In every eukaryotic cell, the genomic information coded in the DNA is packed into the small nuclear volume as chromatin, a complex of DNA and proteins. The ensemble of molecular mechanisms that organize chromatin compaction and allow the specific expression of the portions of genome useful for cell’s biological functions is known as the epigenome. As a result of epigenome activity, chromatin is folded and positioned in the nucleus in a cell-specific manner, generating areas of highly compacted, repressed, heterochromatin and areas of decondensed, gene-rich and transcriptionally active, euchromatin. In our work, we describe chromatin organization in different cell populations and analyse some of its implications in the physiological functions and pathological dysfunctions of the cell. In the first project, we focus on murine muscle stem cells lacking the nuclear structural protein Lamin A/C. We show their irregular differentiation program, due to a spreading of Polycomb group (PcG) of proteins repressors from their target genes over the flanking regions. The consequent alteration in gene expression cause premature exhaustion of quiescent stem cells and accumulation of intramuscular fat, resulting in accelerated senescence and muscular dystrophy progression. On the other hand, the progressive accumulation of a Lamin A aberrant form, Progerin, in Hutchinson-Gilford progeria syndrome (HGPS) also leads to chromatin structure disruption. In particular, it interferes with Lamina Associated Domains (LADs), the peripheral heterochromatin structures associated to the nuclear lamina. For our second project, we develop a new method, SAMMY-seq, based on high-throughput sequencing of chromatin fractions of different solubility. Thanks to this technology, we highlight early changes in heterochromatin accessibility in human HGPS primary fibroblasts. This early structural changes do not alter the deposition of the H3K9me3 heterochromatin mark but are associated with site-specific variations in the PcG-dependent transcriptional regulation. Finally, further improving SAMMY-seq technology, in our third project we describe an unconventional genome organization in resting human CD4+ T lymphocytes extracted from the peripheral blood of healthy donors. In these cells, heterochromatin is sensitive to DNAse digestion while euchromatin is resistant to serial processes of extraction. Preliminary analysis of the content of these compartments suggests that euchromatin contains, beside the actively transcribed genes, also inactive genes specific for lymphocyte activation. Further studies will elucidate the role of this unconventional chromatin organization in lymphocytes functions.

Nei nuclei delle cellule eucarioti, l'informazione genetica codificata nel DNA è concentrata nel microscopico volume nucleare in forma di cromatina, un complesso di DNA e proteine. I meccanismi molecolari che gestiscono la compattazione e il ripiegamento della cromatina e che consentono l'espressione mirata delle porzioni di genoma necessarie alle attività della cellula sono noti come ‘epigenoma’. L’azione dell’epigenoma determina un avvolgimento e un posizionamento nucleare della cromatina specifico per ogni tipo cellulare, con aree dense e trascrizionalmente inattive (eterocromatina) ed aree meno dense, ricche di geni e trascrizionalmente attive (eucromatina). In questo nostro lavoro descriviamo l'organizzazione della cromatina nel nucleo di diverse popolazioni cellulari e ne analizziamo alcuni aspetti fisiologici e patologici. Innanzitutto, studiando le cellule staminali muscolari di topi privi della proteina strutturale nucleare lamina-A/C, descriviamo un irregolare processo di differenziamento dovuto alla redistribuzione dei repressori trascrizionali del gruppo Polycomb (PcG proteins), che dai i loro geni target si diffondono verso regioni cromatiniche fiancheggianti. La conseguente alterazione nell’espressione genica causa l’esaurimento prematuro della riserva di cellule staminali quiescenti e l’accumulo di grasso intramuscolare, portando a una senescenza accelerata e alla distrofia muscolare. D’altro canto, anche il progressivo accumulo di una forma aberrante di lamina-A, la progerina, caratteristica della sindrome di Hutchinson-Gilford (HGPS), causa gravi alterazioni nella struttura della cromatina. In particolare, la progerina interferisce con le strutture eterocromatiniche periferiche associate alla lamina nucleare, i Lamina Associated Domains (LADs). Per il nostro secondo progetto abbiamo sviluppato un nuovo metodo, SAMMY-seq, basato sull’high-throughput sequencing di frazioni di cromatina con diversa solubilità. Tramite questa tecnologia, individuiamo alterazioni nella solubilità dell’eterocromatina in fibroblasti primari derivanti da pazienti progerici in uno stadio precoce di malattia. I cambiamenti strutturali osservati a questo stadio non alterano la deposizione del marcatore eterocromatinico H3K9me3, ma sono associati a variazioni sito-specifiche nella regolazione trascrizionale di geni target delle PcG proteins. Infine, ottimizzando ulteriormente il protocollo di SAMMY-seq, nel nostro terzo progetto mostriamo un’organizzazione non convenzionale della cromatina nei linfociti T CD4+ quiescenti derivanti da sangue periferico di donatori. In queste cellule, l’eterocromatina risulta sensibile alla digestione enzimatica operata dalla DNAsi, mentre l’eucromatina si rivela resistente a diversi processi di estrazione. Un’analisi preliminare del contenuto di questi compartimenti indica la presenza, nell’eucromatina, dei geni specifici per l’attivazione linfocitaria, oltre che dei geni attivi. Ulteriori studi chiariranno il ruolo di questa organizzazione non convenzionale della cromatina nella funzione cellulare linfocitaria.

(2020). Unconventional nuclear architecture in CD4+ T lymphocytes uncouples chromatin solubility from function. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).

Unconventional nuclear architecture in CD4+ T lymphocytes uncouples chromatin solubility from function

LUCINI, FEDERICA
2020

Abstract

In every eukaryotic cell, the genomic information coded in the DNA is packed into the small nuclear volume as chromatin, a complex of DNA and proteins. The ensemble of molecular mechanisms that organize chromatin compaction and allow the specific expression of the portions of genome useful for cell’s biological functions is known as the epigenome. As a result of epigenome activity, chromatin is folded and positioned in the nucleus in a cell-specific manner, generating areas of highly compacted, repressed, heterochromatin and areas of decondensed, gene-rich and transcriptionally active, euchromatin. In our work, we describe chromatin organization in different cell populations and analyse some of its implications in the physiological functions and pathological dysfunctions of the cell. In the first project, we focus on murine muscle stem cells lacking the nuclear structural protein Lamin A/C. We show their irregular differentiation program, due to a spreading of Polycomb group (PcG) of proteins repressors from their target genes over the flanking regions. The consequent alteration in gene expression cause premature exhaustion of quiescent stem cells and accumulation of intramuscular fat, resulting in accelerated senescence and muscular dystrophy progression. On the other hand, the progressive accumulation of a Lamin A aberrant form, Progerin, in Hutchinson-Gilford progeria syndrome (HGPS) also leads to chromatin structure disruption. In particular, it interferes with Lamina Associated Domains (LADs), the peripheral heterochromatin structures associated to the nuclear lamina. For our second project, we develop a new method, SAMMY-seq, based on high-throughput sequencing of chromatin fractions of different solubility. Thanks to this technology, we highlight early changes in heterochromatin accessibility in human HGPS primary fibroblasts. This early structural changes do not alter the deposition of the H3K9me3 heterochromatin mark but are associated with site-specific variations in the PcG-dependent transcriptional regulation. Finally, further improving SAMMY-seq technology, in our third project we describe an unconventional genome organization in resting human CD4+ T lymphocytes extracted from the peripheral blood of healthy donors. In these cells, heterochromatin is sensitive to DNAse digestion while euchromatin is resistant to serial processes of extraction. Preliminary analysis of the content of these compartments suggests that euchromatin contains, beside the actively transcribed genes, also inactive genes specific for lymphocyte activation. Further studies will elucidate the role of this unconventional chromatin organization in lymphocytes functions.
LANZUOLO, CHIARA
Genome conformation; T lymphocyte; Cell identity; Nuclear architecture; Chromatin sequencing
Genome conformation; T lymphocyte; Cell identity; Nuclear architecture; Chromatin sequencing
BIO/11 - BIOLOGIA MOLECOLARE
English
11-feb-2020
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
32
2018/2019
open
(2020). Unconventional nuclear architecture in CD4+ T lymphocytes uncouples chromatin solubility from function. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/262913
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