Maintenance of the hematopoietic system is dependent upon the proper regulation and orchestrated functions of the hematopoietic stem cell (HSC) pool. A number of extrinsic signaling pathways and intrinsic regulators have been found to regulate HSC processes. However a full understanding of the ability of HSC to balance the processes of self-renewal, quiescence, and lineage specification is not yet clear. We therefore set out to identify novel HSC regulators by comparative gene expression analysis of whole genome transcriptomes generated for long-term (LT)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34-), short-term (ST)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34+), and non-HSC (Hoechst+) of the bone marrow. These studies identified IGF2 as one of the most differentially expressed genes within LT-HSC, suggesting a potential role for IGF2 in the regulation of HSC. Using a combination of lentiviral-mediated overexpression and knockdown experiments, we found IGF2 to confer enhanced self-renewal in vitro and in vivo. Overexpression of IGF2 resulted in an increased percentage of multi-lineage colonies within colony-forming unit (CFU) assays without affecting lineage specification. In vivo, serial bone marrow transplantation revealed that IGF2 within HSC enhances short-term and long-term donor contribution. Analysis of the expression of key cell cycle regulators revealed that IGF2 induced upregulation of p57 expression specifically within HSC. This upregulation could be attributed to differences in the methylation status of the p57 promoter in HSC compared to other progenitor and mature blood cell populations. p57, a member of the Cip/Kip family of cyclin dependent kinase inhibitors, has recently been shown to be required for the regulation of HSC quiescence and long-term self-renewal. Analysis of bone marrow obtained from primary and secondary transplant recipients showed that overexpression of IGF2 resulted in an increased percentage of quiescent HSC. Treatment of HSC overexpressing IGF2 with LY294002, a PI3K-Akt inhibitor, prevented IGF2-mediated upregulation of p57 expression. These findings demonstrate that within HSC, IGF2 induces p57 expression through activation of the PI3K-Akt pathway to regulate HSC quiescence. We have identified a novel role for IGF2 in HSC function, providing new insights into the biology of HSC and opening potential platforms for the development of better therapies involving HSC-mediated hematopoietic reconstitution.

Chez les mammifères, l'ADN des cellules interphasiques s'organise en une fibre chromatinienne confinée à l'intérieur de « territoires chromosomiques ». Ce confinement autorise l'établissement d'interactions à longue distance permettant une régulation fine des fonctions génomiques. Toutefois, l'organisation et la dynamique de la chromatine à l'échelle dite supranucléosomale (10 à 500 kb) reste méconnue. Afin d'étudier la chromatine à cette échelle, nous avons utilisé la méthode dite de 3C-qPCR qui permet de mesurer les fréquences d'interactions entre deux portions génomiques. Dans un premier temps, nous avons analysé les collisions aléatoires afin de déterminer l'organisation intrinsèque de la chromatine à l'échelle supranucléosomale. Nos résultats indiquent que, en l'absence d'interactions spécifiques, les collisions aléatoires dans les régions riches en gènes présentent une modulation d'une périodicité d'environ 90kb. Cette modulation semble être sous-jacente à de nombreuses interactions spécifiques et avoir des répercutions sur leur positionnement génomique contribuant ainsi à l'évolution des génomes. Des modèles, dérivés de la physique des polymères, suggèrent que la chromatine s'organise dans ces régions en une hélice statistique. Dans un second temps, nous avons abordé l'organisation tridimensionnelle du locus murin Igf2/H19 soumis à l'empreinte génomique parentale. Les interactions spécifiques identifiées entre des « enhancers » endodermiques et certaines portions du locus ont confirmé l'existence d'une hiérarchie des interactions et ont permis la découverte d'un nouveau locus soumis à l'empreinte (PIHit). Ce locus produit un ARN non codant que nous avons caractérisé mais dont la fonction exacte reste à déterminer.Finalement, mes travaux de thèse ont aussi conduit à la mise au point d'une nouvelle technologie (HRS-SEQ) qui permettra d'aborder l'organisation génomique globale par le biais des séquences récupérées à haut-sel (HRS). / In mammal, the DNA of interphasic cells is organized into the chromatin fiber which is itself confined inside “chromosome territories”. This compact organization allows the establishment of long-range interactions involved in the fine regulation of genomic processes. However, the organization and the dynamic of the chromatin at the so-called supranucleosomal scale (10 to 500kb) remain unclear. In order to study the chromatin at this scale, we used the 3C-qPCR method that allows to measure interaction frequencies between two genomic regions. Firstly, we have analyzed random collisions in order to determine the intrinsic organization of the chromatin at the supranucleosomal scale. Our data indicates that, in the absence of specific interactions, random collisions in gene-rich regions show a periodic modulation of about 90kb. This modulation seems to be underlying numerous locus-specific interactions and have repercussions on their genomic location, thus contributing to genome evolution. Models, derived from polymers physics, suggest that, in these regions, the chromatin is shaped in a statistical helix. Secondly, we have investigated the tridimensional organization of the Igf2/H19 mouse locus which is subject to genomic imprinting. Specific interactions identified between endodermic enhancers and some regions of the locus have confirmed the existence of a hierarchy of interactions and allowed the discovery of a new imprinted locus (PIHit). This locus produces a non-coding RNA that we have characterized but for which the function remains to be determined.Finally, my work also led to the development of a new technology (HRS-SEQ) that allows to study global genome organization through mapping of high-salt recovered sequences (HRS).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20077号 / 医博第4170号 / 新制||医||1018(附属図書館) / 33193 / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 斎藤 通紀, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The growth of vertebrates is primarily regulated by the growth hormone-insulin-like growth factor (GH-IGF) system, but not in isolation. The central question of this dissertation was how do other hormones peripheral to the GH-IGF system interact with the system, including feedbacks by GH and IGF themselves on various tissues in rainbow trout (Oncorhynchus mykiss)? The representative hormones selected were thyroxine, cortisol, and the sex steroids testosterone and estrogen, along with GH and IGF. These hormones were chosen because they are known to affect overall growth and development during specific life events, but exactly what target genes and what mechanisms are involved are only at the early stages of being delineated in fish. Liver and gill tissues were selected as representative tissues to assess the in vitro effects on growth-related genes of the GH-IGF system. A total of more than thirty experiments were conducted, including time- and concentration-response, inhibitory studies, hormone combination studies, and radio-receptor binding assays. Hormones were applied to whole tissue cultures and real-time quantitative-PCR was used to measure hormonal effects on GHR, IGF, and IGFR1 genes. Microsomal preparations were treated with selected hormones and radio-labeled GH or IGF. A gamma counter was used to measure receptor-ligand activity. GH and IGF were found to possess autocrine and/or paracrine actions in self-regulating target growth genes. Thyroxine had no direct effects on targeted growth genes but may interact with other molecules or hormones to elicit its effects on growth and development. Cortisol directly influenced target growth genes in a tissue-specific and isoform-specific manner. Finally, sex steroids differentially regulated the growth genes: estradiol inhibited growth genes while testosterone directly stimulated growth genes. These findings contribute to understanding how hormones peripheral to the GH-IGF system interact with the growth system. / National Science Foundation grant IOS 0920116 to Dr. Mark Sheridan