Ixchelt Cuaranta-Monroy

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.

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.

Is the mouse a good model of human PPARγ-related metabolic diseases?

With the increasing number of patients affected with metabolic diseases such as type 2 diabetes, obesity, atherosclerosis and insulin resistance, academic researchers and pharmaceutical companies are eager to better understand metabolic syndrome and develop new drugs for its treatment. Many studies have focused on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a crucial role in adipogenesis and lipid metabolism. These studies have been able to connect this transcription factor to several human metabolic diseases. Due to obvious limitations concerning experimentation in humans, animal models—mainly mouse models—have been generated to investigate the role of PPARγ in different tissues. This review focuses on the metabolic features of human and mouse PPARγ-related diseases and the utility of the mouse as a model.

Dynamic transcriptional control of macrophage miRNA signature via inflammation responsive enhancers revealed using a combination of next generation sequencing-based approaches

MicroRNAs are important components of the post-transcriptional fine-tuning of macrophage gene expression in physiological and pathological conditions. However, the mechanistic underpinnings and the cis-acting genomic factors of how macrophage polarizing signals induce miRNA expression changes are not well characterized. Therefore, we systematically evaluated the transcriptional basis underlying the inflammation-mediated regulation of macrophage microRNome using the combination of different next generation sequencing datasets. We investigated the LPS-induced expression changes at mature miRNA and pri-miRNA levels in mouse macrophages utilizing a small RNA-seq method and publicly available GRO-seq dataset, respectively. Next, we identified an enhancer set associated with LPS-responsive pri-miRNAs based on publicly available H3K4 mono-methylation-specific ChIP-seq and GRO-seq datasets. This enhancer set was further characterized by the combination of publicly available ChIP and ATAC-seq datasets. Finally, direct interactions between the miR-155-coding genomic region and its distal regulatory elements were identified using a 3C-seq approach. Our analysis revealed 15 robustly LPS-regulated miRNAs at the transcriptional level. In addition, we found that these miRNA genes are associated with an inflammation-responsive enhancer network. Based on NFκB-p65 and JunB transcription factor binding, we showed two distinct enhancer subsets associated with LPS-activated miRNAs that possess distinct epigenetic characteristics and LPS-responsiveness. Finally, our 3C-seq analysis revealed the LPS-induced extensive reorganization of the pri-miR-155-associated functional chromatin domain as well as chromatin loop formation between LPS-responsive enhancers and the promoter region. Our genomic approach successfully combines various genome-wide datasets and allows the identification of the putative regulatory elements controlling miRNA expression in classically activated macrophages.

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.

Genomewide effects of peroxisome proliferator-activated receptor gamma in macrophages and dendritic cells--revealing complexity through systems biology.

Systems biology approaches have become indispensable tools in biomedical and basic research. These data integrating bioinformatic methods gained prominence after high‐throughput technologies became available to investigate complex cellular processes, such as transcriptional regulation and protein–protein interactions, on a scale that had not been studied before. Immunology is one of the medical fields that systems biology impacted profoundly due to the plasticity of cell types involved and the accessibility of a wide range of experimental models.

The IL-4/STAT6/PPARγ signaling axis is driving the expansion of the RXR heterodimer cistrome, providing complex ligand responsiveness in macrophages

Abstract Retinoid X receptor (RXR) is an obligate heterodimeric partner of several nuclear receptors (NRs), and as such a central component of NR signaling regulating the immune and metabolic phenotype of macrophages. Importantly, the binding motifs of RXR heterodimers are enriched in the tissue-selective open chromatin regions of resident macrophages, suggesting roles in subtype specification. Recent genome-wide studies revealed that RXR binds to thousands of sites in the genome, but the mechanistic details how the cistrome is established and serves ligand-induced transcriptional activity remained elusive. Here we show that IL-4-mediated macrophage plasticity results in a greatly extended RXR cistrome via both direct and indirect actions of the transcription factor STAT6. Activation of STAT6 leads to chromatin remodeling and RXR recruitment to de novo enhancers. In addition, STAT6 triggers a secondary transcription factor wave, including PPARγ. PPARγ appears to be indispensable for the development of RXR-bound de novo enhancers, whose activities can be modulated by the ligands of the PPARγ:RXR heterodimer conferring ligand selective cellular responses. Collectively, these data reveal the mechanisms leading to the dynamic extension of the RXR cistrome and identify the lipid-sensing enhancer sets responsible for the appearance of ligand-preferred gene signatures in alternatively polarized macrophages.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory

Graphical Abstract Figure. No caption available. SUMMARY Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPAR&ggr; drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)‐4. The functions of PPAR&ggr; on macrophage polarization in this setting were independent of ligand binding. Ligand‐insensitive PPAR&ggr; bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL‐4 re‐stimulation. Ligand‐insensitive PPAR&ggr; binding controlled the expression of an extracellular matrix remodeling‐related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL‐4 and PPAR&ggr; in the affected tissue. Thus, a predominantly ligand‐insensitive PPAR&ggr;:RXR cistrome regulates progressive and/or reinforcing macrophage polarization. HIGHLIGHTSLigand‐insensitive PPAR&ggr; sites are highly abundant in alternatively polarized MQsPPAR&ggr; is recruited to the genome in a ligand‐independent manner upon polarizationLigand‐insensitive PPAR&ggr; alters chromatin structure and facilitates IL‐4 signalingLigand‐insensitive PPAR&ggr; drives progressive polarization via transcriptional memory &NA; Daniel et al. describe that the nuclear receptor PPAR&ggr; has a significant ligand‐insensitive, genome‐bound fraction that affects local chromatin structure upon macrophage polarization. Ligand‐insensitive PPAR&ggr; mediates the expression of a hidden gene set upon repeated IL‐4 exposure, providing transcriptional memory and an epigenomic ratchet mechanism to support progressive polarization.