Heterochromatin dynamics during epithelial-to-mesenchymal transition

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Universitat Pompeu Fabra Tesi doctoral 2014

Heterochromatin dynamics in

Epithelial-to-Mesenchymal Transition

Dissertation presented by Alba Millanes Romero for the degree of Doctor of Philosophy

Work carried out under the supervision of Drs. Sandra Peiró Sales and Antonio García de Herreros Madueño in the Epithelial-to-Mesenchymal Transition and Tumor Progression Group in the Cancer Research Program in the Institut Hospital del Mar d’Investigacions Mèdiques (IMIM)

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“La humilitat més sincera per a un científic és acceptar

que res no és impossible”

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vii

ABBREVIATIONS xv

ABSTRACT xix

RESUM xx

INTRODUCTION 1

1. Chromatin structure and organization 3

2. Genome organization and transcription 6

2.1. Chromatin dynamics 6

2.2. Histone modifications and DNA methylation 9

3. Euchromatin vs. heterochromatin 14

3.1. Heterochromatin protein 1 (HP1) 15

3.2. Pericentromeric heterochromatin 19

3.2.1. Pericentromeric heterochromatin formation 23 3.2.2. Pericentromeric heterochromatin transcription 25

4. Cancer and tumor progression 29

4.1. Epithelial-to-Mesenchymal Transition (EMT) 30

4.1.1. Physiological EMT 32

4.1.2. Pathological EMT 34

4.1.3. Signaling EMT 37

4.1.4. EMT inducers 38

4.1.4.1. Snail1 transcription factor 38

4.1.4.2. Lysyl oxidase-like 2 (LOXL2) 41

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viii

RESULTS 49

1. Snail1 is essential for pericentromeric heterochromatin

maintenance and organization in mesenchymal cells 51 2. Snail1 regulates pericentromeric transcription 57 3. Snail1 is enriched in pericentromeric regions and interacts with

HP1α 59

4. Pericentromeric transcription is linked to H3 oxidation 63 5. Pericentromeric transcription is tightly regulated during EMT 66 6. Pericentromeric transcription regulation is essential for a complete

EMT 74

DISCUSSION 81

Snail1 and heterochromatin organization 83

Snail1 and heterochromatin transcription 85

Major satellite regulation during EMT 91

HP1 dynamics during EMT 95

Snail1 pericentromeric regulation in other cell contexts 97

Chromatin dynamics and cancer 100

CONCLUDING REMARKS 103

MATERIALS AND METHODS 107

Cell Lines 109

Transfection procedures 110

Retroviral and lentiviral Infection 111

Cloning procedures 112

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ix

Karyotypes 114

Salt Extraction Experiments 115

Subcellular Fractionation 115

Chromatin Immunoprecipitation (ChIP) experiments 116

Western Blot 118

Co-immunoprecipitation assays 118

Genomic DNA extraction 119

RNA extraction procedures 119

Real-time RT-PCR 120

Microarray gene expression analysis 121

Migration and Invasion assays 122

REFERENCES 123

ANNEX 147

ACKNOWLEDGEMENTS 151

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xi INTRODUCTION

Figure I1. Atomic structure of the nucleosome. ... 3

Figure I2. Chromatin organization in the mammalian nucleus ... 4

Figure I3. Conserved role of insulators in nuclear organization ... 8

Figure I4. Summary of histone modifications and histone modifying enzymes... 10

Figure I5. Map of main PTMs in histone tails ... 11

Figure I6. Models for the functional outcomes of reading modified histones ... 12

Figure I7. Properties of euchromatic and heterochromatic regions ... 15

Figure I8. Mouse HP1 domains and interaction partners ... 16

Figure I9. Examples of HP1 interacting partners ... 18

Figure I10. Structural and functional elements of the centromere region ... 20

Figure I11. Comparative organization of centromeric (CT) and pericentromeric (PCT) regions in fission yeast, mouse and human chromosomes ... 21

Figure I12. Chromocenters in mouse cells ... 22

Figure I13. Mechanisms for the initiation of heterochromatin assembly 24 Figure I14. Epithelial and mesenchymal cell features ... 32

Figure I15. Examples of primary EMT ... 33

Figure I16. Acquisition of the metastatic phenotype ... 35

Figure I17. Potential roles of EMT and MET in carcinoma progression ... 36

Figure I18. Overview of the molecular networks that regulate EMT ... 37

Figure I19. Structural domains of Snail1 transcription factor ... 39

Figure I20. Model of Snail1 mediated transcriptional repression ... 40

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xii

RESTULTS

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xiii Figure R19. Major satellite overexpression blocks HP1α release from chromatin during EMT ... 75 Figure R20. Genes differently regulated by TGFβ in NMuMG-Major cells are mainly associated to cancer and EMT related pathways ... 76 Figure R21. Genes involved in EMT are differently regulated by TGFβ in NMuMG-Major cells. ... 77 Figure R22. Major satellite cells have decreased migration and invasion properties ... 78 Figure R23. HP1α and major satellite transcripts knock-down does not affect mesenchymal genes induction after TGFβ treatment ... 79

DISCUSSION

Figure D1. Snail1 ChIP-Seq predicted binding sites analysis ... 90

CONCLUDING REMARKS

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xv 3C: chromosome conformation capture

ATRx: alpha thalassemia/mental retardation syndrome BER: base excision repair

BRG1: SWI/SNF related transcriptional activator BSA: bovine serum albumin

CAF-1p150: chromatin assembly factor-1 p150 subunit CBX: chromobox

CD: chromodomain CDH1: E-cadherin gene

ChIP: chromatin immunoprecipitation ChIP-Seq: ChIP sequencing

CLDN: claudin gene

Clr4: cryptic loci regulator 4 CSC: cancer stem cell

CSD: chromoshadow domain CT: control

CTCF: CCCTC-binding factor

DAPI: 4',6-diamidino-2-phenylindole DNA: deoxyribonucleic acid

Dnmt3a/Dnmt3b: DNA methyltransferase 3a and 3b DSP: desmoplakin gene

ECM: extracellular matrix EGF: epithelial growth factor

EMT: Epithelial-to-Mesenchymal Transition ETaR: endothelin-A receptor

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FAK: focal adhesion kinase FBS: fetal bovine serum FGF: fibroblast growth factor GFP: green fluorescent protein GSK3β: glycogen-synthase kinase-3β HAT: histone acetyltransferase HDAC: histone deacetylase HDM: histone demethylase HGF: hepatocyte growth factor HIF: hypoxia-inducible factor HMT: histone methyltransferase HP1: heterochromatin protein 1 HSF1: heat-shock factor 1 IAP1: intracisternal A particle 1 IgG: immunoglobulin

iMEF: immortalized mouse embryonic fibroblast INCENP: inner centromere protein

Kap-1/Tif1β: Kruppel-associated box (KRAB)-associated protein /transcriptional intermediary factor 1β

Ki-67: cell proliferation antigen of monoclonal antibody Ki-67 KO: knock out

Ku70: 70K autoantigen LAD: lamin-associated domain

LAP2: lamina-associated polypeptide 2

LEF-1/TCF4: lymphoid-enhancer-binding factor/T-cell factor-4 LINE: long interspersed nuclear domain

lncRNA: long non-coding RNA

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xvii MET: mesenchymal-to-epithelial transition

MITR: myocyte enhancer factor2 (MEF2) interacting transcription repressor

MMP: matrix metalloproteinase MSC: mesenchymal stem cell NAD: nucleolus-associated domain ncRNA: non-coding RNA

NER: nucleotide excision repair NES: nuclear export signal NF-κB: nuclear factor-κB NL: nuclear lamina OCLN: occluding gene

ORC1-6: origin recognition complex 1-6 PAR6: partitioning-defective protein-6 Pc: polycomb

PCNA: proliferating cell nuclear antigen PCR1/2: polycomb repressive complex 1/2 PI3K: phosphatidyl-inositol 3-kinase

PIM1: proviral integration site 1 (pim-1) oncogene PKB: protein kinase-B

pMEF: primary mouse embryonic fibroblast Psc3: cohesion subunit Psc3

PTM: posttranslational modification RA: retinoic acid

Rb: retinoblastoma protein

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RNAi: interfering RNA ROS: reactive oxygen species satIII: human satellite III repeat SD: standard deviation

SINE: short interspersed nuclear domain siRNA: small interfering RNA

SP100: nuclear autoantigenSpeckled 100 kD SRCR: scavenger receptor cysteine rich

Suv39h: suppressor of variegation 3-9 homolog Suz12: suppressor of zeste 12

Swi6: switching gene 6. Refers to the S. pombe HP1 ortholog TAD: topological associated domain

TAFII130: TATA-binding protein associated factor p130 TAK1: TGFβ- activated kinase-1

TF: transcription factor

TFGβ: transforming growth factor β TGFβR: TGFβ receptor

tonEBP: tonicity enhancer-binding protein Wnt: wingless-related integration site WntR: Wnt receptor

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RESUM

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1.

Chromatin structure and organization

In eukaryotic cells, DNA exists in the nucleus forming the chromatin, a complex of DNA and a particular group of proteins called histones. The basic repeating structural and functional unit of chromatin is the nucleosome, which consists in 147 base pairs (bp) of DNA wrapped around the nucleosome core particle that contains one (H3)2-(H4)2

tetramer and two H2A-H2B histone dimers 1 (Figure I1). The linker histone H1 binds to the core particles and protects an additional 20 bp of DNA.

Figure I1. Atomic structure of the nucleosome. Each strand of DNA is shown in different colors. The DNA makes 1.7 turns around the histone octamer. Nucleosome core particle is composed by a tetramer of H3 (green) and H4 (yellow), and two dimers of H2A (red) and H2B (pink)2.

Nucleosomes are joined one to each other by the DNA that runs between them, which is known as “linker” DNA. Its length ranges between 20 and 90 bp and varies among different species and tissues3; variation in the length of linker DNA is important for the diversity of gene regulation4.

The DNA-nucleosome complex forms a 10 nm diameter fiber resembling “beads on a string”5(Figure I2-e). Classically, it was proposed that the 10

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(Figure I2-d). However, although it has been extensively studied in vitro, evidence for the existence of the 30 nm fiber in vivo is limited. In recent years, an increasing amount of data suggests that chromatin organization above the 10 nm fiber probably does not exist in most mammalian cells and can only be found in very particular biological contexts, where heterochromatic transcriptional repression and compaction prevail6. Alternatively, the “polymer melt” model has been proposed to explain chromatin organization inside the nucleus of mammary cells as an alternative to the 30 nm fiber, and every day more evidences seem to support it. In this model, chromatin is described as a dynamic disordered state comparable with a “polymer melt”, where nucleosomes that are not linear neighbors on the DNA strand interact within a chromatin region7 (Figure I2-d) and organize into a series of small globules to form a

highly compact state termed “fractal globule”8(Figure I2-c).

Figure I2. Chromatin organization in the mammalian nucleus.(a) Chromosomes are organized in chromosome territories. (b) Chromosomes territories are comprised of fractal globules, and fractal globules from adjacent chromosome territories can interdigitate. (c) Chromatin fibers interact (i) within a fractal globule (frequent), (ii) between fractal globules of the same chromosome territory (rare), or (iii) between adjacent chromosome territories (very rare). (d)

Chromatin may form a 30 nm fiber with a solenoid or zigzag structure, or it may present “polymer melt” organization. (e) Chromatin is resolved as a 10 nm “beads on a string” fiber consisting of nucleosomes 6.

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5 IN T R O D U C T

genome is intimately linked to its biological function has been for many years poorly understood.

Historically, chromatin was divided into two domains: euchromatin and heterochromatin, on the basis of differential compaction at interphase. Heterochromatin was initially defined as the portion of the genome that retains deep staining with DNA-specific dyes as the dividing cell returns to interphase from metaphase. On the other hand, euchromatin was defined as the actively transcribed chromatin9. However, apart from being subdivided into functional domains, microscopic studies and other type of experiments started to suggest the idea that, in the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories10(Figure I2-a).

Recent advances in genomic technologies, such as the development of the Chromosome Conformation Capture (3C)11 and 3C-related

genome-wide techniques, and even more recently the development of Hi-C technique12, have let to rapid advances in the study of three-dimensional genome organization in vivo, based on the information they give about

cis and trans chromatin interactions. As a result, the idea that chromosomes organize in chromosome territories has been recently further reinforced by the tendency of distant loci on the same chromosome to be near one another in space, as has been shown by these long-range genome-wide chromatin interaction techniques8. This can be easily understood if each chromosome, rather than being intertwined, occupies its own distinct region in the nucleus6.

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conserved across species, indicating that they are an inherent property of mammalian genomes13.

However, other types of domains have been lately described, and not all of them seem to be conserved and maintained in different cell types. Chromatin interactions within these domains have been shown to change depending on the cell context, and seem to be associated to specific transcriptional programs14,15.

2.

Genome organization and transcription

Cell type specific transcriptional regulation is crucial in order to maintain cell identity throughout the lifetime of an organism, but it must be flexible enough to allow for responses to endogenous and exogenous stimuli. This regulation is mediated not only by molecular factors such as transcription factors and histone and DNA modifications, but also at the level of chromatin and genome organization.

2.1. Chromatin dynamics

Chromatin dynamics and nuclear organization are recently arising as novel transcriptional key regulators, since gene movement and localization seem to be tightly linked with transcriptional activity.

Thus, although TADs are conserved in different cell types and even between species, it has also been proposed that specific regions within TADs may be dynamic, potentially taking part in cell-type specific regulatory events13. Like this, although TADs as a whole do not change,

the internal TAD contacts rearrange upon ES cell differentiation, affecting differentially regulated genes, supporting the link between chromatin structure and transcription16.

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associated to active or silent transcription. Local and long-range contacts, together with clustering of specific subsets of proteins are responsible for the formation of these sub-domains.

Polycomb (Pc) group proteins and H3K27me3 form particular domains, known as “Polycomb bodies” and are responsible for gene clusters silencing during development. However, they are dynamic structures that, depending on cell type, can be partially or entirely reexpressed15,17.

Furthermore, large organized chromatin K9-modifications (LOCKs) are enriched in H3K9me2 and are highly conserved between human and mouse, but are cell type and tissue specific. Interestingly, genes differently located within or outside LOCKS in different tissues were differently expressed, being largely silenced the ones within LOCKs and showing a broad range of expression the ones outside them18.

Lamin-associated domains (LADs) are formed by genomic regions that interact with the nuclear lamina (NL) and are characterized by low gene density and transcriptional repression19. NL interaction pattern has been shown to be in part cell type specific20, contributing again to the idea that dynamic genomic subdomains reorganize based on cell specific transcriptional requirements. Similar to LADs, nucleolus-associated domains (NADs) preferentially contain repressed genes and show enrichment for repressive histone marks21,22.

Interestingly, not only repressed domains have been described in eukaryotic nuclei, but also transcription has been shown to be spatially organized into discernible nuclear structures termed “transcriptional factories”23, but how genes are dynamically targeted to them remains

poorly understood24,25.

Understanding how all these domains are formed and how do they change in particular cell contexts is still something under study. Insulators, which are multi-protein DNA complexes that mediate long-range physical interactions in the nucleus seem to be key elements in the partition of chromatin into structural and functional domains25 (Figure

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Figure I3. Conserved role of insulators in nuclear organization. Insulators in yeast (orange), Drosophila (green and yellow), and mammals (brown and orange) mediate long-range inter- and intrachromosomal interactions important for gene regulation; they are associated to Polycomb (Pc) body repression (blue), to transcription factories (transcription factors in pink) and to lamina-associated domains (LADs)25.

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2.2. Histone modifications and DNA methylation

Although all cells have the same genome, cell identity is established by the expression of a particular transcriptome, which means that each cell type expresses and represses particular subsets of genes.

In a specific cell context, genes can be either actively transcribed or silenced, which has been associated with different states of chromatin compaction. In this way, “open” or decondensed chromatin is usually associated to active transcription, whereas “closed” or condensed chromatin is associated to silenced or repressed transcription.

In the last decades, epigenetics have arisen as one of the main regulators of chromatin transcription. By definition, epigenetics enclose those processes that ensure the inheritance of variation (“-genetic”) above and beyond (“epi-”) changes in the DNA sequence. In other words, the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence26. Two of the main epigenetic mechanisms in mammals are histone modifications and DNA methylation, which influence each other during development and during other cell processes.

The ability of chromatin to condense can be regulated in part by posttranslational modification (PTM) of the N-terminal tails of the histones, which overhang from the DNA and become exposed to chromatin modifying enzymes27. These enzymes are normally highly

specific and catalyze specific residue modifications at particular amino acid positions in histone tails (Figure I4).

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Figure I4. Summary of histone modifications and histone modifying enzymes. (a) Histone acetylation at lysines, generally an active mark, is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetlyases (HDACs). (b) Histone methylation at lysine and arginine residues is catalyzed by histone methyltransferases (HMTs) and removed by histone demethylases (HDMs). Histone methylation has been linked to both transcriptional activation and transcriptional repression. Methylation can occur in mono-, di-, or even tri-methylated states. (c) Histone phosphorylation at serine residues, generally linked to transcriptional activation, is catalyzed by protein kinases, whereas phosphorylation marks are removed by protein phosphatases29.

Methylation, acetylation and phosphorylation are the modifications that have been more extensively described and characterized (Figure I5). However, histones can suffer many other modifications including ubiquitylation, proline isomerization, propionylation, butyrylation, formylation, sumoylation, citrullination, hidroxilation, ADP ribosylation and crotonylation28. Moreover, deamination of lysine has been recently

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Figure I5. Map of main PTMs in histone tails. Acetylation (blue), methylation (red), phosphorylation (yellow) and ubiquitylation (green). The number in grey under each amino acid represents its position in the sequence31.

As previously mentioned, histone modifications are associated with both gene silencing and activation, depending on the nature of the modification and the specific amino acid modified. In this way, two major mechanisms are thought to contribute to the regulation of chromatin transcription by histone modifications32. On one hand, histone PTMs may

have a structural role based on the fact that resultant charge density in histone tails may impact on their interactions with DNA. Thus, acetylated histone tails would be expected to propagate a more open chromatin state. Moreover, PTMs may alter inter-nucleosomal interactions, thereby regulating chromatin structure and the access of DNA-binding proteins such as transcription factors33.

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modified histones via specialized structural folds such as bromodomains and chromodomains among others, which bind acetylated and methylated lysine residues, respectively35,36. Alternatively, histone PTMs can also function by inhibiting the interaction of specific binders with chromatin. The functional readout of a particular PTM will depend on the specific readers and effectors that are recruited there, and its particular activity. Based on their functional outcome, readers are classified in four groups34 (Figure I6).

Figure I6. Models for the functional outcomes of reading modified histones.

PTM readers are classified in four groups: architectural proteins, chromatin remodelers, chromatin modifiers and adaptors34.

Histone modifications induce changes in protein interactions between chromatin and its binding partners. These changes contribute to the establishment of a particular chromatin environment that correlates with different transcriptional states, which in turn translate into different biological outcomes.

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in the cytosine base and the majority of these modifications are present exclusively at CpG dinucleotides37. DNA methylation associates with

stable long-term repression in comparison to histone tail methylation, which is responsible for reversible local formation of heterochromatin38. The DNA methylation pattern is erased in the early embryo and then re-established in each individual at approximately the time of implantation39,40.

De novo DNA methylation is carried out by the DNA methyltransferase enzymes DNMT3A and DNMT3B41. On the other hand, DNMT1 is considered the maintenance DNMT and is required to methylate hemimethylated sites that are generated during semiconservative DNA replication37. In contrast, DNA demethylation can be achieved either passively, by simply not methylating the new DNA strand after replication, or actively, by replication-independent processes associated to base excision repair (BER) and nucleotide excision repair (NER) pathways42.

DNA methylation does not seem to be as dynamic as histone modifications. Therefore, the possible combinations between DNA methylation and different types of repressive histone modifications will establish several repressed chromatin states, which will be more or less easily reversible depending on their epigenetic component.

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3.

Euchromatin vs. heterochromatin

Euchromatin and heterochromatin have different patterns of histone modifications, are associated with different modes of nucleosome packaging and therefore, have differences in higher-order packaging and nuclear organization.

Euchromatin is less condensed, more accessible and generally more easily transcribed. It is mainly located at chromosome arms, is enriched in gene sequences, replicates in early S-phase and suffers recombination during meiosis (Figure I7).

On the other hand, heterochromatin is typically highly condensed and more inaccessible43 and it can be subdivided into constitutive and facultative heterochromatin. In higher eukaryotes, only regions important for genome integrity such as telomeres and centromeres, as well as repetitive and noncoding sequences are kept stably heterochromatinized and referred to as constitutive heterochromatin, which replicates in late S-phase and is protected against meiotic recombination (Figure I7).

In contrast, facultative heterochromatin can be molecularly defined as condensed transcriptionally silent chromatin regions that decondense and allow transcription depending on developmental states, specific cell-cycle stages or nuclear localization changes from the center to the periphery or vice versa. This means that facultative heterochromatin is transcriptionally silent but retains the potential to interconvert between heterochromatin and euchromatin44.

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Figure I7. Properties of euchromatic and heterochromatic regions. Cluster of general important properties of euchromatin and heterochromatin are specified, though there are exceptions in every instance. Not all centromeric and telomeric domains exhibit these characteristics, but they are especially consistently observed in pericentromeric heterochromatin, found in the regions that flank the centromeres of many eukaryotic chromosomes. Adapted9.

3.1. Heterochromatin protein 1 (HP1)

HP1 is a highly conserved protein, which has homologues in various organisms, ranging from S. pombe (Swi6) to mammals, in which three HP1 isoforms, HP1α, HP1β and HP1γ have been identified45. The HP1 family of proteins is encoded by a class of genes known as the chromobox (CBX) genes: HP1α is encoded by the Chromobox homolog 5 (CBX5), HP1β by CBX1 and HP1γ by CBX346.

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These proteins are small (around 25 kDa) and contain a conserved N-terminal region that is known as the chromodomain (chromatin-organization modifier), followed by a variable hinge region and a conserved C-terminal chromoshadow domain (Figure I8-b).

All proteins containing chromodomains (CD) can characteristically alter the structure of chromatin. In particular, the CD of HP1 binds specifically to methylated lysine 9 of histone H3 (H3k9me3)48,49. The hydrophobic

pocket of the CD provides the appropriate environment for docking onto this methylated residue50,51.

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The linker or hinge region is the least conserved domain of HP1 proteins, which may explain why distinct HP1 isoforms are targeted to different locations. This hinge region is responsible of HP1α RNA-binding properties52, but it has no obvious homology to known RNA-binding proteins. In addition, the hinge region of HP1 can also bind DNA and chromatin without obvious sequence specificity53,54.

Finally, the chromoshadow domain (CSD) is involved in homo- and/or heterodimerization and interaction with other proteins. The overall structure of the CSD domain is very similar to that of the CD. However, it has a particular domain that is involved in dimerization, with conserved residues that are unique to the CSD and localize at the dimer interface. As a result, this dimer structure creates a nonpolar groove that can accommodate HP1-interacting proteins containing the consensus sequence PXVXL55. Like this, almost all HP1 partners described interact

through the CSD.

So far, many proteins have been shown to interact with HP1 family of proteins (Figure I9). Some of them, like Suv39h1, Dnmt3a and Dnmt3b are clearly involved in the most common of HP1 functions that is the formation of heterochromatin. Suv39h1 is the histone methyltransferase (HMT) that catalyzes H3K9 trimethylation56. Interestingly, Suv39h1 is able

to interact with HP1 through its CSD domain57,58. In this way, it is proposed that, once Suv39h1 methylates H3K9, HP1 binds there and is able to recruit more Suv39h1 establishing a ‘self-sustaining’ loop that would explain how heterochromatin may propagate once an initiating site has been established48,49. This model has been also extended to DNA

methylation, as both HP1 and Suv39h1 recruit DNMTs59.

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Protein HP-1 variant Domain

Transcriptional regulators of chromatin-modifying proteins

Histone H1 HP1 ND

Histone H3 HP1, HP1Mmα, HP1Mmβ, HP1Mmγ CD

Methyl K9 Histone H3 Swi6, HP1, HP1α, HP1β, HP1γ CD

Histone H4 HP1, HP1Mmα CSD

SUV39H1 HP1, HP1α, HP1β, HP1γ CSD

Polycomb HP1Hsα, HP1Hsγ CSD

Dnmt3a HP1Mmα ND

Dnmt3b HP1α, HP1β ND

Kap-1/Tif1β HP1α, HP1β, HP1γ CSD

Rb HP1Hsγ ND

MITR HP1Mmα Linker

BRG1 HP1Mmα CSD

ATRx HP1Mmα, HP1Mmβ CSD

TAFII130 HP1Hsα, HP1Hsγ CSD

PIM1 HP1Hsγ CSD

RNA HP1Mmα, HP1Mmγ Hinge

DNA replication and repair

CAF-1 p150 HP1α, HP1β CSD

Ku70 HP1Hsα, phosphoS83-HP1Hsγ CSD, Linker

ORC1-6 HP1 CD, CSD

Other chromosome-associated proteins

Psc3 Swi6 CD

INCENP HP1Hsα, HP1Hsγ Linker

Hsk1/CDC7 Swi6 ND

Ki-67 HP1Mmα, HP1Mmβ, HP1Mmγ CSD

SP100 HP1Hsα, HP1Hsβ, HP1Hsγ CSD

Nuclear structure proteins

Nuclear envelope HP1Mmα, HP1Mmβ, HP1Mmγ CD

Lamin B receptor HP1Hsα, HP1Hsβ, HP1Hsγ CSD

Lamin B HP1Mmβ CD

LAP2β HP1Mmβ CD

Figure I9. Examples of HP1 interacting partners. “HP1” alone refers to Drosophila HP1; HP1α, HP1β and HP1γ refer to both mouse and human unless specified (Mm, mouse; Hs, human); Domain abbreviations: CD, chromodomain; CSD, chromoshadow domain; ND, not determined. Adapted46.

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Lately, it has been shown that each HP1 isoform can suffer several PTMs, such acetylation, phosphorylation, ubiquitilation and sumoylation, in a similar way to histones61,62.

Many of these modifications are found in both the CD and the CSD, as well as in the linker domain, suggesting that they may have an important role in modulating HP1 interactions or functions. In addition, different modifications such as acetylation and methylation can occur in the same residue, which suggests that specific residues of HP1 proteins may act as “switches”, and may be directly involved in the regulation of HP1 functions.

3.2. Pericentromeric heterochromatin

Among the different constitutive heterochromatin domains, centric heterochromatic, also known as pericentromeric heterochromatin, is the most abundant in the genome.

Centromeres were originally defined as a cytologically visible “primary” constriction in the chromosome, and later as chromosomal sites that were essential for normal inheritance, which suffer greatly reduced or absent meiotic recombination. Nowadays, centromere is defined as the site of kinetochore formation, a proteinaceous structure on each chromosome that is responsible for their attachment to and movement along microtubules. The centromere is therefore essential for chromosomal plateward prometaphase and poleward anaphase movements63. However, in terms of terminology, the “centromere

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Figure I10. Structural and functional elements of the centromere region. The centromere/inner kinetochore, outer kinetochore, centric heterochromatin and chromosome arms, and associated functions, are shown63.

Centromeric chromatin is mainly characterized by the incorporation of the centromeric histone H3 variant (cenH3) within its nucleosomes, and the presence of specific centromeric proteins required for kinetochore formation. On the other hand, pericentromeric heterochromatin is enriched in epigenetic repressive marks such as DNA methylation, hypoacetylated histones and H3K9 and H4K20 methylation. Moreover, pericentromeric heterochromatin is also enriched in the three isoforms of HP1, especially α and β, and in an RNA component that seem to have a crucial role in its formation and maintenance45. Interestingly, pericentromeric heterochromatin also has a role in proper segregation of sister chromatids, since its deregulation is associated to chromosome mis-segregation and genome instability65–67.

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of repetitive elements, which can differ in sequence and size in different species, but are enriched with the same type of proteins (Figure I11).

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In S. pombe, centromeric regions are formed by a central core domain which contains a unique AT rich sequence of ~4 kb (central core) flanked by imperfect repeats (imr) of ~5 to 6 kb each. Pericentromeric regions are made of two types of outer repeats: dh and dg repeats of ~5 kb each. In mouse, centromeric and pericentromeric regions have not been fully characterized. Centromeric regions of ~600 kb are made of a repetition of AT-rich minor satellite motifs of 120 pb68, whereas pericentromeric

regions of ~6 Mb are made of a repetition of AT-rich major satellite motifs of 234 bp.

In humans, centromeric regions of about ~240 kb to 5 Mb, depending on the chromosome considered, are made of repetition of AT-rich alpha satellite motifs of 171 bp. The size and structure of pericentromeric regions, which also varies between chromosomes, are made of satellite repeats of three types: type I (0.5% of the genome), type II (2% of the genome) and type III (1.5% of the genome)69.

Pericentromeric heterochromatin of several chromosomes can cluster to form chromocenters, a highly condensed structure characterized by classical heterochromatin epigenetic traits. Chromocenters are especially visible in mouse cells, were they become clearly defined just by DAPI staining. In addition, they can also be easily visualized by HP1α, H3K9me3 or H4K20me3 immunofluorescence, among others, as well as with DNA FISH against major satellite repeats (Figure I12).

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The number and size of chromocenters per cell varies between cell types71,72. Interestingly, chromocenters tend to be smaller when they are

very abundant in a cell, and seem to be bigger when a lower number of chromocenters is found per cell. For instance, lymphocyte nuclei show a strong clustering of pericentromeric regions reflected by the small number of chromocenters compared to fibroblasts. Accordingly, chromocenters of lymphocytes appear to be much bigger than the ones found in fibroblasts72.

Therefore, different number of chromosomes may participate in the formation of a chromocenter in different cell contexts. This implies a certain degree of dynamism and flexibility to these highly condensed domains, which may be able to reorganize under specific circumstances, such as during the differentiation to a particular and specialized cell type.

3.2.1.

Pericentromeric heterochromatin formation

The core of heterochromatin assembly pathway found in Drosophila and mammals is conserved in S. pombe9. It involves posttranslational modifications of histones and a common set of structural proteins. In addition to histone deacetylation, heterochromatin assembly requires methylation of H3K9 that provides binding site for the HP1 family of chromodomain proteins73. In S. pombe, Chp1, Chp2, and Swi6 bind through their chromodomain to H3K9me, which is methylated by Clr4 (homolog of human Suv39h), establishing a heterochromatic context. However, the strategies used to initiate heterochromatin assembly may differ depending on the chromosomal context (Figure I13)43. In particular,

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Figure I13. Mechanisms for the initiation of heterochromatin assembly.

Heterochromatin structures can be nucleated by factors that recognize specific DNA sequences, such as transcription factors (TF), or by the RNAi machinery that targets repetitive DNA elements. Both mechanisms recruit histone-modifying enzymes such HMTs and HDACs to nucleate heterochromatin at a specific site. Swi6/HP1 binds to modified histone tails and allows heterochromatin to spread to surrounding sequences. Red flag, H3K9me; blue flag, H3K4me43.

Heterochromatin domains replicate during S-phase and the newly synthetized DNA must conserve epigenetic heterochromatin traits. To do so, pericentromeric regions may decondense to allow the entrance of the DNA replication machinery but, once heterochromatin has been replicated, it has to be re-silenced again. Interestingly, yeast pericentromeric transcription increases during S-phase. These transcripts are processed by the RNAi machinery and originate siRNA that target RITS complex and other heterochromatin factors such as Clr4 to pericentromeric domains, which will proceed to re-establish heterochromatin structure and silencing75,76.

In mammals, pericentromeric repeats are also transcribed and some evidences suggest that the transcripts generated may be involved in heterochromatin formation and silencing by a mechanism similar to the once described in yeast. In this way, mouse cells express RNA transcripts of nearly unknown function that are homologous to both strands of the major satellite repeats, as well as an RNA component (“structural” RNA) that is implicated in the maintenance of pericentromeric heterochromatin organization45. However, how these transcripts are

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Nevertheless, it has been recently shown that de novo targeting of HP1α in mouse pericentromeric regions is mediated by major satellite transcripts. SUMOylated HP1α interacts with forward major RNAs providing specificity to the initial targeting of HP1α to these domains. Then, HP1α is stabilized by the recognition of H3K9me3, and together with Suv39h is able to spread by a “self-enforcing” loop70.

3.2.2.

Pericentromeric heterochromatin transcription

Although classically it was thought that heterochromatin is completely repressed, nowadays it has become evident that, despite its “silenced – like” state, it is actively transcribed.

In particular, transcripts derived from pericentromeric repeats have been detected in a wide variety of cell types and they represent a new subtype of ncRNAs. Indeed, they should be considered as long ncRNAs (lncRNAs) at least in mammalian cells, where long pericentromeric transcripts of up to 8Kb have been detected64. However, little is known about their function in the cell, beyond its role in the formation and maintenance of heterochromatin and genome stability.

Interestingly pericentromeric transcripts are differently expressed in different cell types and their expression is modulated in response to specific stimuli, which suggests that tightly regulation of these transcripts may be essential in particular cell contexts.

One of the most dramatic examples of transcriptional activation of pericentromeric specific sequences is that which occurs in response to cell stress. In numerous normal primary and cancer human cell lines, heat-shock has been shown to induce satellite III sequence (satIII) transcription primarily from the 9q12 locus, although transcription from other pericentromeric regions has also been observed particularly in tumor cells, independent of the cell cycle77,78. In addition, induction of

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and, interestingly, transcript levels and orientation vary according to the nature of the stress signal79.

Sense and antisense pericentromeric transcription is also spatially and temporally regulated throughout mouse embryonic development. As early as during the 2-cell stage, there is a strand-specific burst in major satellite transcription that rapidly decreases by the 8-cell stage. Interestingly, both up- and down-regulation of major satellite transcripts are necessary events for proper chromocenter organization and developmental progression80.

Major satellite transcripts are also detected in more advanced stages of mouse embryonic development. In 11.5-15.5 dpc (day post coitum) embryos, pericentromeric transcripts in sense orientation, and in antisense in minor extent, are ubiquitously distributed in various tissues, especially in the central nervous system. In adult tissues, sense expression of pericentromeric sequences is detected in liver and testis but not in other tissues such as brain, colon, spleen, heart and lung, thus revealing a specific pattern of expression, not only with regard to embryonic stage, but also with regard to cell type81. Expression of

pericentromeric transcripts has also been described in human testis, and it is thought that they might play an essential role in the differentiation of germinal cells82.

In agreement with this, transcription from major satellite regions is significantly increased during neuronal differentiation, both in vitro and in vivo, and is accompanied by an enrichment of H3K4me3 in heterochromatin domains, as well as increased nuclease sensitivity83.

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These conflicting results suggest that major satellite transcription regulation may be more complex than initially thought, and that it may not only depend on changes in histone modifications in pericentromeric regions, but also on cell specific additional factors.

In concordance, retinoic acid (RA) treatment causes different effects on major satellite transcription depending on cell type. Major satellites were strongly repressed by RA in HeLa and P19 cells81, but were up-regulated

in ES cells induced to form embryonic bodies by RA treatment85.

So, although the function of major satellite transcripts is still rather enigmatic, strong evidences point that there is a link between major satellite transcription and differentiation and cell type specification. However, many efforts remain to be done to further understand how major satellite transcripts may be able to specify or determine cell fate decisions.

Major satellite transcription has also been shown to be cell cycle dependent. In proliferative mouse cells, two different populations of major satellite transcripts accumulate at different times during cell cycle. Small RNA species are exclusively synthesized during mitosis and are rapidly eliminated during mitotic exit, suggesting they may be involved in reinforcing the heterochromatin structure during the late stages of mitosis or assisting the reloading of HP1 during anaphase. Alternatively, a more abundant population of large, heterogeneous transcripts is induced late in G1-phase and their synthesis decreases during mid S-phase, which is coincident with pericentromeric heterochromatin replication86. This observation strongly suggests that, similar to what is described in S.

pombe75, pericentromeric transcripts may also play a role in the re-formation of pericentromeric heterochromatin after replication in mammalian cells64.

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the latter could be generated from long precursors through post-transcriptional mechanisms involving splicing.

Furthermore, the mechanism through which pericentromeric transcription is ultimately regulated is still poorly understood. The impossibility of defining conventional promoter regions in heterochromatin domains, has classically nearly discarded the possibility of transcription factors regulating pericentromeric transcription. However, several transcription factors have been shown to bind pericentromeric heterochromatin and regulate its transcription.

The first transcription factors identified to be involved in human pericentromeric transcription were Heat-Shock Factor 1 (HSF1)87 and

tonicity Enhancer-Binding Protein (tonEBP)79. Upon stress, HSF1 directly

binds to satIII sequences at the 9q12 locus and promotes transcription, since its absence prevents accumulation of satIII transcripts in heat-shocked cells. TonEBP also accumulates at the 9q12 locus and is required for induction of SatIII RNAs during hyper-osmotic stress.

More recently, it has been shown that the transcription factors Pax3 and Pax9 act as redundant regulators of mouse heterochromatin, as they repress major satellite repeats by associating with DNA within pericentromeric heterochromatin. Interestingly, this study also shows that all HMT Suv39h–dependent heterochromatic repeat regions in the mouse genome present a high concordance with the presence of transcription factor binding sites, and suggests transcription factor dependent heterochromatin formation as a general mechanism 88.

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4.

Cancer and tumor progression

Today, cancer is a leading cause of death worldwide. The word “cancer” includes a broad group of diseases, since more than 100 different types of cancers have been described. All of them are characterized by the following alterations in cell physiology: self-sufficient growth, insensitivity to anti-growth signals, evasion of apoptosis and immune response, and ability for sustained angiogenesis, tissue invasion and metastasis, among others89. Moreover, a highly heterogeneity among the cancer cells within a tumor is also a common hallmark of all cancer types90.

Carcinoma, originated in epithelial cells, is the most prevalent form of cancer, and it includes colorectal, breast, prostate and lung cancer among others91. Although diagnosis and prognosis of these cancers is improving every day, metastasis is still responsible for as much as 90% of cancer-associated mortality. So further understanding of this process is essential to progress in the fight against cancer.

Still nowadays, it is not clear how tumors initiate and progress. Two current ideas describe the establishment and maintenance of tumor heterogeneity, which are the clonal evolution model and the cancer stem cell hypothesis. Both of them propose that tumors originate from a single cell that has acquired multiple mutations and has gained unlimited proliferative potential, but through different mechanisms92.

The clonal evolution model states that, when cancer cells acquire genetic alterations, each of them confers one or another form of increased fitness that triggers clonal expansion. This genetic drift and stepwise natural selection favors the most aggressive cells and drives progression. Based in this model, any tumor cell that acquires the capacity of self-renew has the potential to contribute to tumor progression.

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renewal and differentiation capability is responsible for tumor heterogeneity.

Both paradigms of tumor propagation are likely to exist in human cancers since they are not mutually exclusive, and CSC themselves may undergo clonal expansion92.

As previously mentioned, carcinomas arise in epithelial tissues. Usually, normal epithelial cells are tightly bound to neighboring cells and to underlying basement membranes by a complex junctional network. However, as the tumor progresses, carcinoma cells lose these associations, acquire mesenchymal traits and increased migration and invasion capability, being able to dissolve extracellular matrix and move through the surrounding tissue towards blood and lymphatic vessels, establishing the first step for metastasis progression93.

The process by which epithelial cells undergo all this morphological and molecular changes is referred as epithelial-to-mesenchymal transition (EMT). Its importance in cancer is now widely accepted by pathologists and the cancer research field considers this process as one of the most important to understand.94

4.1. Epithelial-to-Mesenchymal Transition (EMT)

The EMT program describes a series of events during which epithelial cells lose many of their epithelial characteristics and take on properties that are typical of mesenchymal cells. Accordingly, cells engaged in the EMT program undergo complex changes in cell architecture and behavior95. The reverse process, known as mesenchymal to epithelial transition (MET) has also been reported96.

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Epithelial cells are apico-basal polarized, with the apical and basal surfaces serving different functions.

E-cadherin represents the best characterized molecular marker expressed in epithelial cells. It is a transmembrane protein localized to the adherens junctions, with an extracellular domain that mediates calcium-dependent homotypic interactions with E-cadherin molecules on adjacent cells, and an intracellular domain that binds cytosolic catenins and provides a link to the actin cytoskeleton. Loss of E-cadherin is by itself a hallmark of EMT since it is associated with loss of epithelial phenotype. However, many other epithelial markers are down-regulated during EMT, such as claudins and occludins, located in tight junctions and acting as barriers that inhibit lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains97,98.

Mesenchymal cells, on the other hand, do not have stable intercellular junctions and possess an elongated morphology with front-to-back asymmetry that facilitates motility and locomotion. Upon EMT, many mesenchymal markets become expressed, such as integrin family receptors that localize in the filapodial extensions at the leading edge of the mesenchymal cells and interact with the extracellular matrix99, or

matrix metalloproteinases (MMP) that digest basement membranes and promote invasion100.

Mesenchymal cells also have increased expression of mesenchymal proteins, such as the intermediate filament protein vimentin, or other cytoskeletal proteins, including smooth muscle actin, as well as extracellular matrix components such as fibronectin and collagen precursors101(Figure I14).

These changes in protein expression, and many others, are associated with changes in transcription101,102. Therefore, activation and repression of specific sets of genes during this process must be tightly regulated to establish the particular transcriptome of mesenchymal cells, which clearly differs from the one typical of epithelial cells.

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organization may be occurring during EMT. Activation and repression of transcription has been associated to gene movement, but is still unknown whether clusters of epithelial and mesenchymal genes move and re-localize to specific sub-nuclear domains during EMT. What seems obvious, although not yet demonstrated, is that nuclear architecture and organization may notably differ in mesenchymal cells compared to epithelial cells, since physiological requirements of each type of cell is completely different.

Figure I14. Epithelial and mesenchymal cell features. Epithelial cells are characterized by their apico-basal polarity and their cell-cell and cell-ECM contacts (adherens junctions, tight junctions and desmosomes). Upon EMT, epithelial cells acquire mesenchymal properties as loss of cell contacts, expression of mesenchymal markers such as vimentin and fibronectin, and increased cell motility.

4.1.1.

Physiological EMT

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both vertebrates and invertebrates. Indeed, several rounds of EMT and MET are necessary for the final differentiation of specialized cell types and the acquisition of the complex three-dimensional structure of internal organs. Accordingly, these sequential rounds are referred to as primary, secondary, and tertiary EMT. Examples of primary EMT include mesoderm formation during gastrulation and neural crest delamination, whereas liver and pancreas result from secondary and heart from tertiary EMT103.

The earliest example of EMT during embryonic development is the generation of the mesoderm, which marks the beginning of gastrulation. After invagination of the epithelial cells from the epiblast (primitive ectoderm) around the primitive streak, the basement membrane breaches locally and cells lose their tight cell-cell adhesions and remain attached to neighboring cells only by disperse focal contacts. After completing EMT they migrate along the narrow extracellular space underneath the ectoderm to form the mesoderm104(Figure I15).

Figure I15. Examples of primary EMT. The first EMT after implantation is that undergone by the mesendodermal progenitors during gastrulation, whereas the delamination of neural crest cells from the dorsal neural tube is a later event103.

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structures, most of the peripheral nervous system, some endocrine cells, and melanocytes103(Figure I15).

Although EMT has been largely described during development, similar processes occur in adult tissues. The classical example is skin wound healing, during which keratinocytes at the border of the wound migrate to close it. To do so, they acquire a “metastable” state, characterized by rearrangement of their actin cytoskeleton, extended lamellipodia and loss of both cell–cell contacts and hemidesmosomes. In addition, they alter the expression of integrin receptors and express various proteases to degrade connective tissue. But they do no undergo a complete EMT since they retain some intercellular junctions and express epidermal keratins but not vimentin105.

4.1.2.

Pathological EMT

EMT has an important role in the development of many tissues during embryogenesis, but similar cell changes are recapitulated during pathological processes, such as fibrosis and cancer.

During progression to metastatic competence, carcinoma cells acquire mesenchymal gene-expression patterns and properties. This results in changed adhesive properties and activation of proteolysis and motility, which allows the tumor cells to metastasize and establish secondary tumors at distant sites95.

Activation of an EMT program during tumorigenesis often requires signaling between cancer cells and neighboring stromal cells104. Islands of

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operate in a small fraction of cancer cells that are in intimate contact with the adjacent reactive stroma.

Furthermore, EMT can induce non-CSC to enter into a CSC-like state. As such, the EMT confers on epithelial cells precisely the set of traits (invasion, apoptosis resistance…) that would empower them to disseminate from primary tumors and seed metastases. This implies that not only intrinsic CSC but also induced subtypes of CSC within a tumor would be able to acquire a metastatic phenotype (Figure I16)93.

Figure I16. Acquisition of the metastatic phenotype. Intrinsic CSCs are thought to exist in primary tumors from the very early stages of tumorigenesis and may be the oncogenic derivatives of normal-tissue stem or progenitor cells. Induced CSCs may arise as a consequence of the EMT. In this case, carcinoma cells initially recruit a variety of stromal cells that create a reactive microenvironment that releases factors that cause the neighboring cancer cells to undergo the EMT and acquire CSC-like characteristics93.

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maintenance of some epithelial properties may somehow favor metastasis. In addition, observation that metastases appear histologically similar to the primary tumor from which they are derived suggests that migrating cells may undergo MET once they reach a secondary site to establish a micrometastasis. Thus, acquisition of mesenchymal characteristics may be transitory and favor invasion and intravasation of cancer cells, but may undergo a reversal during later tumorigenesis to allow establishment and development of metastasis at distal organs (Figure I17).

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4.1.3.

Signaling EMT

Many signaling pathways trigger EMT in both embryonic development and in normal and transformed cell lines. These signaling pathways include those triggered by soluble factors such as different members of the TGFβ superfamily, Wnts, Notch, other tyrosine kinase receptors family members (EGF, HGF and FGF), HIF, and many others103,104. Also

components of the extracellular matrix (ECM), such as collagen and hyaluronic acid108 are able to induce EMT95(Figure I18).

Figure I18. Overview of the molecular networks that regulate EMT. Selection of signaling pathways activated by regulators of EMT and limited representation of their crosstalk. Activation of receptor tyrosine kinases (RTKs) induces EMT but it often requires co-activation of integrin receptors. TGFβ triggers EMT, but there is also a mutual regulation of the TGFβ and NOTCH pathways during EMT. Other signaling pathways could have an important role in EMT, including G-protein-coupled receptors and matrix metalloproteinases (MMPs) that can also trigger EMT through as-yet-undefined receptors95.

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response to TGFβ, Smad2 and Smad3 are activated and form complexes with Smad4, which then regulate transcription of target genes. The expression of three families of transcription factors (Snail, ZEB and bHLH families) is induced in response to TGFβ, either through a Smad-dependent mechanism (in the case of Snail proteins) or indirectly through activation of other transcription factors or relief of repression. Upon activation, these transcription factors repress epithelial markers gene expression and activate mesenchymal gene expression109.

A central target of these transcriptional regulators is the repression of the E-cadherin gene (CDH1), an important caretaker of the epithelial phenotype. Its down-regulation abolishes E-cadherin-mediated sequestering of β-catenin in the cytoplasm and as a result, β-catenin localizes to the nucleus and feeds into the Wnt signalling pathway by activating transcription of LEF-1/TCF4 target genes95, which in turn favors invasion110.

4.1.4.

EMT inducers

E-cadherin repressors can be classified into two groups depending on their effects on the CDH1 promoter: Snail/Slug, Zeb1/Zeb2, E47, and KLF8 bind to and repress the activity of the CDH1 promoter111–117, whereas factors such as Twist, Goosecoid, E2.2, and FoxC2 repress CDH1

transcription indirectly118–121.

4.1.4.1.

Snail1 transcription factor

Snail1 is one of most widely studied effectors of EMT and CDH1

repression. Besides CDH1, Snail1 represses several other epithelial genes such as occluding (OCLN) and claudin (CLDN), but it is also involved in the regulation of genes related to cell cycle progression or apoptosis122.

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at least in certain conditions, it may work as a direct activator together with p65-NFκB124.

Snail1 belongs to the Snail superfamily of transcription factors, which is subdivided into the Snail and Scratch families. Three members of the Snail family have been described in vertebrates to date: Snail (SNAI1), Slug (SNAI2) and Smuc (SNAI3). Members of the Snail family are zinc-finger transcription factors that share a common organization: a highly conserved C-terminal region that contains from four to six zinc C2H2 type

fingers and a divergent N-terminal region125.

In particular, Snail1 has 4 zing fingers in its C-terminal domain, which function as DNA binding domains and bind to specific sequences called E-boxes: 5’-CACCTG-3’ or 5’-CAGGTG-3’126,127 located in the promoters of

its target genes (Figure I19).

Figure I19. Structural domains of Snail1 transcription factor. Snail1 has an N-terminal SNAG domain involved in co-repressor interaction. The central region comprises a serine-proline domain, a destruction box and a nuclear export signal domain (NES) and is important for protein localization and stability. The C-terminal domain has 4 zing fingers responsible for direct binding to E-boxes in the DNA. Adapted128.

The central region of Snail1 comprises a nuclear export signal (NES), a destruction box domain, and a serine-proline rich region128. This central

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targeted to proteasome degradation by β-TrCP1130. By contrast, phosphorylation of Snail1 by Pak1 retains it in the nucleus enhancing its repressive function131. Fbxl14, another ubiquitin ligase is also able to ubiquitylate Snail1 in this central region and target it to degradation132.

Finally, Snail1 has a SNAG (Snail/Gfi-1) domain in its N-terminal domain that is important for co-repressor interaction and repressive capacity126.

Thus, Snail1 binds the promoter of its target genes through its zing finger domain, and interacts with histone modifying enzymes through the SNAG domain. In this way, Snail1 recruits the co-repressor Sin3A together with HDAC1/2 and induces deacetylation of histones H3 and H4133. It also recruits Polycomb repressive complex 2 (PRC2) which trimethylates H3K27134 and LSD1, which is found as a component of the HDAC1/

2-containing co-repressor complex CoREST and removes mono- and dimethyl marks on H3K4135(Figure I20).

Figure

Figure I3. Conserved role of insulators in nuclear organization. Insulators in yeast (orange), Drosophila (green and yellow), and mammals (brown and orange) mediate long-range inter- and intrachromosomal interactions important for gene regulation; they are

Figure I3.

Conserved role of insulators in nuclear organization. Insulators in yeast (orange), Drosophila (green and yellow), and mammals (brown and orange) mediate long-range inter- and intrachromosomal interactions important for gene regulation; they are p.28
Figure I4. Summary of histone modifications and histone modifying enzymes. histone acetyltransferases (HATs) and removed by histone deacetlyases (HDACs)

Figure I4.

Summary of histone modifications and histone modifying enzymes. histone acetyltransferases (HATs) and removed by histone deacetlyases (HDACs) p.30
Figure I5. Map of main PTMs in histone tails. Acetylation (blue), methylation (red), phosphorylation (yellow) and ubiquitylation (green)

Figure I5.

Map of main PTMs in histone tails. Acetylation (blue), methylation (red), phosphorylation (yellow) and ubiquitylation (green) p.31
Figure I7. Properties of euchromatic and heterochromatic regions. Cluster of that flank the centromeres of many eukaryotic chromosomes

Figure I7.

Properties of euchromatic and heterochromatic regions. Cluster of that flank the centromeres of many eukaryotic chromosomes p.35
Figure I8. Mouse HP1 domains and interaction partners. (a) Amino-acid

Figure I8.

Mouse HP1 domains and interaction partners. (a) Amino-acid p.36
Figure I9. Examples of HP1 interacting partners. “HP1” alone refers to Drosophila HP1; HP1α, HP1β and HP1γ refer to both mouse and human unless specified (Mm, mouse; Hs, human); Domain abbreviations: CD, chromodomain; CSD, chromoshadow domain; ND, not dete

Figure I9.

Examples of HP1 interacting partners. “HP1” alone refers to Drosophila HP1; HP1α, HP1β and HP1γ refer to both mouse and human unless specified (Mm, mouse; Hs, human); Domain abbreviations: CD, chromodomain; CSD, chromoshadow domain; ND, not dete p.38
Figure I10. Structural and functional elements of the centromere region. The centromere/inner kinetochore, outer kinetochore, centric heterochromatin and chromosome arms, and associated functions, are shown63

Figure I10.

Structural and functional elements of the centromere region. The centromere/inner kinetochore, outer kinetochore, centric heterochromatin and chromosome arms, and associated functions, are shown63 p.40
Figure I11. Comparative organization of centromeric (CT) and pericentromeric

Figure I11.

Comparative organization of centromeric (CT) and pericentromeric p.41
Figure I14. Epithelial and mesenchymal cell features. Epithelial cells are epithelial cells acquire mesenchymal properties as loss of cell contacts, characterized by their apico-basal polarity and their cell-cell and cell-ECM contacts (adherens junctions,

Figure I14.

Epithelial and mesenchymal cell features. Epithelial cells are epithelial cells acquire mesenchymal properties as loss of cell contacts, characterized by their apico-basal polarity and their cell-cell and cell-ECM contacts (adherens junctions, p.52
Figure I18. Overview of the molecular networks that regulate EMT. Selection of is also a mutual regulation of the TGFβ and NOTCH pathways during EMT

Figure I18.

Overview of the molecular networks that regulate EMT. Selection of is also a mutual regulation of the TGFβ and NOTCH pathways during EMT p.57
Figure I20. Model of Snail1 mediated transcriptional repression. Snail1 binds LSD1 through its SNAG domain

Figure I20.

Model of Snail1 mediated transcriptional repression. Snail1 binds LSD1 through its SNAG domain p.60
Figure R1. Heterochromatin organization is compromised in the absence of Snail1. (A) Western blot for Snail1 in pMEFs Snail1F/F/Cre- and Snail1F/F/Cre+ treated with tamoxifen

Figure R1.

Heterochromatin organization is compromised in the absence of Snail1. (A) Western blot for Snail1 in pMEFs Snail1F/F/Cre- and Snail1F/F/Cre+ treated with tamoxifen p.72
Figure R2. Snail1 is required for S-phase progression. (A) A representative image of iMEFs CT and KO for Snail1 stained with DAPI and PCNA (replication marker) to show typical early replication staining (PCNA diffused in the nucleus) and mid-late replicati

Figure R2.

Snail1 is required for S-phase progression. (A) A representative image of iMEFs CT and KO for Snail1 stained with DAPI and PCNA (replication marker) to show typical early replication staining (PCNA diffused in the nucleus) and mid-late replicati p.74
Figure R4. Snail1 depletion induces genome instability. (A) Distribution of number of chromosomes per cell observed in CT and KO pMEFs methaphases

Figure R4.

Snail1 depletion induces genome instability. (A) Distribution of number of chromosomes per cell observed in CT and KO pMEFs methaphases p.76
Figure R5.  Snail1 regulates pericentromeric heterochromatin transcription. (A)

Figure R5.

Snail1 regulates pericentromeric heterochromatin transcription. (A) p.78
Figure R6. Snail1 binds pericentromeric regions in mouse and human cells. (A)

Figure R6.

Snail1 binds pericentromeric regions in mouse and human cells. (A) p.80
Figure R7. Snail1 SNAG domain interacts with HP1α chromoshadow domain. (A)Western blotting using the indicated antibodies

Figure R7.

Snail1 SNAG domain interacts with HP1α chromoshadow domain. (A)Western blotting using the indicated antibodies p.82
Figure R8. LOXL2 favors nucleosomal compaction. Nuclei isolated from HEK293T transfected with LOXL2 wild type (wt), inactive mutant (mut) or an empty vector (Ø) were digested with micrococcal nuclease (MNase) for 5 or 10 min

Figure R8.

LOXL2 favors nucleosomal compaction. Nuclei isolated from HEK293T transfected with LOXL2 wild type (wt), inactive mutant (mut) or an empty vector (Ø) were digested with micrococcal nuclease (MNase) for 5 or 10 min p.83
Figure R9. There is less oxidized H3 in major satellite regions of Snail1 KO

Figure R9.

There is less oxidized H3 in major satellite regions of Snail1 KO p.84
Figure R11. LOXL2 binds major satellite sequences and oxidizes H3. CT iMEFs were infected with a shRNA control or a shRNA against LOXL2, and LOXL2 ChIP (left panel), H3K4me3 ChIP (right panel) and oxidized H3 Re-ChIP conducted as in Figure R9 (middle panel

Figure R11.

LOXL2 binds major satellite sequences and oxidizes H3. CT iMEFs were infected with a shRNA control or a shRNA against LOXL2, and LOXL2 ChIP (left panel), H3K4me3 ChIP (right panel) and oxidized H3 Re-ChIP conducted as in Figure R9 (middle panel p.86
Figure R12. HP1α delocalizes from chromocenters after TGFβ treatment.  µm. Inset shows magnified representative nucleus, scale bar 5 µm

Figure R12.

HP1α delocalizes from chromocenters after TGFβ treatment. µm. Inset shows magnified representative nucleus, scale bar 5 µm p.87
Figure R14. Release of HP1α from chromocenters is Snail1 and LOXL2 dependent. (A)transfected with shRNA control or shRNA against LOXL2

Figure R14.

Release of HP1α from chromocenters is Snail1 and LOXL2 dependent. (A)transfected with shRNA control or shRNA against LOXL2 p.90
Figure R17. Snail1 is responsible for major satellite down-regulation during EMT. (A) Western blot showing Snail1 induction upon TGFβ treatment in siControl NMuMG cells, and absence of Snail1 up-regulation in siSnail1 cells

Figure R17.

Snail1 is responsible for major satellite down-regulation during EMT. (A) Western blot showing Snail1 induction upon TGFβ treatment in siControl NMuMG cells, and absence of Snail1 up-regulation in siSnail1 cells p.92
Figure R16. Snail1 binding to major satellites after TGFβ treatment correlates with increased H3 oxidation

Figure R16.

Snail1 binding to major satellites after TGFβ treatment correlates with increased H3 oxidation p.92
Figure R18. LOXL2 regulates pericentromeric transcription during EMT through H3 oxidation

Figure R18.

LOXL2 regulates pericentromeric transcription during EMT through H3 oxidation p.93
Figure R19. Major satellite overexpression blocks HP1α release from chromatin

Figure R19.

Major satellite overexpression blocks HP1α release from chromatin p.95
Figure R21. Genes involved in EMT are differently regulated by TGFβ in NMuMG-Major cells

Figure R21.

Genes involved in EMT are differently regulated by TGFβ in NMuMG-Major cells p.97
Figure R23. HP1α and major satellite transcripts knock-down does not affect mesenchymal genes induction after TGFβ treatment

Figure R23.

HP1α and major satellite transcripts knock-down does not affect mesenchymal genes induction after TGFβ treatment p.99
Figure D1. Snail1 ChIP-Seq predicted binding sites analysis. (A) Presence of E-boxes across the predicted binding sites, where canonical Snail1 E-boxes are 5’-CACCTG-3’ and 5’-CAGGTG-3’, non-canonical Snail1 E-boxes are 5’-CACGTG-3’ and 5’-CAGCTG-3’ and Sn

Figure D1.

Snail1 ChIP-Seq predicted binding sites analysis. (A) Presence of E-boxes across the predicted binding sites, where canonical Snail1 E-boxes are 5’-CACCTG-3’ and 5’-CAGGTG-3’, non-canonical Snail1 E-boxes are 5’-CACGTG-3’ and 5’-CAGCTG-3’ and Sn p.110
Figure CR1.  Model for pericentromeric heterochromatin reorganization during EMT. Upon TGFβ induction of EMT, Snail1 is rapidly up-regulated, binds to pericentromeric regions and recruits LOXL2 to oxidize H3 and repress major satellite transcription

Figure CR1.

Model for pericentromeric heterochromatin reorganization during EMT. Upon TGFβ induction of EMT, Snail1 is rapidly up-regulated, binds to pericentromeric regions and recruits LOXL2 to oxidize H3 and repress major satellite transcription p.126

Referencias

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