The discovery of human pluripotent stem cells (hPSCs) has opened a new field called regenerative medicine that offers new strategies for curing diseases and drug discovery. It also provides the means of regenerating disease-relevant cells in vitro for disease modeling, and the possibility of cell replacement therapy. Among the most promising applications of hPSCs technology is the generation of blood cells that can be used for engraftment or transfusion in the clinic. Generating engraftable hematopoietic stem cells from hPSCs in vitro can fulfill the promise of using hPSCs to cure human diseases. Making functional HSCs in vitro from hPSCs remains an elusive goal. There are key pathways that are misregulated during hPSCs differentiation, which could impair the engraftment potential of hPSCs. WNT signaling is needed in the early phase of differentiation. However, evidence from mouse models and human development show that WNT signaling is downregulated during the maturation of HSCs. Therefore, we hypothesize that mimicking the dynamics of WNT signaling temporally during the differentiation could improve the functional maturation of differentiated HPCs. To this end, we have established an inducible gene activation system based on dCas9-VPR that can activate endogenous loci. We performed targeted activation of negative regulators of WNT. The system has shown promise in specific activation of WNT negative regulators, AXIN2 and APC2, but it needs further optimization to be able to steer cell fate and obtain functional HSCs.

Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

FOXL2 constitue un gène majeur de la différenciation et de la fonction ovarienne. Chez l’Homme, l’haploinsuffisance de ce gène induit des malformations palpébrales qui peuvent être associées ou non à une insuffisance ovarienne prématurée (BPES de type I ou II). Dans certains cas, des anomalies de l’expression de FOXL2 sont liées à des délétions de régions situées très en amont du gène, témoignant de la présence d’activateurs distaux. L’existence de cette régulation à longue distance a aussi été mise en évidence dans l’espèce caprine par notre équipe. La mutation naturelle PIS (Polled Intersex Syndrome) induit à l’état homozygote l’absence d’expression du gène FOXL2 dans les gonades XX, conduisant au développement de testicules à la place d’ovaires. Ainsi, au cours de mon doctorat j’ai été amenée à travailler sur deux aspects différents : (i) l’analyse des cibles/voies activées par FOXL2 dans la gonade, et (ii) l’étude de la régulation à distance de l’expression de FOXL2.En ce qui concerne le premier point, l’analyse du rôle d’un gène candidat, Dmxl2, chez la souris a nécessité la mise en place d’une invalidation conditionnelle de ce gène (Knock-out ou KO) dans les gonades (le KO total étant létal à la naissance). Chez les mâles, une diminution de 60% de la production de spermatozoïdes a été observée à la puberté pendant la première vague de spermatogénèse.En ce qui concerne le second point, l’étude d’une région régulatrice potentielle de l’expression de FOXL2 a permis de définir des éléments très conservés présentant un profil épigénétique caractéristique de régions de type « enhancers ». J’ai ensuite établi un modèle in vitro « d’édition de l’épigénome » du locus FOXL2, en utilisant la technologie CRISPR/dCas9-p300 pour modifier la marque épigénétique H3K27ac et activer l’expression de ce gène. A long terme, ces travaux pourraient aboutir à la création de « médicaments épigénétiques » pour soutenir l’expression de FOXL2 et rétablir la fertilité des patientes atteintes d’une mutation de ce gène. / FOXL2 is a major gene for ovarian differentiation and functions. In humans, FOXL2 haploinsufficiency induces eyelid malformations with or without premature ovarian failure (BPES type I or II). In some cases, abnormalities of FOXL2 expression are related to deletions of regions located far upstream of this gene, indicating the presence of distal activators. The existence of this long-range regulation has also been demonstrated in the goat species by our laboratory. The natural mutation PIS (Polled Intersex Syndrome) when homozygous induces the silencing of FOXL2 expression in XX gonads, leading to the development of testes instead of ovaries. Thus, during my PhD, I worked on two different aspects: (i) the analysis of FOXL2-activated targets/pathways in the gonad, and (ii) the study of the long-range regulation of FOXL2 expression.Regarding the first point, gene function analysis of the candidate gene Dmx12 required the establishment of a conditional knock-out in the mouse gonad (Dmxl2 total KO is lethal at birth). In males, a 60% decrease in sperm production was observed at puberty during the first wave of spermatogenesis.Regarding the second point, the study of a putative regulatory region of FOXL2 expression allowed to define highly conserved elements harbouring typical enhancer epigenetic profile. Then, I established an in vitro model of FOXL2 locus “epigenome editing”, using the CRISPR/dCas9-p300 technology to modify the epigenetic mark H3K27ac. In the long term, this work may lead to the development of "epigenetic drugs" to support the expression of FOXL2 and restore the fertility of patients with a mutation of this gene.

Non-coding DNA in the human genome is widely studied to investigate its effect on coding DNA and gene expression. Non-coding DNA contains cis-regulatory elements that influence transcription of genes upstream, downstream, or nearby. These regulatory elements have largely been studied as enhancers that promote the transcription of genes. To explore these regulatory elements as silencers, we chose validated bifunctional elements to study their silencing capability and their chromatin markers. We used chromatin immunoprecipitation methods with dCas9 to target these elements using single-guide RNAs (sgRNAs). We experimented with various cloning methods to insert dCas9 into the pUAS vector. We initially planned to use the Gibson Assembly method, but after no success, we tried site-directed mutagenesis and traditional cloning with restriction enzymes. We were able to successfully insert dCas9 into the pUAS vector with traditional cloning, and we were then able to inject the construct into Drosophila melanogaster. We designed sgRNAs to target desired elements of DNA that we chose to study as cis-regulatory elements. The sgRNA sequences were cloned into the pCFD5 vector and injected into another line of flies. The transgenic flies containing the pUAS/dCas9 plasmid will then be crossed with the flies containing the pCFD5/sgRNA to develop offspring that express the target elements and could undergo chromatin pulldown to examine the bifunctional regulation of these DNA elements in cells. Results from a quantitative PCR (qPCR) assay on Drosophila expressing the cloned pUAS vector with dCas9 and a sgRNA for the white gene showed chromatin pulldown efficiency and successful transfection. The Drosophila chromatin targeted by the sgRNAs will be pulled down, solubilized, and then analyzed on a western blot to screen for chromatin modifications, primarily histone modifications. We can then identify chromatin markers associated with elements when they act as silencers in the mesoderm versus when they act as non- mesodermal enhancers. We can also determine if the silencer acts by interacting with a promoter or with an enhancer to repress gene expression. If ENCODE can profile the data found in this project, the chromatin markers can act as a predictive tool for the identification of silencers.

La méthylation de l’ADN est une modification épigénétique sur les cytosines des dinucléotides CpG catalysée par les enzymes DNMT. Les cellules cancéreuses présentent des hyperméthylations aberrantes sur les promoteurs de gènes dits suppresseurs de tumeurs, ce qui contribue à leur répression transcriptionnelle et favorise la progression tumorale. De par sa nature réversible, la méthylation de l’ADN est une cible de choix pour des thérapies épigénétiques ; cependant, les inhibiteurs de DNMT ont une action de déméthylation globale du génome qui conduit à une forte toxicité. Mon travail a consisté à développer des stratégies de déméthylation ciblée sur des régions spécifiques du génome. Premièrement, j’ai validé une stratégie induisant une reprogrammation épigénétique spécifique et durable du gène suppresseur de tumeurs SERPINB5 dans des cellules de cancer du sein. Deuxièmement, j’ai optimisé des stratégies d'édition de l’épigénome comme outil en recherche fondamentale. / DNA methylation takes place on cytosines of CpG dinucleotides in mammals and is catalysed by DNMT enzymes. Cancer cells are characterised by frequent promoter hypermethylation leading to transcriptional repression of tumor suppressor genes and favouring tumor progression. Because of its reversible nature, DNA methylation is a target of choice in epigenetic therapies. However, current DNMT inhibitors act in a global and non-specific manner, leading to side effects and toxicity in normal cells. During my thesis I have developed strategies to perform targeted demethylation in specific regions of the genome without affecting global methylation. First, I have validated a strategy inducing the specific and durable epigenetic reprogramming of the tumor suppressor gene SERPINB5 in a breast cancer cell line, which can pave the way to further biomedical research. Second, I have optimised epigenome editing strategies as a regular tool in basic research.

Die Besonderheit der DNA-Nanotechnologie liegt in der Verwendung von DNA als Konstruktionsmaterial für die Herstellung von artifiziellen Strukturen im Nanometermaßstab (Seeman, 1982; Winfree et al., 1998). Diese DNA-Nanoobjekte, wie beispielsweise DNA-Nanoröhren, offerieren innovative und vielversprechende Anwendungsmöglichkeiten in verschiedenen Bereichen wie der Elektronik oder Medizin. Eine Herausforderung für die dauerhafte Verwendung von DNA-Nanoröhren stellt deren Stabilität dar. Einflussfaktoren wie die DNA-Nukleaseaktivitäten, die Ionenstärke und hohe Temperaturen können dabei eine langfristige Anwendung limitieren. Als ein Lösungsansatz für die Erhöhung der Beständigkeit wird eine gezielte Mineralisierung der DNA-Nanoröhren durch spezifische Fusionsproteine angestrebt. Ziel dieser Arbeit ist die dafür notwendige Herstellung und Charakterisierung der DNA-bindenden Fusionsproteine mit Mineralisierungsdomänen vorzustellen. Das umfasst das Auswählen geeigneter DNA-Bindungsproteine als Bestandteil der Fusionsproteine. In dieser Arbeit wurden die DNA-Bindungsproteine MutH und SBB aus Escherichia coli (E. coli) sowie Yku70p aus Saccharomyces cerevisiae (S. cerevisiae) aufgrund ihrer spezifischen Bindungseigenschaften dafür identifiziert. Damit können die Fusionsproteine sequenzspezifische oder -unspezifische Bindungen mit doppelsträngiger (engl. double stranded DNA, dsDNA) oder einzelsträngiger DNA (engl. single stranded DNA, ssDNA) eingehen. Es ist mittels Klonierung gelungen, verschiedene Fusionskonstrukte mit den genannten DNA-Bindungsproteinen zu generieren. Diese beinhalten ebenfalls das enhanced green fluorescent protein sowie den His6-Tag für den Expressionsnachweis und die Proteinreinigung. Weitere Varianten der Fusionskonstrukte bestehen zusätzlich aus der tobacco etch virus-Protease-Erkennungssequenz zur Entfernung des His6-Tags und der Domänen R5-Peptid (R5P) oder Poly-L-Arginin (PLR) für die Mineralisierung. Bestandteil dieser Arbeit sind Western-Blot-Analysen und mikroskopische Aufnahmen, welche die erfolgreiche heterologe Expression aller Fusionskonstrukte nachweisen. Aus den Ergebnissen der Expressions- und Löslichkeitsanalysen lässt sich schlussfolgern, dass insbesondere das Expressionslevel und die Synthese löslicher Proteine mit den Mineralisierungsdomänen eine Herausforderung darstellen. Ebenfalls in dieser Arbeit sind Versuche zur Optimierung mit verschiedenen Expressionsstämmen (E. coli und S. cerevisiae) und Expressionsparametern (Temperatur und Induktor-Konzentration) enthalten. Den Ergebnissen nach eignen sich besonders die Fusionsproteine MutH-EGFP-His6 und SSB-EGFP-His6 für die weiteren Experimente. Die Untersuchungen der DNA-Bindungseigenschaften erfolgten mittels Electrophoretic mobility shift assay (EMSA) und Rasterkraftmikroskopie (engl. atomic force microscopy, AFM). Diese Methoden mussten für jedes Fusionsprotein zuvor etabliert und optimiert werden. Zu Beginn stand das MutH-Fusionsprotein im Focus, wobei die durchgeführten EMSA-Untersuchungen die Spezifität zur GATC-Erkennungssequenz sowie zur dsDNA betrachteten. Die Charakterisierung mittels AFM diente als weitere Möglichkeit zur Analyse der DNA-Bindungseigenschaften. Zusätzlich kam in dieser Arbeit eine Variante des CRISPR/Cas9-Systems als Fusionsprotein für eine sequenzspezifische Adressierung von dsDNA zum Einsatz. Die EMSA- und AFM-Analysen deuteten dabei auf eine Interaktion von dem dCas9-Fusionsproteins und dsDNA hin. Weiterhin war das SSB-Fusionsprotein Bestandteil der Untersuchungen. Die Bindungsanalysen mittels EMSA zeigten, dass es bevorzugt mit ssDNA interagiert und nur eine geringe Affinität zu dsDNA vorliegt. Die Bindung zu ssDNA konnte ebenfalls erfolgreich anhand von AFM-Untersuchungen gezeigt werden. Zusammenfassend bestätigen die Ergebnisse die Funktionalität des MutH- und SSB-Fusionsproteins. Es konnten zudem erste Hinweise erbracht werden, die eine spezifische Bindung der Fusionsproteine an dsDNA oder ssDNA belegen. Mit dieser Arbeit ist es gelungen, Proteinderivate zu generieren und charakterisieren, wodurch eine entscheidende Grundlage für die gezielte Mineralisierung von DNA-Konstrukten geschaffen wurde.

Of the 12 million people who inject drugs worldwide, 13% are chronically infected with Human Immunodeficiency Virus (HIV), i.e., they live with HIV. Chronic opioid use affects the host immune system and increases an individual’s susceptibility to HIV infection. However, it is unclear how opioid use changes the course of HIV pathogenesis. Particularly, there is a gap in understanding how opioids impact HIV latency. Latency results in a reservoir of infected quiescent cells that evade antiviral immune responses, are not targeted by antiretroviral therapy (ART), and allow HIV viremia to rebound upon treatment interruption. While in vitro studies show that opioids modulate the activity of transcription factors involved in T-cell activation and HIV transcription, few studies have investigated whether opioid use impacts HIV latency in vivo in HIV-infected people. In this research, peripheral blood mononuclear cells (PBMCs) were utilized from People with HIV (PWH) with or without recent opioid use or opioid use disorder (OUD) who were enrolled in the Linking Infection and Narcology Care-Part II (LINC-II) and Studying Partial Agonists for Ethanol and Tobacco Elimination in Russians with HIV (St PETER HIV ARCH) studies conducted in St. Petersburg, Russia. Intact proviral DNA digital droplet PCR (ddPCR) assays were performed on PBMCs from antiretroviral treated PWH, with (n=8) or without (n=11) current OUD, to quantify intact and defective proviral genomes. Samples from ART-treated PWH with OUD compared to those without OUD had similar levels of intact and defective proviruses. To evaluate latency reversal, PBMCs from ART-treated PWH with or without OUD, were activated with anti-CD3/28 beads and RT-ddPCR assays were performed to measure HIV LTR-gag RNA. A variable response in PWH without OUD was seen where half of the samples showed an increase in HIV RNA upon activation. Interestingly, only 1 of 8 samples from PWH with OUD showed an increase in HIV transcription. However, no suppression of HIV reactivation was found in vitro from latent cells generated using a primary CD4+ T-cell latency model in the presence or absence of morphine. Similarly, no differences in HIV integration and transcription in vitro were observed between morphine and control conditions. Additionally, expression of opioid receptors was not detected in primary PBMCs, CD4 T cells, or macrophages. These results show that PWH with OUD have a pool of persistent HIV proviruses that are refractive to reactivation, although opioids did not affect HIV replication and latency reactivation in vitro. The discrepancy in these in vitro and in vivo results and the lack of expression of opioid receptors in immune cells suggests that while opioids do not directly impact HIV replication, latency, and reactivation in target CD4+ cells, opioids could indirectly shape the HIV reservoir in vivo by modulating general immune functions, neuroderived factors or other cells that are responsive to opioids. Eradication of the latent HIV reservoir is necessary to achieve a cure for HIV/AIDS. One approach for latency eradication is the “shock and kill” approach that entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. An alternative approach is to permanently repress HIV expression precluding viral rebound after ART discontinuation. Here, a nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB) was used to epigenetically silence the proviral DNA. I show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, which are histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies. The research presented here provides evidence of opioid-mediated modulation of HIV-1 latency reactivation in PWH with opioid dependency. Additionally, we show that HIV-1 reactivation can be suppressed by epigenetic remodeling of the HIV-1 promoter using a repurposed CRISPR/Cas9 system.

A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing. Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology.

The definitive endoderm gives rise to several specialized organs, including the thymus. Improper development of the definite endoderm or its derivatives can lead to human disease; in the case of the thymus, immunodeficiency or autoimmune disorders. Human pluripotent stem cells (hPSCs) have emerged as a system to model human development, as study of their differentiation allows for elucidation of the molecular basis of cell fate decisions, under both healthy and impaired conditions. Here, we first developed a CRISPR-effector system to control endogenous gene expression in hPSCs, a novel approach to manipulating hPSC state. Next, the human-specific, loss-of-function phenotypes of candidate transcription factors driving hPSC-to-definitive endoderm differentiation were analyzed through combined CRISPR-perturbation and single-cell RNA-sequencing. This analysis revealed the importance of TGFβ mediators in human definitive endoderm differentiation as well as identified an unappreciated role for FOXA2 in human foregut development. Finally, as the differentiation of definitive endoderm to thymic epithelial progenitors (TEPs) is of particular interest, a single-cell transcriptomic atlas of murine thymus development was generated in anticipation of identifying factors driving later stages of TEP differentiation. Taken together, this dissertation establishes a CRISPR-effector system to interrogate gene and regulatory element function in hPSC differentiation strategies, details the role of specific transcription factors in human endoderm differentiation, and sets the groundwork for future investigations to characterize hPSC-derived TEPs and the factors driving their differentiation.

No description available.