Programming and reprogramming cellular identity

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.Every cell in the human body contains the same genetic information, with few exceptions, yet each cell type enacts a distinct gene expression program to allow for highly specialized functions. These tightly controlled programs are the results of transcriptional regulation, by transcription factors and chromatin regulators, as well as post-transcriptional regulation, mediated in part by microRNAs (miRNAs). Additionally, cells must respond to external cues, and signal transduction pathways converge on gene regulatory machinery to shape cellular identity. The work presented here focuses on the mechanisms by which transcription factors, chromatin regulators, miRNAs and signal transduction pathways coordinately regulate two particular medically important gene expression programs: (1) the program that controls pluripotency in embryonic stem (ES) cells, giving these cells the capacity to differentiate into every adult cell type, and (2) the program that allows regulatory T (Treg) cells to prevent autoimmunity by suppressing the response of self-reactive conventional T cells. Genomic investigations of the core regulatory circuitry of each of these cells types presented here provide new insight into the genetics of pluripotency and autoimmunity, and suggest a strategy for reprogramming based on chemical manipulation of the cellular programs that control cell identity.by Alexander Marson.Ph.D

Spinal neurons activated in response to pudendal or pelvic nerve stimulation in female rats

The overlapping distribution of spinal neurons activated with either pudendal sensory nerve or pelvic nerve stimulation was examined in the female rat using c-fos immunohistochemistry. Pudendal sensory nerve stimulation resulted in a significant increase in fos-positive cells in the ipsilateral dorsal horn and bilaterally in the medial, lateral and intermediate gray of L5-S1. Pelvic nerve stimulation resulted in significant increases of c-fos immunoreactive nuclei in the ipsilateral dorsal horn, lateral and intermediate gray and bilaterally in the medial gray of L5-S1. Co-distribution of fos immunoreactive nuclei with the vesicular glutamate transporters (VGlut2 and VGlut3) and neurokinin I receptors were found in distinct regions of the dorsal horn, medial and lateral gray. Specific areas in the medial dorsal horn, dorsal gray commissure, laminae VI and X and dorsal lateral gray were activated after stimulation of the pudendal sensory and pelvic nerves, suggesting these areas contain spinal neurons that receive both somatomotor and visceral inputs and are part of the intraspinal circuit that regulates sexual and voiding function

Landscape of stimulation-responsive chromatin across diverse human immune cells.

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells

A large CRISPR-induced bystander mutation causes immune dysregulation.

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic bystander mutations that escape detection by routine targeted genotyping assays

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

CRL4^(AMBRA1) targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling

Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4^(AMBRA1) (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4^(AMBRA1) is required to disrupt the assembly and attenuate the ligase activity of human CRL5^(SOCS3) and HIV‐1 CRL5^(VIF) complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4^(AMBRA1) modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5^(SOCS3) and CRL5^(VIF), respectively. Thus, by discovering a substrate of CRL4^(AMBRA1), ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway

Small-Molecule RORγt Antagonists Inhibit T Helper 17 Cell Transcriptional Network by Divergent Mechanisms

SummaryWe identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity

Transcriptional role of cyclin D1 in development revealed by a “genetic-proteomic” screen

Author manuscript: 2010 September 22.Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers[superscript 1, 2]. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas—an organ that critically requires cyclin D1 function[superscript 3, 4]—cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term ‘genetic–proteomic’, can be used to study the in vivo function of essentially any protein

Specific Immunosuppression with Inducible Foxp3-Transduced Polyclonal T cells

Forkhead box p3 (Foxp3)-expressing regulatory T cells are key mediators of peripheral tolerance suppressing undesirable immune responses. Ectopic expression of Foxp3 confers regulatory T cell phenotype to conventional T cells, lending itself to therapeutic use in the prevention of autoimmunity and transplant rejection. Here, we show that adoptive transfer of polyclonal, wild-type T cells transduced with an inducible form of Foxp3 (iFoxp3) can be used to suppress immune responses on demand. In contrast to Foxp3-transduced cells, iFoxp3-transduced cells home “correctly” into secondary lymphoid organs, where they expand and participate in immune responses. Upon induction of iFoxp3, the cells assume regulatory T cell phenotype and start to suppress the response they initially partook in without causing systemic immunosuppression. We used this approach to suppress collagen-induced arthritis, in which conventional Foxp3-transduced cells failed to show any effect. This provides us with a generally applicable strategy to specifically halt immune responses on demand without prior knowledge of the antigens involved