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Restriction-Modification and CRISPR-Cas Systems: Cooperation Between Innate and Adaptive Immunity in Prokaryotes
Full text linkBacteria have evolved numerous mechanisms to resist the constant assault of viruses (called bacteriophages, or simply phages) that can infect and kill them. Restriction-modification (RM) systems represent one such strategy. Generally, these systems provide defense by coordinating the activities of two distinct enzymes: a restriction endonuclease and a methyltransferase. Both enzymes recognize the same short DNA sequences. The methyltransferase modifies these target sites in the host chromosome, which prevents the restriction endonuclease from cleaving the host\u27s own DNA. In contrast, foreign phage DNA is usually not methylated at these sequences. Consequently, upon injection into the host, the viral DNA is recognized and cleaved by the restriction endonuclease, preventing the progression of the phage\u27s life cycle. Therefore, RM systems are considered a part of the innate immune response because they can provide defense against any phage, including ones that have never been encountered previously, as long as they harbor RM target sites. Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated genes (cas) form another defense system that destroys foreign DNA. The CRISPR array consists of a series of repetitive DNA sequences separated by unique DNA sequences known as spacers. During phage infection, short DNA fragments are taken from the viral DNA and integrated into the CRISPR locus to form new spacers. These sequences are then transcribed into CRISPR RNAs (crRNAs). In type II-A CRISPRCas systems, the crRNAs guide the Cas9 nuclease to a matching viral DNA target for cleavage. As such, unlike RM systems, CRISPR-Cas systems represent an adaptive immune response because they require an initial exposure to a virus in order to become successfully immunized through the acquisition of new spacer sequences. CRISPR-Cas and RM are two of the most prevalent types of defense systems found in bacteria and often co-exist together in a single host. Yet, how they may interact with each other in the context of immunity during bacteriophage infection is poorly understood. Here, in my thesis work, I investigate the interplay between RM and type II-A CRISPR-Cas systems. First, I demonstrate that RM systems provide a weak and temporary protection that stimulates CRISPR spacer acquisition, enabling the cells to survive the viral infection. Then, I go on to show that the restriction activity of the RM system is critical for this process and that the rate of spacer acquisition is correlated to the number of RM target sites in the phage genome. To further uncover the mechanistic link between restriction and the acquisition of new spacers, I implement next-generation sequencing to demonstrate that spacers are preferentially extracted at the dsDNA breaks (DSBs) generated by the restriction endonuclease. Additionally, I show that the host DNA repair complex, AddAB, can process these breaks, which further enhances spacer acquisition. Finally, I follow the dynamics between RM and CRISPR-Cas during the chain of events that occur upon viral infection. I demonstrate that although the RM system provides an immediate line of defense due to its ability to recognize a broad range of foreign invaders, it is ultimately overcome by the rapid emergence of methylated phages, resulting in the death of much of the bacterial population. However, the early RM immune response creates substrates for spacer acquisition by the CRISPR-Cas system in a subset of cells. By using these newly acquired spacers which specify the viral sequences for lethal cleavage by Cas9, these cells can now extinguish the methylated phages, resulting in the survival and regrowth of the population. Collectively, my thesis reveals the molecular mechanisms connecting RM and CRISPR-Cas systems in providing a synergistic anti-phage defense. Reminiscent of eukaryotic immunity, I demonstrate that RM systems provide an initial, short-lived innate immune response, which stimulates a secondary, more robust adaptive immune response by CRISPR-Cas. This work highlights an example of cooperation between RM and CRISPR-Cas, which are two of the most common bacterial defense systems. However, prokaryotes have been shown to harbor a multitude of other putative antiphage defense systems, which can often exist together in a single host. I predict that future studies will likely uncover many more fascinating instances of immune interaction among other sets of defense systems
Structural Studies of the Nucleolar Stages of Ribosome Biogenesis in Yeast
Full text linkThe ribosome is the RNA-protein machine responsible for the essential task of translating mRNA into proteins. Ribosomes are heterodimers made up of a small subunit (SSU, 40S) and a large subunit (LSU, 60S). At the interface of these subunits, the mRNA is decoded by the small subunit and peptide bond formation is catalyzed by the rRNA of the large subunit. The cell therefore requires the timely and accurate assembly of functional ribosomal subunits, a complex process termed ribosome biogenesis. In addition to the ribosomal RNAs and ribosomal proteins that make up the mature subunits, eukaryotic cells require over 200 trans-acting factors to assemble ribosomes. This process begins in the nucleolus- a subcompartment of the nucleus- where the ribosomal DNA is transcribed to produce pre-ribosomal RNAs (pre-rRNAs). The newly transcribed pre-rRNAs recruit many assembly factors essential to their early folding, but many of these factors exact roles were largely unknown at the start of this work. Additionally, as the rRNA forms the active centers of the ribosome, the initial steps of rRNA folding and chaperoning are of great interest. Structural biology studies have elucidated snapshots of later stages of assembly, but structural understanding of the very early stages of ribosome assembly were limited at the start of this work, and therefore our understanding of early assembly factor function and rRNA folding was restricted. To gain insight into the earliest stages of large subunit assembly, we aimed to isolate and structurally characterize an early nucleolar stage of large subunit assembly. The structures of the nucleolar pre-60S provided insights into the roles of many nucleolus-specific assembly factors. Additionally, we observed that the pre-60S rRNA is largely disordered at this stage in assembly. The rRNA domains making up the solvent exposed side of the LSU are in a nearmature conformation, while the rRNA domains that will eventually form the active centers are largely disordered and prevented from folding (Chapter 2). Assembly factors enforce the open architecture of the rRNA while also preventing premature recruitment of later assembly factors. Initial findings towards the study of an even earlier stage of LSU assembly are also described here (Chapter 4). Among the hundreds of assembly factors are several essential RNA helicases. Their functions and RNA targets are a particularly poorly understood aspect of ribosome biogenesis. One of the most well studied helicases necessary for small subunit assembly is Dhr1. Dhr1 is responsible for removing the U3 small nucleolar RNA (U3 snoRNA) from one of the earliest stable precursors of small subunit assembly called the SSU processome. Structures of the SSU processome revealed that the U3 snoRNA is a key architectural feature of this assembly intermediate. Basepairing between the U3 snoRNA and the small subunit rRNA prevents folding between the subdomains of the SSU rRNA, ensuring they mature separately at this stage of assembly. Characterization of Dhr1 and its co-activator Utp14 in biochemical and structural studies has laid the foundation for understanding how the activity of this key enzyme is regulated (Chapter 3). Overall, this work has contributed to the understanding of nucleolar stages of both small and large subunit biogenesis. The studies of the nucleolar pre-60S have elucidated the structures of several nucleolar specific assembly factors and revealed the architecture of early LSU rRNA folding. Biochemical and structural characterization of Dhr1 provided insight into how this RNA helicase is regulated in small subunit biogenesis. Together, these works have expanded our knowledge of the high level of control the cell exhibits over RNA folding and enzyme activity during the earliest stages of ribosome assembly
Ultra-Fast and Multi-Dimensional Face Processing in a New Face Area
Full text linkFaces contain a plethora of information crucial for social interactions. Facial information could be transmitted either through facial shape or motion. The last two decades have established that a network of face-selective areas in the temporal lobe of macaque monkeys supports the visual processing of faces. Most of these studies focused on the processing of static facial shape. They found that each area within the face network contains a large fraction of face-selective cells. And each area encodes facial identity and head orientation differently. A recent brain imaging study discovered a new face area outside of this classic network, the medio-dorsal face area (MD). This finding offers the opportunity to determine whether coding principles revealed inside the core network would generalize to face areas outside the core network. We investigated the encoding of static faces and objects, facial identity, and head orientation, dimensions which had been studied in multiple areas of the core faceprocessing network before, as well as facial expressions and gaze in MD. We found that MD populations form a face-selective cluster with a degree of selectivity comparable to that of areas in the core face-processing network. MD encodes facial identity, expression, and head orientation robustly and independently from each other. Furthermore, MD also encodes the direction of gaze, in addition to head orientation. Thus MD contains a heterogeneous population of cells that establish a multi-dimensional code for static facial shape. Faces could also move in a highly nonlinear fashion, displaying both complex motion patterns and changes in facial shape. Previous brain imaging studies suggest that MD is a face-motion area, but direct evidence from electrophysiology is still missing. We recorded single-unit activities from MD and tested if MD cells are selective to face motion. We found MD cells which only respond to the simultaneous presence of facial shape and motion, thus are truly integrating the two. We found no evidence for two separate MD populations in which one is selective only to facial shape and the other to general motion. Interestingly, MD cells represent face motion in a higher-dimensional space than the optic flow of the stimuli and are highly sensitive to physically subtle face motion, like the shifting of gaze direction. Further, MD cells encode face motion utilizing multiple reference frames to separate the motion of the entire head from that of facial parts. Thus, MD is a bonafide face-motion selective area and might create representation for face motion using canonical neural computations. Finally, we found that MD responds with much a shorter delay than any other face areas. It starts providing facial information as early as 30 ms. We then performed connectivity experiments to study where MD sends such fast information. We found MD is connected to a variety of high-level areas in the prefrontal and other cortices, showing a connectivity pattern strikingly different from those of the other face areas. In sum, MD packs multiple computations into a single area and enables rapid multidimensional face analysis. It also sends such information to downstream areas, possibly for high-level cognitive and social processing. This makes MD an ideal area to support real-life facial interactions
Exhibit details
No full textIdea, design - Olga Nilova
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1005/thumbnail.jp
First Dissertations
No full textFirst dissertations from the Markus Library special collection
Since 1959 The Rockefeller University has awarded over one thousand Ph.D.’s
See also Student Theses and Dissertationshttps://digitalcommons.rockefeller.edu/objects-tell-stories/1023/thumbnail.jp
Centennial Medal
No full textRU Hospital Centennial medal, 2010
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/objects-tell-stories/1033/thumbnail.jp
Endogenous Neurosteroid Hormone Production and Early Oligodendricyte Development
Full text linkThe field of neuroendocrinology grew immensely with the realization that steroid hormone production is not confined to the adrenal and reproductive glands but also occurs in the central nervous system (CNS). Steroids synthesized de novo in the brain and spinal cord are referred to as neurosteroid or neuroactive hormones and encompass estrogens, androgens, glucocorticoids, and mineralocorticoids. Though all substrates and enzymes required for neurosteroid biosynthesis exhibit CNS expression, a thorough comprehension of their functionality is lacking. In addition to mediating stress responses, neurosteroids influence CNS-specific processes known to regulate neural development and pathology. Multiple sclerosis (MS), a debilitating neurodegenerative disorder classified by rampant demyelination, exemplifies the neuroendocrine crosstalk facilitated by CNS-resident steroids. Local steroid production from cholesterol, which also happens to be the primary lipid component of myelin sheaths, is critical to myelin repair. Progesterone in particular is implicated in expediting remyelination following demyelinating insults in animal models via an unknown mechanism. Despite the established effect of progesterone on myelin regeneration, its impact on early myelinogenesis remains unclear. This observation inspired the work presented here, in which I investigated a potential role for progesterone in embryonic oligodendrocyte development. Applying the synthetic progestin Nestorone to mouse cerebellar slice cultures, I found that progesterone stimulates the expression of the mature myelin protein, myelin basic protein. Curious as to whether this phenomenon mirrors progesterone-induced remyelination at the molecular level, I implemented the same experimental system in mice genetically altered to delete expression of the nuclear progesterone receptor. Unexpectedly, removal of this receptor from cerebellar slices led not only to an increase in myelin basic protein expression but more robust oligodendrocyte maturation into myelinating cells actively extending processes to axons and participating in fiber formation. The fact that this surprising effect could not be mediated by the nuclear progesterone receptor prompted me to examine potential caveats to studying individual hormones like progesterone in isolation. Due to the existence of multiple non-canonical progesterone receptors expressed in the CNS, the rapid metabolism of progesterone under physiological conditions, and endogenous astrocytic progesterone production, I opted to transition to a mouse devoid of all neurosteroid hormones. To this end, I obtained a transgenic strain with a mutation inhibiting activity of the enzyme that initiates steroid hormone biosynthesis from cholesterol. Histological analyses of E18.5 embryos revealed no differences between wild-type and knockout general anatomy, CNS architecture, or oligodendrocyte quantity and distribution in the brain and spinal cord. I therefore concluded that access to maternal steroid hormones is sufficient to sustain the knockout mice until inevitable perinatal lethality. Unconvinced that the apparent histological normalcy of the knockout CNS represented the full extent of oligodendrocyte functionality, I performed spinal cord explant experiments in which uniform segments of spinal cords dissected from E12.5 embryos were cultured for four days with no exposure to steroid hormones. In this protocol, fluorescent staining with oligodendrocyte transcription factor Nkx2.2 and myelin basic protein revealed a novel CNS phenotype attributed to neurosteroid deficiency. Diffuse Nkx2.2 and myelin basic protein staining was evident across genotypes. However, the explants from knockout embryos also contained a significantly elevated concentration of myelin basic protein-positive cells along the entirety of the midline in the tissue. While further studies are necessary to determine the nature of this abnormality, the results described here suggest excess oligodendrocyte production and/or proliferation defects in spinal cord tissue deprived of neurosteroid hormones may be at play. With our limited understanding of the intricacies governing oligodendrocyte development from neural precursors, relevant neurosteroid activity undoubtedly warrants ongoing pursuit. Taken together, the data holds diverse implications for neurosteroid involvement in oligodendrocyte development, distinct from the myelin repair so urgently needed in MS patients. Spontaneous remyelination proceeds relatively normally in early acute lesions, but progressively declines over time as lesions become chronic and prone to glial scarring and accumulation of inflammatory cellular debris. My finding that unlike in myelin regeneration, progesterone-induced effects on developmental myelin formation do not rely on signaling through the nuclear progesterone receptor, reaffirms the notion that simply recapitulating the process of early oligodendrocyte maturation is inadequate to treat degenerative disorders like MS. The capacity of a sex hormone like progesterone to exert such remarkable effects on oligodendrocyte behavior is in accordance with the striking gender differences characterizing MS, which is nearly three times more common in women but typically more severe in men. Gender-based variations in steroid hormone levels over the course of a lifetime certainly must be considered. Conceivably, men and women may respond uniquely to various pharmacological interventions; thus, MS treatment modalities cannot be approached with a one size fits all mentality. Future therapeutic improvements are incumbent on accounting for the explicit sexual dimorphism of MS, to which neurosteroid hormones assuredly contribute
A Developmental Pathway for Epithelial-to-Motoneuron Transformation in C. Elegans
Full text linkDuring animal development, neurons and neuron-like cells are generated from progenitor cells that often line tubes. For example, in the developing spinal cord, basal processes of radial glial stem cells line the fluid-filled spinal canal. Notch/Delta signaling is implicated in the differentiation of these cells into motoneurons through control of their proliferative state. Down-regulation of the Notch-dependent Hes1 and Hes5 transcriptional inhibitors in differentiating cells induces expression of the proneural bHLH transcription factors Olig2 and Ngn2, leading to adherens junction loss, delamination from the epithelium, cell migration, and neuronal maturation. A similar sequence of events characterizes formation of pancreatic insulin-secreting β-cells, which are innervated and express genes also active in motoneurons. Here, epithelial progenitor cells lining pancreatic ducts express high levels of Notch, which activates Hes1 repressor, in turn blocking Ngn3 expression. Down-regulation of Notch drives delamination, cell migration, and subsequent differentiation by lifting inhibition of Ngn3 and by allowing Notch to directly activate Ngn3 expression. The Olig family bHLH factor bHLHb4 is expressed in delaminating/migrating cells, and may be involved in β-cell differentiation. The dynamics of gene expression and the regulatory interactions among genes driving motoneuron formation are not fully understood. Furthermore, distinguishing whether genes governing this differentiation event control cell division or cell fate acquisition can be challenging. To address these issues, I have studied a similar transformation process in the genetically amenable nematode, Caenorhabditis elegans. The C. elegans Y cell is one of six epithelial cells that line the rectal tube in first-larval-stage (L1) animals. In L2 animals, Y loses apical junctions with neighboring cells, migrates anterodorsally, and transforms into the PDA motoneuron, which innervates the intestinal and anal depressor muscles. Remarkably, this transformation takes place without cell division. Forward and reverse genetic studies identified a number of genes required for the Y-to-PDA transition, including lin-12/Notch, sem-4/Sall, egl-5/Hox, and several chromatin remodeling genes. Thus, the Y-to-PDA transition is an excellent setting in which to identify regulators that specifically affect motoneuron progenitor-cell differentiation, and not cell division. Here, I present my identification of novel regulators of Y-to-PDA transformation and explore the interactions among them and previously identified genes. I find that loss of lin-12/Notch blocks PDA neuron formation, and gain of lin-12/Notch function generates a precocious PDA neuron. Thus, lin-12/Notch acts as a timing rheostat for motoneuron generation in a cell-division-independent capacity. lin-12/Notch functions, at least in part, by regulating ngn-1/Ngn, through control of the bHLH gene hlh-16/Olig. ngn-1/Ngn basal expression levels are set early on by sem-4/Sall, egl-5/Hox, and hlh-16/Olig. Coincident with the onset of Y-cell morphological changes and migration, I find an increase in ngn-1/Ngn gene expression, governed by sem-4/Sall and egl-5/Hox, but not hlh-16/Olig. Y-cell migration is accompanied by retrograde extension of a process that remains anchored at the rectal slit. The tip of this process serves as a growth point for the PDA axon. Following axonal growth, I identify the axonal cytoskeleton genes unc-119, unc-44/Ank, and unc-33/Crmp as targets of hlh-16/Olig and ngn-1/Ngn, and demonstrate a previously unappreciated role for these genes in regulating the expression of motoneuron terminal differentiation genes. My results highlight intriguing similarities with spinal-cord motoneuron and pancreatic islet formation, suggesting a conserved module for the differentiation of tube-lining cells into motoneuron/motoneuron-like progeny
Characterization of a Novel Sensing Mechanism Governing Antigenic Variation in P. Falciparum
Full text linkPlasmodium falciparum expresses a multi-copy gene family called var in the intraerythroyctic stages of its life cycle in a mutually exclusive manner. var genes encode the chief antigenic and virulence determinant of P. falciparum, PfEMP1, and switching between active genes results in antigenic variation, allowing the parasite to evade the human immune system and cause chronic infections. The molecular mechanisms that control activation and silencing of individual var genes, as well as coordination of the switching process, presently remain incompletely defined. P. falciparum contains only ~60 var gene family members in its genome. Consequently, the question remains as to how this parasite can maintain an antigen-switch rate that allows for the emergence of a new variant when necessary, without rapidly exhausting all 60 members, to sustain chronic infections. The currently held paradigm proposes that antigenic variation follows an intrinsic, programed switching rate, operating independently of any external stimuli. In the following thesis I will present results suggesting the novel possibility that P. falciparum possesses cellular machinery capable of sensing changes in the environment of its host and is able to respond by altering antigen expression. It has been shown that changes in the transcription state of a var gene are controlled epigenetically. The methylation state of histone marks, deposited at active and silent var genes by histone methyltransferases (HMTs), play prominent roles in var gene regulation. Previously, Ukaegbu et al., 2015 showed that manipulating deposition of these marks had a striking impact on var gene expression. Metabolism and epigenetic control of gene expression are linked, as HMT activity is dependent on the intracellular concentrations of methyl donors, which can fluctuate based on nutrient availability. Various studies in other organisms have shown that there is a direct link between the level of intracellular S-adenosylmethionine (SAM), the principle methyl donor in biological methylation modifications, and histone methylation. I explored this connection between metabolism and var gene expression in P. falciparum. Parasites were cultured in growth media containing altered concentrations of nutrients involved in SAM metabolism. Bulk RNA was extracted from cultures, used as a template to synthesize cDNA, and analyzed by qPCR to determine the var gene expression at the population level. Conditions believed to increase SAM pools induced a coordinated switch to one particular var gene, var2csa, over time, phenocopying the results from Ukaegbu et al., 2015. This hypothesis was further tested by modifying expression of key enzymes involved in SAM metabolism. Once again, modifications thought to increase the intracellular level of SAM were found to induce a coordinated switch at the population level to var2csa. Conversely, modifications that lower the level of SAM did not induce expression of var2csa, but instead activated many vars at once across the population. These observations directly challenge the stochastic var switching paradigm by instead suggesting P. falciparum possesses the ability to sense environmental changes. After recognition of a pathogen, activated macrophages modify their microenvironments in various ways. I next tested the effect of two of these immune responses, depletion of amino acids and release of polyamines, on var expression of parasites in vitro. Both perturbations altered var expression, again specifically inducing var2csa. Taking these results together, I propose and discuss two possible models of antigenic variation in P. falciparum. The first centers on intracellular SAM metabolism in describing a promoter competition model governing var switching through var2csa. The second suggests that P. falciparum can sense when the host immune system first begins to recognize it via environmental cues resulting from antibody recognition, and respond by switching var gene expression. This would allow parasites to switch expression of var genes exactly when needed, allowing the most efficient utilization of their limited var gene repertoir
Five Rockefeller Trailblazers
No full textA new display in the Markus Library features Drs. Marie Daly, Rebecca Lancefield, Louise Pearce, Gertrude Perlmann, and Florence Sabin, researchers who held significant tenures at Rockefeller or went on to have distinguished careers at other institutions after training at the university. All were among the first women in their respective fields and are recognized for their pioneering contributions to science.https://digitalcommons.rockefeller.edu/five-rockefeller-trailblazers/1000/thumbnail.jp