Genome-wide mapping of chromatin landscape and regulatory networks in decidualizing human endometrial stromal cells and cultured mesenchymal stem cells

Decidualization denotes the differentiation of endometrial stromal cells (EnSCs) into specialized decidual cells that control embryo implantation. This process can be recapitulated in culture upon treatment of primary EnSCs with cyclic AMP analogue and progesterone. In this work, I subjected undifferentiated and decidualizing human EnSCs to Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. ATAC-seq is a newly developed technique that utilizes the highly active transposase Tn5 to interrogate accessibility of the genome and map open chromatin regions. These putative cis-regulatory DNA regions can be further explored for “footprints” of transcription factor (TF) binding. In this study, I optimized ATAC-seq and used this technique first to investigate the regulatory mechanisms underlying decidualization of the human endometrial cells. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility within 10 kb of transcription start sites upon was strongly associated with differential gene expression in decidualizing EnSCs. Analysis of 1,533 opening as well as closing chromatin regions revealed overrepresentation of DNA binding motifs for known decidual TFs and identified putative new regulators, including RAR related orphan receptor A (RORA), aryl hydrocarbon receptor nuclear translocator like (ARNTL) and Meis homeobox 1 (MEIS1). Conversely, downregulation of runt related transcription factor 1 and 2 (RUNX1/RUNX2), SRY-box 12 (SOX12), transcription factor 3 (TCF3), and ETS Proto-Oncogene 1 (ETS1) upon decidualization corresponded to loss of corresponding high-affinity binding sites differentiating EnSCs. Because of its dynamic nature, cyclic human endometrium is a rich source of adult stem cells that could be exploited for clinical purposes. However, clinical use of eMSCs is hampered by differentiation and loss of proliferative capacity of cells in prolonged cultures. As part of my Monash-Warwick alliance studentship, I joined the laboratory of Professor Gargett in Melbourne, Australia, and applied integrated ATAC-seq and RNA-seq analyses to study the impact of TGF-β receptor inhibition on endometrial mesenchymal stem cells (eMSCs) maintained in prolonged culture. I demonstrated that culturing of eMSCs in the presence A83-01, a small molecule inhibitor the of TGF-β receptor, maintains the proliferative capacity and attenuates the loss of stemness features of eMSCs in extended cultures. Furthermore, integrated ATAC-seq and RNA-seq revealed that A83-01 modifies the chromatin accessibility of 5,967 loci and alters the expression 1,463 genes. Mining and cross-referencing of these data sets revealed that A83-01 not only maintains selected stemness-associated genes but also that it represses multiple genes involved in extracellular matrix (ECM) deposition and metabolism. Furthermore, my analysis indicated that induction nuclear receptor subfamily 4 group A member 1 (NR4A1, also known as NUR77) may be an important TF that mediate the repression of ECM genes in response to A83-01 treatment. In summary, by integrating advanced genome-wide expression and DNA accessibility profiling techniques, my work has advanced our understanding of the dynamic changes in the cis-regulatory DNA landscape underpinning decidualization of EnSCs and in the maintenance of a stem-like phenotype of eMSCs in prolonged cultures. Analyses of these two large data sets revealed novel transcriptional regulators in cycling endometrium and putative new targets that could be exploited to accelerate clinical translation of autologous eMSC therapies for a variety of reproductive disorders. Furthermore, the data sets generated during the course of my investigations constitute an important resource to interrogate fundamental molecular questions pertaining to human endometrial cell biology

Analysis of chromatin accessibility in decidualizing human endometrial stromal cells

Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif-basic leucine zipper-as part of a triple motif that also comprised the estrogen receptor and Pax domain binding sites. Without exception, triple motifs were located within Alu elements, which suggests a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally under-represented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression

Impact of sustained transforming growth factor-β receptor inhibition on chromatin accessibility and gene expression in cultured human endometrial MSC

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-β receptor (TGFβ-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFβ-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFβ-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARβ). Selective RARβ inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture

Genome-wide mapping of chromatin landscape and regulatory networks in decidualizing human endometrial stromal cells and cultured mesenchymal stromal cells.

<div>Decidualization denotes the differentiation of endometrial stromal cells (EnSCs) into specialized decidual cells that control embryo implantation. This process can be recapitulated in culture upon treatment of primary EnSCs with cyclic AMP analogue and progesterone. In this work, I subjected undifferentiated and decidualizing human EnSCs to Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. ATAC-seq is a newly developed technique that utilizes the highly active transposase Tn5 to interrogate accessibility of the genome and map open chromatin regions. These putative cis-regulatory DNA regions can be further explored for “footprints” of transcription factor (TF) binding. In this study, I optimized ATAC-seq and used this technique first to investigate the regulatory mechanisms underlying decidualization of the human endometrial cells. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility within 10 kb of transcription start sites upon was strongly associated with differential gene expression in decidualizing EnSCs. Analysis of 1,533 opening as well as closing chromatin regions revealed overrepresentation of DNA binding motifs for known decidual TFs and identified putative new regulators, including RAR related orphan receptor A (RORA), aryl hydrocarbon receptor nuclear translocator like (ARNTL) and Meis homeobox 1 (MEIS1). Conversely, downregulation of runt related transcription factor 1 and 2 (RUNX1/RUNX2), SRY-box 12 (SOX12), transcription factor 3 (TCF3), and ETS Proto-Oncogene 1 (ETS1) upon decidualization corresponded to loss of corresponding high-affinity binding sites differentiating EnSCs.</div><div><br></div><div>Because of its dynamic nature, cyclic human endometrium is a rich source of adult stem cells that could be exploited for clinical purposes. However, clinical use of eMSCs is hampered by differentiation and loss of proliferative capacity of cells in prolonged cultures. As part of my Monash-Warwick alliance studentship, I joined the laboratory of Professor Gargett in Melbourne, Australia, and applied integrated ATAC-seq and RNA-seq analyses to study the impact of TGF-β receptor inhibition on endometrial mesenchymal stem cells (eMSCs) maintained in prolonged culture. I demonstrated that culturing of eMSCs in the presence A83-01, a small molecule inhibitor the of TGF-β receptor, maintains the proliferative capacity and attenuates the loss of stemness features of eMSCs in extended cultures. Furthermore, integrated ATAC-seq and RNA-seq revealed that A83-01 modifies the chromatin accessibility of 5,967 loci and alters the expression 1,463 genes. Mining and cross-referencing of these data sets revealed that A83-01 not only maintains selected stemness-associated genes but also that it represses multiple genes involved in extracellular matrix (ECM) deposition and metabolism. Furthermore, my analysis indicated that induction nuclear receptor subfamily 4 group A member 1 (NR4A1, also known as NUR77) may be an important TF that mediate the repression of ECM genes in response to A83-01 treatment. In summary, by integrating advanced genome-wide expression and DNA accessibility profiling techniques, my work has advanced our understanding of the dynamic changes in the cis-regulatory DNA landscape underpinning decidualization of EnSCs and in the maintenance of a stem-like phenotype of eMSCs in prolonged cultures. Analyses of these two large data sets revealed novel transcriptional regulators in cycling endometrium and putative new targets that could be exploited to accelerate clinical translation of autologous eMSC therapies for a variety of reproductive disorders. Furthermore, the data sets generated during the course of my investigations constitute an important resource to interrogate fundamental molecular questions pertaining to human endometrial cell biology.<br></div