Author Correction: Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality.

Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner.

Modulation of chimeric antigen receptor surface expression by a small molecule switch.

Background: Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells.

An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis.

Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes.

Enhancer modeling uncovers transcriptional signatures of individual cardiac cell states in Drosophila.

Here we used discriminative training methods to uncover the chromatin, transcription factor (TF) binding and sequence features of enhancers underlying gene expression in individual cardiac cells. We used machine learning with TF motifs and ChIP data for a core set of cardiogenic TFs and histone modifications to classify Drosophila cell-type-specific cardiac enhancer activity. We show that the classifier models can be used to predict cardiac cell subtype cis-regulatory activities.

Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification.

The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). Here, we used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities.

Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)).

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos.

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos.

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network.

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns.

Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway.

The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other. During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic, and computational strategy for identifying genes expressed in the developing Drosophila heart. Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor and in symmetric cell divisions that produce yet a third type of heart cell.

A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis.

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart.

Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity.

A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh).

Toward a systems-level understanding of developmental regulatory networks.

Developmental regulatory networks constitute all the interconnections among molecular components that guide embryonic development. Developmental transcriptional regulatory networks (TRNs) are circuits of transcription factors and cis-acting DNA elements that control expression of downstream regulatory and effector genes. Developmental networks comprise functional subnetworks that are deployed sequentially in requisite spatiotemporal patterns.

Expression-guided in silico evaluation of candidate cis regulatory codes for Drosophila muscle founder cells.

While combinatorial models of transcriptional regulation can be inferred for metazoan systems from a priori biological knowledge, validation requires extensive and time-consuming experimental work. Thus, there is a need for computational methods that can evaluate hypothesized cis regulatory codes before the difficult task of experimental verification is undertaken. We have developed a novel computational framework (termed "CodeFinder") that integrates transcription factor binding site and gene expression information to evaluate whether a hypothesized transcriptional regulatory model (TRM; i.

An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed.

An increased frequency of autoantibody-inducing CD4+ T cells in pre-diseased lupus-prone mice.

Pathogenic autoantibody production in murine models of lupus is dependent on autoreactive CD4+ helper T cells. However, the mechanisms which permit the selection and maintenance of this autoantibody-inducing CD4+ T-cell repertoire are currently unknown. We hypothesized that the peripheral CD4+ T-cell repertoire of lupus-prone mice was enriched with autoantibody-inducing specificities.

Activation of diverse repertoires of autoreactive T cells enhances the loss of anti-dsDNA B cell tolerance.

CD4+ helper T cells play a critical role in the production of the antinuclear autoantibodies that characterize systemic lupus erythematosus in mice and humans. A key issue is whether this help is derived from a diverse repertoire of autoreactive CD4+ T cells or from a select number of T cells of limited specificity. We used the chronic graft-versus-host disease model to define the diversity of the CD4+ T cell repertoire required to induce the autoantibody response.

Antigen presentation by keratinocytes directs autoimmune skin disease.

The antigen-presenting cells that initiate and maintain MHC class II-associated organ-specific autoimmune diseases are poorly defined. We now describe a new T cell antigen receptor (TCR) transgenic (Tg) model of inflammatory skin disease in which keratinocytes activate and are the primary target of autoreactive CD4(+) T cells. We previously generated keratin 14 (K14)-A(beta)b mice expressing MHC class II only on thymic cortical epithelium.