Jessica Chang

Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells

A complex network makes simple pores Stomata, the pores found on the surface of plant leaves, form at intervals from stem cells. Development of stomata is controlled by the SPEECHLESS transcription factor. Lau et al. surveyed the genes that SPEECHLESS itself controls. Targets include genes involved in hormone signaling, control of cell proliferation, and the specification of asymmetric cell fates. Despite the apparent simplicity of a single pore, the genetic network that generates that pore is anything but simple. Science, this issue p. 1605 The molecular pathways that regulate an essential adult stem cell lineage in plant stomata are dissected. 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.

Transcriptome dynamics of the stomatal lineage: birth, amplification and termination of a self-renewing population

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.

Development of a Comprehensive Genotype-to-Fitness Map of Adaptation-Driving Mutations in Yeast

Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.

The Molecular Mechanism of a Cis-Regulatory Adaptation in Yeast

Despite recent advances in our ability to detect adaptive evolution involving the cis-regulation of gene expression, our knowledge of the molecular mechanisms underlying these adaptations has lagged far behind. Across all model organisms, the causal mutations have been discovered for only a handful of gene expression adaptations, and even for these, mechanistic details (e.g. the trans-regulatory factors involved) have not been determined. We previously reported a polygenic gene expression adaptation involving down-regulation of the ergosterol biosynthesis pathway in the budding yeast Saccharomyces cerevisiae. Here we investigate the molecular mechanism of a cis-acting mutation affecting a member of this pathway, ERG28. We show that the causal mutation is a two-base deletion in the promoter of ERG28 that strongly reduces the binding of two transcription factors, Sok2 and Mot3, thus abolishing their regulation of ERG28. This down-regulation increases resistance to a widely used antifungal drug targeting ergosterol, similar to mutations disrupting this pathway in clinical yeast isolates. The identification of the causal genetic variant revealed that the selection likely occurred after the deletion was already present at high frequency in the population, rather than when it was a new mutation. These results provide a detailed view of the molecular mechanism of a cis-regulatory adaptation, and underscore the importance of this view to our understanding of evolution at the molecular level.

XenMine: A genomic interaction tool for the Xenopus community.

The Xenopus community has embraced recent advances in sequencing technology, resulting in the accumulation of numerous RNA-Seq and ChIP-Seq datasets. However, easily accessing and comparing datasets generated by multiple laboratories is challenging. Thus, we have created a central space to view, search and analyze data, providing essential information on gene expression changes and regulatory elements present in the genome. XenMine (www.xenmine.org) is a user-friendly website containing published genomic datasets from both Xenopus tropicalis and Xenopus laevis. We have established an analysis pipeline where all published datasets are uniformly processed with the latest genome releases. Information from these datasets can be extracted and compared using an array of pre-built or custom templates. With these search tools, users can easily extract sequences for all putative regulatory domains surrounding a gene of interest, identify the expression values of a gene of interest over developmental time, and analyze lists of genes for gene ontology terms and publications. Additionally, XenMine hosts an in-house genome browser that allows users to visualize all available ChIP-Seq data, extract specifically marked sequences, and aid in identifying important regulatory elements within the genome. Altogether, XenMine is an excellent tool for visualizing, accessing and querying analyzed datasets rapidly and efficiently.

Deletion of the sclerotome-enriched lncRNA PEAT augments ribosomal protein expression.

Significance The majority of transcription generates noncoding RNAs, most of which are uncharacterized. Using RNA-seq on cultured mouse sclerotome, we identified PEAT, a long-noncoding RNA (lncRNA) adjacent to a key regulator of sclerotome, Pax1. We deleted the entire PEAT-transcribed unit using CRISPR/Cas9 and analyzed RNA-seq from mutant embryos. While some lncRNAs regulate the expression of their proximal genes, our analysis showed Pax1 expression to be unchanged. However, we identified 60 ribosomal proteins with elevated expression, and found evidence that bone morphogenetic protein signaling is slightly elevated in PEAT mutants. This study reveals a role for the lncRNA PEAT in sclerotome development and shows next-generation sequencing to be a powerful tool to reveal surprising functions for lncRNAs. To define a complete catalog of the genes that are activated during mouse sclerotome formation, we sequenced RNA from embryonic mouse tissue directed to form sclerotome in culture. In addition to well-known early markers of sclerotome, such as Pax1, Pax9, and the Bapx2/Nkx3-2 homolog Nkx3-1, the long-noncoding RNA PEAT (Pax1 enhancer antisense transcript) was induced in sclerotome-directed samples. Strikingly, PEAT is located just upstream of the Pax1 gene. Using CRISPR/Cas9, we generated a mouse line bearing a complete deletion of the PEAT-transcribed unit. RNA-seq on PEAT mutant embryos showed that loss of PEAT modestly increases bone morphogenetic protein target gene expression and also elevates the expression of a large subset of ribosomal protein mRNAs.

Transcriptional dynamics of tail regeneration in Xenopus tropicalis

In contrast to humans, many amphibians are able to rapidly and completely regenerate complex tissues, including entire appendages. Following tail amputation, Xenopus tropicalis tadpoles quickly regenerate muscle, spinal cord, cartilage, vasculature and skin, all properly patterned in three dimensions. To better understand the molecular basis of this regenerative competence, we performed a transcriptional analysis of the first 72 h of tail regeneration using RNA‐Seq. Our analysis refines the windows during which many key biological signaling processes act in regeneration, including embryonic patterning signals, immune responses, bioelectrical signaling and apoptosis. Our work provides a deep database for researchers interested in appendage regeneration, and points to new avenues for further study.

Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin

ABSTRACT Changes in nuclear morphology contribute to the regulation of complex cell properties, including differentiation and tissue elasticity. Perturbations of nuclear morphology are associated with pathologies that include progeria, cancer and muscular dystrophy. The mechanisms governing nuclear shape changes in healthy cells remain poorly understood, partially because there are few models of nuclear shape variation in healthy cells. Here, we introduce nuclear branching in epidermal fin cells of Xenopus tropicalis as a model for extreme variation of nuclear morphology in a diverse population of healthy cells. We found that nuclear branching arises within these cells and becomes more elaborate during embryonic development. These cells contain broadly distributed marks of transcriptionally active chromatin and heterochromatin, and have active cell cycles. We found that nuclear branches are disrupted by loss of filamentous actin and depend on epidermal expression of the nuclear lamina protein Lamin B1. Inhibition of nuclear branching disrupts fin morphology, suggesting that nuclear branching may be involved in fin development. This study introduces the nuclei of the Xenopus fin as a powerful new model for extreme nuclear morphology in healthy cells to complement studies of nuclear shape variation in pathological contexts. This article has an associated First Person interview with the first author of the paper. Highlighted Article: Nuclei are highly branched throughout the heterogeneous population of healthy epidermal cells in the Xenopus tail fin periphery, and disruption of nuclear branching results in improper fin morphology.

Abstract 185: p300 and STAT3 drive YAP-independent mechanotransduction during breast cancer invasion

83% of non-invasive breast cancers are diagnosed as ductal carcinoma in situ (DCIS). While some DCIS tumors remain confined in the mammary duct, about half progress into invasive ductal carcinoma (IDC), when carcinoma cells break through the basement membrane (BM) into the type-1 collagen (col-1) rich stroma, a key first step towards metastasis. As 90% of cancer-related deaths are due to metastasis, preventing progression to invasive disease could significantly reduce cancer mortality. However there are no established biomarkers for invasive potential and current diagnostic methods cannot predict which DCIS cases will progress to IDC. Interestingly, increased tissue stiffness correlates with invasion and the transcriptional regulator YAP has been implicated as a mechanotransducer, largely based on 2D culture studies. To identify the drivers of DCIS progression, we encapsulated mammary epithelial cells (MECs) in 3D hydrogels with a range of stiffness and that presented either BM-ligands or col-1 containing microenvironments to the cells. RNA-seq identified the global gene expression changes induced by increased 3D culture stiffness in BM-like environments. 3SEQ analysis of breast cancer patient samples revealed that genes regulated by 3D culture stiffness were upregulated in breast cancer patients, demonstrating the relevance of 3D culture models and suggesting expression of S100A7 as a potential biomarker of breast cancer progression. Interestingly gene expression changes induced by increased 3D stiffness in BM-like environments were distinct from col-1 like environments. Col-1 exposure in stiff gels promotes expression of genes whose protein products remodel the col-1 network including FN1 and LOX. As col-1 remodeling promotes cell dissemination, this suggests that col-1 exposure following BM invasion induces pro-metastatic changes in carcinoma phenotype. Surprisingly, enhanced stiffness induced invasion in MECs independently of YAP activation in both BM-like and col-1 rich 3D hydrogels. Instead, bioinformatic analysis identified transcriptional regulators p300 and STAT3 as mediators of 3D mechanosensing. Inhibition of p300 and STAT3 in stiff 3D BM-like environments and, conversely, overexpression in soft 3D BM-like environments confirm a role during stiffness-induced proliferation. This suggests p300 and STAT3 as possible targets for preventing progression to invasive disease. While genetic alterations initiate transformation, these results reveal the miroenvironment events that initiate breast cancer invasion. Citation Format: Joanna Y. Lee, Jessica Chang, Sungmin Nam, Hong-pyo Lee, Antonia A. Dominguez, Sushama Varma, Lei S. Qi, Robert B. West, Ovijit Chaudhuri. p300 and STAT3 drive YAP-independent mechanotransduction during breast cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 185.

PyBoost: A parallelized Python implementation of 2D boosting with hierarchies

Motivation: Gene expression is controlled by networks of transcription factors that bind specific sequence motifs in regulatory DNA elements such as promoters and enhancers. GeneClass is a boosting-based algorithm that learns gene regulatory networks from complementary paired feature sets such as transcription factor expression levels and binding motifs across conditions. This algorithm can be used to predict functional genomics measures of cell state, such as gene expression and chromatin accessibility, in different cellular conditions. We present a parallelized, Python-based implementation of GeneClass, called PyBoost, along with a novel hierarchical implementation of the algorithm, called HiBoost. HiBoost allows regulatory logic to be constrained to a hierarchical group of conditions or cell types. The software can be used to dissect differentiation cascades, time courses or other perturbation data that naturally form a hierarchy or trajectory. We demonstrate the application of PyBoost and HiBoost to learn regulators of tadpole tail regeneration and hematopoeitic stem cell differentiation and validate learned regulators through an inducible CRISPR system. Availability: The implementation is publicly available here: https://github.com/kundajelab/boosting2D/.