RNA Molecules

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all‐trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.

Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid

Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocytes mimic the gene-expression profile of subcutaneous isolated adipocytes in vivo remarkably well, much closer than 3T3-L1 derived ones. Moreover, the differentiated cells are in a monolayer, allowing a broad range of genome-wide studies in early and late stages of adipocyte differentiation to be performed.

OCT4 acts as an integrator of pluripotency and signal-induced differentiation

Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or β-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses.

PRMT1 and PRMT8 Regulate Retinoic Acid‐Dependent Neuronal Differentiation with Implications to Neuropathology

Retinoids are morphogens and have been implicated in cell fate commitment of embryonic stem cells (ESCs) to neurons. Their effects are mediated by RAR and RXR nuclear receptors. However, transcriptional cofactors required for cell and gene‐specific retinoid signaling are not known. Here we show that protein arginine methyl transferase (PRMT) 1 and 8 have key roles in determining retinoid regulated gene expression and cellular specification in a multistage neuronal differentiation model of murine ESCs. PRMT1 acts as a selective modulator, providing the cells with a mechanism to reduce the potency of retinoid signals on regulatory “hotspots.” PRMT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional coactivator of retinoid signaling at later stages of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression, and altered neuronal activity. Importantly, depletion of PRMT8 results in altered expression of a distinct set of genes, including markers of gliomagenesis. PRMT8 is almost entirely absent in human glioblastoma tissues. We propose that PRMT1 and PRMT8 serve as a rheostat of retinoid signaling to determine neuronal cell specification in a context‐dependent manner and might also be relevant in the development of human brain malignancy. Stem Cells 2015;33:726–741

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

Summary The molecular basis of signal‐dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL‐4‐activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage‐determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL‐4‐repressed genes. In addition, STAT6‐repressed enhancers show extensive overlap with the NF‐&kgr;B p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL‐4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL‐1&bgr; production, and pyroptosis. Thus, the IL‐4‐STAT6 signaling pathway establishes an alternative polarization‐specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli. Graphical Abstract Figure. No Caption available. HighlightsIL‐4‐activated STAT6 acts as a transcriptional repressor in macrophagesIL‐4‐STAT6‐repressed enhancers associate with reduced LDTF and p300 bindingInflammatory responsiveness of the IL‐4‐repressed enhancers is attenuatedIL‐4 limits the LPS‐induced inflammasome activation, IL‐1&bgr; production, and pyroptosis &NA; The molecular bases of repressive transcriptional mechanisms contributing to macrophage polarization are not well understood. Czimmerer et al. show that in alternatively polarized macrophages, IL‐4‐activated STAT6 represses a large set of enhancers modulating the transcriptional program. STAT6‐repressed enhancers are characterized by reduced chromatin accessibility, eRNA expression, LDTF, and p300 binding. IL‐4‐STAT6‐mediated repression limits the inflammatory responsiveness including inflammasome activation, IL‐1&bgr; production, and pyroptosis. Thus, the IL4‐STAT6 pathway establishes an epigenomic signature to selectively repress the macrophage inflammation program.

Nuclear receptors as regulators of stem cell and cancer stem cell metabolism

Cellular metabolism is underpinning physiological processes in all cells. These include housekeeping functions as well as specific activities unique to a particular cell type. A growing number of studies in various experimental models indicate that metabolism is tightly connected to embryonic development as well. It is also emerging that metabolic processes have regulatory roles and by changing metabolism, cellular processes and even fates can be influenced. Nuclear receptors (NRs) are transcription factors, responding to changes in metabolites and are implicated in diverse biological processes such as embryonic development, differentiation, metabolism and cancer. Therefore, NRs are key links between metabolism and cell fate decisions. In this review, we introduce ESRRβ, DAX-1 and LRH-1 as putative regulators of metabolism in pluripotent embryonic stem cells. We also discuss the role of TR4, NGF1β, LXRβ and RARs in stemness. In addition, we summarize our current understanding of the potential roles of NRs in cancer stem cells.

Retinoid Signaling is a Context-Dependent Regulator of Embryonic Stem Cells

Although the beneficial effect of certain foods, such as liver, egg or carrot is known from ancient remedies, one of the common active substances, called Vitamin A, was not identified until 1913, when it has been independently discovered by Elmer McCollum at the University of Wisconsin–Madison, and Lafayette Mendel and Thomas Burr Osborne at Yale University. Since then numerous studies have come to light documenting the effect of vitamin A on the health of the individual from birth to adult age. Hale has demonstrated among the first that deprivation of vitamin A during pregnancy induces congenital ocular malformation (Hale, 1933). Wilson and Warkany later described several other congenital malformations that occurred in fetuses from vitamin A-deficient (VAD) rats affecting the genito-urinary tract, heart and great vessels, ocular and respiratory system (Wilson and Warkany 1947; Wilson and Warkany 1950; Warkany 1954). In 1968 Saunders and Gasseling have shown that grafting a posterior margin zone (called zone of polarizing activity, ZPA) of a chick embryo limb bud to the anterior side is able to induce an extra set of limb structures (Saunders 1968). It suggested that the ZPA region contains a diffusible morphogen. Surprisingly, retinoic acid (RA), a derivative of vitamin A, has been found to have the same effect on the anterior side of the bud (Tickle, Alberts et al. 1982). There was a doubt that retinoic acid is responsible in vivo for the phenomenon, but in 1987 Thaller and Eichele demonstrated the graded distribution of endogenous retinoic acid from posterior to anterior in the limb bud (Thaller and Eichele 1987). This was the time when retinoic acid became known as the first morphogen. Part of the truth that later RA was found to act indirectly, via induction of sonic hedgehog (Shh), and actually Shh is the true morphogen signal peptide produced by the ZPA (Riddle, Johnson et al. 1993). Our understanding on how vitamin A and its derivatives (called retinoids) are able to have such morpho-regulatory effect on the body has dramatically increased in parallel with the evolution of molecular biology methodologies. Development of cloning strategy, establishment of cDNA library made possible the identification of a receptor for the active derivative of vitamin A by the Evans and Chambon laboratories (Giguere, Ong et al. 1987; Petkovich, Brand et al. 1987). This opened the way to clarify the role of retinoids in embryonic development. Several aspects of the retinoid signaling have been described in the last two deacades using genetically modified mice. Excellent reviews are available summarizing these in vivo results (Duester 2008; Dolle 2009; Mark, Ghyselinck et al. 2009).

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

RXR heterodimers orchestrate transcriptional control of neurogenesis and cell fate specification

Retinoid X Receptors (RXRs) are unique and enigmatic members of the nuclear receptor (NR) family with extensive and complex biological functions in cellular differentiation. On the one hand, RXRs through permissive heterodimerization with other NRs are able to integrate multiple lipid signaling pathways and are believed to play a central role to coordinate the development of the central nervous system. On the other hand, RXRs may have heterodimer-independent functions as well. Therefore, a more RXR-centric analysis is warranted to identify its genomic binding sites and regulated gene networks, which are orchestrating the earliest events in neuronal differentiation. Recently developed genome-wide approaches allow systematic analyses of the RXR-driven neural differentiation. Here we applied next generation sequencing-based methodology to track the dynamic redistribution of the RXR cistrome along the path of embryonic stem cell to glutamatergic neuron differentiation. We identified Retinoic Acid Receptor (RAR) and Liver X Receptor (LXR) as dominant heterodimeric partners of RXR in these cellular stages. Our data presented here characterize the RAR:RXR and LXR:RXR-mediated transcriptional program in embryonic stem cells, neural progenitors and terminally differentiated neurons. Considering the growing evidence for dysregulated RXR-mediated signaling in neurodegenerative disorders, such as Alzheimers Disease or Amyotrophic Lateral Sclerosis, the data presented here will be also a valuable resource for the field of neuro(patho)biology.