Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants. Genome-wide identification of SPCH binidng sites
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants. RNA-Seq profiles of inducible SPCH and wild-type upon induction
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.
Project description:Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.
Project description:CD4+CD25+FOXP3+ human regulatory T cells (Treg) are essential for self-tolerance and immune homeostasis. Here, we generated genome-wide maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation for CD4+CD25highCD45RA+ naive and CD4+CD25highCD45RA- memory Treg and their CD25- conventional T cell (Tconv) counterparts after in vitro expansion . In addition we generated genome-wide maps of the transcription factors STAT5, FOXP3, RUNX1 and ETS1 in expanded CD4+CD25highCD45RA+ Treg- and CD4+CD25- Tconv to elucidate their role in cell type-specific gene regulation. ChIP-seq of 2 histone marks and transcription factors ETS1, STAT5, FOXP3 and RUNX1 in expanded T cell subpopulations
Project description:Obesity is a heritable disorder, with children of obese fathers at higher risk of developing obesity.  Environmental factors epigenetically influence somatic tissues, but the contribution of these factors to the establishment of epigenetic patterns in human gametes is unknown. Here, we hypothesized that weight loss remodels the epigenetic signature of spermatozoa in human obesity. Comprehensive profiling of the epigenome of sperm from lean and obese men showed similar histone positioning, but small non-coding RNA expression and DNA methylation patterns were markedly different. In a separate cohort of morbidly obese men, surgery-induced weight loss was associated with a dramatic remodeling of sperm DNA methylation, notably at genetic locations implicated in the central control of appetite. Our data provide evidence that the epigenome of human spermatozoa dynamically changes under environmental pressure, and offers  insight into how obesity may propagate metabolic dysfunction to the next generation. Examination of the DNA methylation status, histone retention and sncRNA expression of the semen of 13 lean and 10 obese individuals; as well as the DNA methylation status of the semen of 6 obese men undergoing Roux-en-Y GBP surgery, at three time points: approximately 1 week before, 1 week after and 1 year after the surgery.
Project description:We present a novel Tandem Mass Tag Solid Phase Amino Labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatised (C18) solid supports. This method can reduce the number of steps required in complex protocols saving time and potentially reducing sample losses. In our global phosphopeptide profiling workflow (SysQuant) we can cut 24 hours from the protocol while increasing peptide identifications (20%) and reducing side-reactions. Solid phase labeling with TMTs does require some modification to typical labeling conditions particularly pH. It has been found that complete labeling equivalent to 2 standard basic pH solution phase labeling for small and large samples can be achieved on C18 resins using slightly acidic buffer conditions. Improved labeling behaviour on C18 compared to standard basic pH solution phase labeling is demonstrated. We analysed our samples for histidine, serine, threonine and tyrosine-labeling to determine the degree of over-labeling and observed higher than expected levels (25% of all Peptide Spectral Matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution phase labeling protocol. Over-labeling at all these sites is greatly reduced (four-fold to 7% of all PSMs) by the low pH conditions used in the TMT-SPAL protocol. Over-labeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT-labeling compared to label-free methods. Our results also highlight the importance of searching data for over-labeling when labeling methods are used.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants.
Project description:Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In higher plants, the initiation and proliferation of stomatal stem cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). The stomatal stem cells and SPCH, which represent an innovation in seed plants, allow flexibility in the production of stomata, but how SPCH generates these stem cells is unclear. Here, we developed a highly sensitive chromatin immunoprecipitation (ChIP) assay and profiled the cell-type specific genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH directly controls key and novel regulators that drive cell fate and asymmetric cell divisions and enhances responsiveness to cell-cell communication. Our results provide molecular insights on how a master transcription factor generates an adult stem cell lineage that contributes to the success of land plants.