Gaël Guibon
email: gael.guibon@inria.fr

Marine Courtin
email: marine.courtin@sorbonne-nouvelle.fr

Kim Gerdes

Bruno Guillaume
email: bruno.guillaume@inria.fr

When Collaborative Treebank Curation Meets Graph Grammars Arborator With a Grew Back-End

Keywords: dependency treebanks, annotation tools, crowd-sourcing, class-sourcing, error-mining, graph banks

In this paper we present Arborator-Grew, a collaborative annotation tool for treebank development. Arborator-Grew combines the features of two preexisting tools: Arborator and Grew. Arborator is a widely used collaborative graphical online dependency treebank annotation tool. Grew is a tool for graph querying and rewriting specialized in structures needed in NLP, i.e. syntactic and semantic dependency trees and graphs. Grew also has an online version, Grew-match, where all Universal Dependencies treebanks in their classical, deep and surface-syntactic flavors can be queried. Arborator-Grew is a complete redevelopment and modernization of Arborator, replacing its own internal database storage by a new Grew API, which adds a powerful query tool to Arborator's existing treebank creation and correction features. This includes complex access control for parallel expert and crowd-sourced annotation, tree comparison visualization, and various exercise modes for teaching and training of annotators. Arborator-Grew opens up new paths of collectively creating, updating, maintaining, and curating syntactic treebanks and semantic graph banks.

Introduction

Dependency treebanks have become the standard resource for training syntactic parsers, and substantial efforts have been undertaken to develop large scale and multi-lingual treebanks. The flagship project is certainly Universal Dependencies (UD) [START_REF] Mcdonald | Universal dependency annotation for multilingual parsing[END_REF], which has served as the input to numerous parsers, text generators, and morphological taggers around various shared tasks [START_REF] Zeman | CoNLL 2018 shared task: Multilingual parsing from raw text to universal dependencies[END_REF][START_REF] Mille | The second multilingual surface realisation shared task (SR'19): Overview and evaluation results[END_REF][START_REF] Mccarthy | The sigmorphon 2019 shared task: Morphological analysis in context and cross-lingual transfer for inflection[END_REF]. The impressive project with more than a hundred treebanks in the same annotation scheme for 90+ languages, combined with great online viewers and query tools have given increased visibility to the project also inside the syntax and typology communities [START_REF] Croft | Linguistic typology meets universal dependencies[END_REF][START_REF] Gerdes | Rediscovering greenberg's word order universals in ud[END_REF]). Yet, for UD as well as for other treebank creation projects, many of the treebanks contain substantial errors and inconsistencies, which can be attributed to three main causes:

1. Many of the UD treebanks are converted from other formats that do not contain all the information needed for a transfer into UD, or the converters are incomplete.

2. Some descriptions in the UD guidelines are underspecified and leave room for different analyses of the same construction, inside a language, a language group, or generally among languages, cf. the constantly active UD discussion group on GitHub and also [START_REF] Gerdes | Dependency annotation choices: Assessing theoretical and practical issues of universal dependencies[END_REF]. Moreover, some UD rules are not well-adapted for specific languages -e.g. the discussion of the direction of coordination in Japanese [START_REF] Kanayama | Coordinate structures in universal dependencies for head-final languages[END_REF].

3. Some treebank creators do not have sufficient time or competence to provide satisfactory analyses of some of the innumerable syntactic phenomena of their language. Moreover some of the treebanks have been abandoned and do not follow the latest updates of the UD specifications.

Generally speaking, dependency parsing and tagging with recent quantitative NLP tools can overcome, to a certain degree, rarely occurring errors in the training corpus. This is particularly true when making use of word vector representation models trained on massive amount of raw textual data [START_REF] Devlin | BERT: Pre-training of deep bidirectional transformers for language understanding[END_REF][START_REF] Peters | Deep contextualized word representations[END_REF]. However, in the use cases of language teaching, the query of counterexamples to syntactic claims, or typological comparative measures of (word order, syntactic relation, construction) distribution tendencies, these errors can influence the results significantly. See for example the differences between treebanks of the same language reported by [START_REF] Chen | Classifying languages by dependency structure. typologies of delexicalized universal dependency treebanks[END_REF]. It has become essential for the UD project not only to facilitate treebank curation for the treebank maintainer but to find ways to open treebank corrections to a wider audience of linguists and language students. All the aforementioned treebank annotation errors come essentially in two flavors: occasional slips of attention of the annotator and systematic discrepancies with the desired correct analysis. The former type of problems (occasional slips of attention of the annotator) can be addressed by means of an easy access to "strange" constructions 1 and myriads of annotators who look at these potential issues and who then either directly fix the error or validate the rare construction as being correct. The later type of problems (systematic discrepancies with the desired correct analysis) needs systematic corrections by means of a graph grammar and non-regression validation2 . Examples of this kind of systematic errors are provided by [START_REF] Haverinen | A dependency-based analysis of treebank annotation errors[END_REF] where the authors found that annotators frequently confused direct objects and nominal modifiers, syntactically readily mistakable. But annotators also confused subjects and adjectival modifiers for the surprising reason that the annotation tool's shortcut keys are placed next to one another on the keyboard. These types of error can easily be detected using Grew [START_REF] Bonfante | Application of Graph Rewriting to Natural Language Pro-Figure 7[END_REF].

Our new tool Arborator-Grew provides support for the whole process of treebank creation, publication, errormining, and curation. It is essentially a front-end editor to the Grew graph rewriting system3 [START_REF] Guillaume | Grew: un outil de réécriture de graphes pour le tal (grew: a graph rewriting tool for nlp)[END_REF][START_REF] Bonfante | Application of Graph Rewriting to Natural Language Pro-Figure 7[END_REF], adding access control, predefined error mining queries, exercises for teaching and annotator training, graphical diff tools, and versioning via GitHub.

Arborator-Grew is under active development and a first public release is planned during Spring 2020. It attempts to replicate the features of the current version of Arborator [START_REF] Gerdes | Collaborative dependency annotation[END_REF], in particular its class-sourcing tools [START_REF] Zeldes | The gum corpus: creating multilayer resources in the classroom[END_REF], while improving and modernizing queries, error-mining, versioning, and collaborative features. It is the first tool to integrate complex graph querying and treebank annotation software. The advantages of this combination will be discussed in Section 4.

Related Work

Quite a few tools exist for dependency treebank development, visualization, and querying, many of which share some of the features of Arborator-Grew. These tools can be divided into three mains groups: dependency visualization tools, dependency annotation tools, and treebank query tools. Without claiming to be exhaustive, we present here the main features of some of these tools, which have led us to the development of Arborator-Grew's specifications.

Dependency visualization. UDAPI [START_REF] Popel | Udapi: Universal API for universal dependencies[END_REF], CoNLL-U viewer4 and TüNDRA [START_REF] Martens | Tündra: A web application for treebank search and visualization[END_REF] [START_REF] Heinecke | Conllueditor: a fully graphical editor for universal dependencies treebank files[END_REF]. The ConlluEditor is noteworthy for its easy token splitting and joining, its stemma-like horizontal visualization, its integration of UD validation scripts, and its interaction with GitHub versioning. One last annotation tool worth mentioning in this context is ZombiLingo [START_REF] Guillaume | Crowdsourcing Complex Language Resources: Playing to Annotate Dependency Syntax[END_REF], a tool for crowd-sourcing of the syntactic annotation process through gamification. Users have to pass rather basic proficiency tests to be allowed to play: they are presented with a sentence at a time, for which they have to determine one single relation, such as finding the subject of a verb, and they can gain points and enter a public wall of fame. The annotations that most players agree on are combined into dependency trees of yet unannotated sentences.

Query tools. The most famous linguistic annotation query tool is arguably Annis [START_REF] Zeldes | Annis: A search tool for multi-layer annotated corpora[END_REF]. Annis proposes its own query language AQL (ANNIS Query Language) which can handle multi-layer annotations, a graphical query builder, chunk, phrase structure tree, and dependency visualization (the latter using Arborator's visualization Javascript library), integration of sound files, and queries on multiple tokenizations thanks to its stand-off format [START_REF] Krause | Multiple tokenizations in a diachronic corpus[END_REF]). Yet, due to its complexity, Annis requires a non-trivial installation and data-insertion process. AQL is rather verbose, and it is not blazingly fast. Other tools are more specifically designed for queries into single-layer dependency treebanks. One of these tools is Dep Search from Turku [START_REF] Luotolahti | dep search: Efficient search tool for large dependency parsebanks[END_REF]. It is very lightweight and fast, with a succinct and quite powerful query language based on TGrep [START_REF] Rohde | Tgrep2 user manual[END_REF], though quite unfamiliar and tricky for users trained on other query languages.

Most of these tools are designed to provide the matching trees alongside a simple count of matches, but they do not include further statistical data about the query results. Grew-match9 goes a step beyond that with the possibility to cluster on any of the nodes or edges of the query results. This includes simple clustering of the form for any lemma, thus providing a list of forms, and also the clustering of the relation between any two parts of speech, providing a list of relations that link the two parts of speech. The integration of this feature into Arborator-Grew and its usage will be explained in Section 3.2. Another remarkable tool that goes a step further is the Trameur [START_REF] Fleury | Trameur: A framework for annotated text corpora exploration[END_REF]. It applies corpus linguistics statistical tools to raw corpora, and, in its online version iTrameur10 also to dependency treebanks. It can therefore show significant over or under-representations of specific sub-trees in one sample compared to another. We intend to study further the possible use of these measures in Arborator-Grew's error-mining tools.

iTrameur/ iTrameur only has a French interface.

One important question in the design for multi-user annotation systems is the status of tokenization. Brat and Web-Anno allow the user to annotate any span of text, most other tools consider that the annotated object is a series of (predefined) tokens. It may seem like a more natural choice to actually annotate texts and to combine the tokenization and syntactic annotation step into one single task. Note however that annotator-based tokenization complicates significantly the computation of inter-annotator agreement, as we jointly observe tokenization and syntactic annotation. And most importantly, tokenization is either trivial and orthography based (i.e. a token is a sequence of letters, possible errors are expressed in the syntactic annotation) or based on lexical and semantic criteria, which makes it a challenging task to reach a satisfying inter-annotator agreement [START_REF] Farahmand | A multiword expression data set: Annotating noncompositionality and conventionalization for english noun compounds[END_REF][START_REF] Savary | The parseme shared task on automatic identification of verbal multiword expressions[END_REF]. Arborator takes a middle stand, making use of its hierarchy of user modes, see Section 4., and allows validators and project owners to modify (delete, add, join, split) tokens, but these changes are then carried out on all trees, whatever the user, of the modified sentence. Such a global modification of a sentence's tokenization can cause other annotators' trees to be disconnected or different from the desired structure. This behavior is the only exception to the basic Arborator rule which states that users can view other annotators' trees, depending on their access level, but can only create or modify their own tree. 11

Architecture

Arborator-Grew is a complete redevelopment that does not share a line of code with the legacy Arborator, beyond the Python CoNLL-U parser. The legacy Arborator is written in Python 2. It is a simple CGI web page and uses a SQLite 12 database with the FTS4 module for fast text searches. User identification is handled via the quite ancient Login Tools 13 , and the front-end runs in Jqueryenhanced Javascript with the rather slow Raphael.js 14 for drawing SVG tree graphs. The new Arborator-Grew consists of three completely separate pieces of software that interact via stateless REST interfaces and follows a Model-View-Controller (MVC) architecture shown in Figure 2. 12 https://www.sqlite.org/index.html 13 http://www.voidspace.org.uk/python/ logintools.html 14 https://github.com/DmitryBaranovskiy/ raphael 15 https://ocaml.org added an API accepting json queries. It is possible to run the Grew API on a different server than the main Arborator-Grew, and this is the configuration that we use for our current development setup.

Back-end and User Persistence. The middleman is a Flask16 application written in Python 3 that uses the Authomatic17 library for social login. The main task of the Flask application is to control read and write access to the storage back-end, handle software logic through services, and manage different resource routes for the front-end. User information and access rules are persisted in a SQLite database interfaced by an object-relation mapping 18 . The Flask application also pre-mashes some of the replies to make it easier for the front-end to quickly access data, and it keeps in memory frequent queries, mainly for tree comparison in the teaching mode, in order to lower the strain on the Grew server. Note that the Grew API provides CoNLL-U data that the Flask application only passes on to the front-end for visualization. Only meta-information such as the user name and time-stamping is accessed and modified at this stage. Hence, the Flask application represents the Model and Controller in the MVC architecture while the Grew server represents the Data Access Objects related to trees from the Model.

Front-end. Lastly, Arborator-Grew's front-end (Figure 1 shows a screenshot) is written using a Javascript based framework named VueJS19 , which facilitates the development of reactive, modular, and flexible front-end user interfaces, and enjoys rising popularity among web developers. In particular, the Arborator-Grew front-end makes the View part of the MVC independent from the back-end. This facilitates the development of mobile or desktop versions because the same base code can be automatically translated using Node ecosystem packages such as Electron and Cordova. The actual dependency graph is now drawn via the Snap20 SVG library. The new tree-drawing algorithm has more configuration options. In particular, trees can now take up different heights depending on the complexity of the tree, in order to avoid labels placed too close to one another. Arborator-Grew also adapts better to UD's syntactic relation system that consists of "universal" relations followed by language-specific sub-relations, such as aux:pass. SUD, the Surface-Syntactic version of UD (Gerdes et al., 2019a), further separates deep-syntactic relations, which can give relations such as comp:obj@x. The combinatorics of universal and other relation names makes it inconvenient to provide the annotator with an exhaustive list of all possible combinations, and Arborator Grew can be configured to show separate choices of universal and secondary relations, see Figure 3.

New Features in Arborator-Grew

The rework of Arborator combined with Grew results in novel ways to build new treebanks and enhance existing annotations. In this section, we focus on two main aspects: classroom collaborative treebank creation (Section 4.1.) and error mining inside an existing treebank using queries (Section 4.2.).

Class Sourcing

One of the key features of Arborator is collaborative annotation, thus controlling access to the resources must be well-organized. The legacy Arborator was laid out for separate instances on separate servers and it was not designed for multiple projects on the same server with different administrators and different people teaching separate classes.

In the new Arborator-Grew, we distinguish the following roles:

1. Guests: A project can be public or private. If it is public, guests can browse and query, but not modify the treebank.

2. Annotators: Each sample has a list of annotators and validators. Annotators can browse and modify the treebank (modify in the sense that a modified tree is saved under their name). They can keep track of their progress by marking trees and whole samples as "ok".

Annotators can be denied access to other annotators' trees, in order to insure independent annotation allowing the computation of inter-annotator agreement.

3. Validators of a sample can see all the trees and choose the correct version if the annotators disagree.

4. Administrators are assigned during the project creation process. They can change the status of the project, and admit and assign other users (administrators, validators, and annotators). They can also upload new samples to the project, create exercises, and determine the POS and relational tagsets.

5. SuperAdmins are determined during the installation of Arborator-Grew, hence they are administrators for the whole system. They can create projects and assign project administrators.

This configuration allows for Class Sourcing of the treebank creation, which is a way to merge treebank creation and teaching of academic students, interns, or colleagues willing to learn about syntax. The precision that can be obtained in the setting of class-sourcing has been analyzed in [START_REF] Gerdes | Collaborative dependency annotation[END_REF], and the actual treebank creation has been demonstrated with the GUM Corpus [START_REF] Zeldes | The gum corpus: creating multilayer resources in the classroom[END_REF], using the legacy version of Arborator for the syntactic component of the corpus.

Annotating actual texts is a great way of studying the syntax of the students' mother tongues as well as of foreign languages where the students have a high degree of proficiency. Arborator, right from its legacy version, was conceived to be used in the classroom, in particular for undergraduate and graduate students.

Exercises modes. Beyond the simple exploration of an existing treebank by means of searches and in-class discussion of various structures, Arborator-Grew allows the configuration of exercises. Exercises have four modes: graphical feedback (the student can click on a "check" button and wrong categories or relations are marked in red), percentage (the student receives a feedback as a percentage of correct categories and relations, without the indication of where the errors are localized in the sentence), teacher visible (the students can see the reference tree, but cannot modify it directly -they have to redraw the tree from an empty annotation), and no feedback where only the teacher can receive the student's score. Teachers need to have an administrator status in the project in order to set up an exercise. For the sample to be used as an exercise, they simply have to choose an exercise mode and determine a reference tree per sentence. Then, the reference tree will be used to provide students with the desired feedback and finally, to compute the students' scores that the teacher can export into a spreadsheet at any time. Arborator's exercises have been tested on various levels and in various countries, and the feedback has been positive from the teachers as well as from students. Syntax exercises seem to feel more like a computer game, in particular the percentage exercise mode, where completist students try hard to reach a 100% score.

Treebank construction in the classroom. Training is an essential first step to class-sourcing, and one can go one step further with a class of students pre-trained on a set of exercises: They can be asked to annotate samples where no reference annotation exists. Depending on the number, the level, and the syntactic and linguistic proficiency of the students, various setups have been tried out. In small groups of graduate students, with interns employed for the task, and among colleagues that want to develop a treebank, the most common configuration is that we provide a first draft of an annotation guide, and we assign a sample to one or two annotators. They annotate the sample and note difficulties that are discussed in a group meeting. Together, the annotation guidelines are updated and specified in order to answer the questions the annotators had.

In the setting of a larger undergraduate class, samples can be distributed to larger sets of students, which allows to compute trees that obtain the highest "votes" by the students in a ROVER-like fashion [START_REF] Fiscus | A post-processing system to yield reduced word error rates: Recognizer output voting error reduction (rover)[END_REF]. The evaluation of the students has then to be done manually, for example by randomly sampling a few of the student's trees.

To automatize this step and in order to avoid students that have been assigned the same sample working together, we use a script (not yet included into Arborator) that combines reference sentences from a gold-standard treebank with yet to be annotated pre-parsed sentences. Optionally, errors can be inserted in both types of trees, using the parser's confusion matrix to make "plausible" errors. For each student an individual set of sentences is prepared, that is given as an assignment. Then, scores can be automatically computed for the student evaluation, but also for a better ROVER vote, where the students' grades are used as a confidence score in the computation [START_REF] Gerdes | Collaborative dependency annotation[END_REF].

Error Mining

Most treebank developers have already stumbled upon errors in their own, or other people's, treebanks while browsing through the trees. If time allows, the tree has then to be looked up in the latest version of the samples, corrected, and uploaded as a new version. It would be better if the annotator could then check whether similar trees have a similar error. These steps have been quite cumbersome up to now, and this difficulty is the central motivation for the merging of Arborator and Grew.

Pattern matching. The central feature of Grew-match is precisely the query of syntactic patterns in a treebank project. Grew has its own query language which is easy to learn and very powerful. This query language is welldocumented 21 and a tutorial is made available on the Grewmatch site 22 . The pattern matching system can match subtrees based on forms, lemmas, part-of-speech tags, presence of a morphological feature, value of the morphological feature, incoming and outgoing dependencies (typed or not, enhanced or syntactic), linear order between nodes and any combination of these features, which can result in very complex queries (see an example of a more complex query in Figure 4). It can also filter out these results based on negative patterns (patterns that must not appear in the graph).

The nodes that match the pattern are then highlighted in the trees on the results page. See an example of a query on comp:aux relations 23 in the SUD treebank of Old French in Figure 5. But once the faulty tree has been found, it cannot be modified inside Grew. The next logical step was to inte- grate Grew as a perfect querying system inside Arborator, thus allowing treebank creators and users to easily find syntactic patterns inside their projects and to directly correct them. In Arborator-Grew, it is now possible to open the Grew-match component (see Figure 6) in a project or in a sample. The result page, containing all the matching trees, possibly from different samples, can then directly be edited and saved.

Clustering the results. Grew also has the ability to cluster the results of a query based on one or several features, thus making it easier to quickly sort through the results. For example, one could look for all the verbs with an object and cluster them based on the part-of-speech of the object. This functionality can be used to build a relation

Distribution

Arborator-Grew is currently accessible through an early version, thus we keep two separate access paths. One for the legacy Arborator24 , another for the new Arborator-Grew25 . The latter follows the same principle as the former and is distributed in two versions:

• Aborator Draft: a simple visualisation Javascript standalone plugin which aims to replace Arborator Quick for a faster visualization tool efficient for large treebanks. Available freely with no account or set up required.

• Arborator-Grew: the new server version of Arborator merged with Grew functionalities. It uses an enhanced version of Arborator Draft to handle tree visualization and graphical editing.

In fine we aim at completely replacing the legacy Arborator with the new one. Legacy and reworked Arborator softwares are licenced under the GNU Affero General Public License v3.0 while Grew server is licence under the CeCILL Licence v2.126 .

Future Work

The development of Arborator-Grew is currently under heavy development, and we are planning on integrating other features that have been suggested by colleagues working on treebank development.

In particular, we are performing tests to switch the ongoing Naija 27 and Old French 28 treebank development projects to the new Arborator-Grew. 27 NaijaSynCor is a French national project on treebank development of spoken Naija, the Nigerian Pidgin-Créole with about 100 million speakers [START_REF] Caron | A surface-syntactic ud treebank for naija[END_REF] 28 Profiterole is another French national project for the diachronic treebank development of Old French [START_REF] Regnault | Challenges of language change and variation: towards an extended treebank of medieval french[END_REF] It will also be possible to integrate Grew rewriting grammars in Arborator-Grew where changes are made across a sample or a whole project. This would allow the parallel development of two versions of a treebank which are transferable into one another by rewriting rules. For the development of SUD and UD grammars, where Grew rewriting grammars exist in both directions, annotators can then choose in which view they would like to annotate and propose corrections -the other treebank version being just a click away. One point that has become very apparent is the need for a better handling of versioning, to allow treebank developers to return to previous states of the annotation, should they need it. The graphical Javascript editor has an undo functionality that does not persist on reload of the editing page. In order to integrate versioning we plan to rely on the GitHub social login integration: A user logged in via GitHub will be able to commit and push a version to his or her GitHub repository. Reverting to previous versions can then be done directly on the GitHub page, making use of the convenient GitHub user interface and also possibly external tools that the user may want to apply to the CoNLL files. At any point, the user will be able to pull the latest version from the repository into Arborator-Grew. To facilitate error-mining and treebank validation, we are also planning to integrate a validation script that would describe forbidden patterns. This practice has now become part of the Universal Dependencies project, where all treebanks must pass through a validation script to be accepted in the new releases that occur every 6 months, so as to maintain the overall quality of the annotation. Finally, thanks to the modular architecture of Arborator-Grew with the separation of the front-end from the two (Flask and Grew) REST server engines, laid out in Section 3., it is straightforward to create a mobile version of the front-end part, sharing most of the features with the current desktop version.

Figure 1 :

 1 Figure1: A screenshot of the user interface that gives access to the different annotations of a sentence, with one dependency tree per user. The sentence is drawn from the ongoing treebank annotation project of spoken Naija (Nigerian Pidgin-Créole). The transcribed sentence has a so-called macro-syntactic markup instead of standard punctuation and can roughly be translated by I really like maize dumplings.

Figure 2 :

 2 Figure 2: Software architecture of Arborator-Grew

Figure 3 :

 3 Figure 3: Interface for the relation selection for a SUD project configuration.

Figure 4 :

 4 Figure 4: Pattern to look for potential errors on verbs without subjects

  table which summarizes all dependencies within a project, based on the part-of-speech of the governor and dependent. Having this relation table easily accessible is a great way to look for rare structures and potential errors inside a treebank, and to get an overview of the existing structures. In Arborator-Grew, the user can open this table (see Figure7) and access directly the trees that match the pattern to see if the analysis is correct, and update it if they find an error. Work on this distributional relation table can easily be integrated in a teaching context where each student has to check a different set of relations. Grew also allows to directly modify samples and whole treebanks by means of a graph rewriting grammar. Access to this feature is planned but not yet realized in Arborator-Grew, as it requires good versioning and feedback in order to avoid the intrusion of unforeseen errors, see Section 6.

In the simple sense of being rare or in the sense of not corresponding to the prediction of a parser that has been trained on the rest of the corpus.

This can by realized by means of a set of correct target trees that have to be attained by the conversion grammar.

http://grew.fr

https://github.com/rug-compling/ conllu-viewer

https://d3js.org/

https://github.com/Arborator/ arborator-draft

https://arborator.github.io/live.html

https://arborator.ilpga.fr/q.cgi

http://match.grew.fr

http://www.tal.univ-paris3.fr/trameur/

https://palletsprojects.com/p/flask/

https://authomatic.github.io/authomatic/ For the moment we use Google and GitHub social login, and with Authomatic it is easy to add further social logins to the system. The social login is an important progress for Arborator, as caring for login problems of classes of up to a 100 students can take up considerable time for the teacher.

We used the ORM framework SQLAlchemy: https:// www.sqlalchemy.org/

We used https://vuejs.org/ associated to multiple components mostly coming from the Quasar VueJs Framework: https://quasar.dev/

http://snapsvg.io/ Snap is also a faster and lighter redevelopment of Raphael.js by the same authors.

https://github.com/Arborator/ arborator-server

https://arborator.github.io/ arboratorgrew.html

http://cecill.info/licences/Licence_ CeCILL_V2-en.html

Acknowledgements

This work was made possible thanks to the NaijaSynCor French project ANR-16-CE27-0007 (2017-2020) directed by Bernard Caron. The Grew API is hosted in the CPER LCHN (Contrat de Plan État-Région -Langues, Connaissances et Humanités Numériques) infrastructure.


Marco Bruschi

Laure Garnier

Elouan Cleroux

Alicia Giordano

Anaïs Michael Dumas

Flore Bardet

Thomas Kergrohen

Stanislas Quesada

Pierre Cesses

Michael Weber

Anaïs Flore Bardet

François Gerbe
email: francois.gerbe@igf.cnrs.fr

Philippe Jay
email: philippe.jay@igf.cnrs.fr

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5 + Intestinal Stem Cells

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Although colorectal cancer (CRC) remains one of the leading causes of cancer-related death in developed countries [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF], the mechanisms involved in its initiation remain only partially understood. The vast majority (80-90%) of CRCs is initiated by the constitutive activation of the Wnt pathway due to inactivating mutations in the Adenomatous polyposis coli (APC) tumor suppressor gene. Mouse models with germline or inducible Apc deletion recapitulate the early stages of tumorigenesis [START_REF] Su | Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene[END_REF][START_REF] Colnot | Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers[END_REF]. In such models, adenomatous growth occurs upon bi-allelic mutation of Apc in the stem cell compartment, but not in differentiated cells, which are short lived, highlighting the key role of stem cells in tumorigenesis [START_REF] Barker | Crypt stem cells as the cells-of-origin of intestinal cancer[END_REF]. Intestinal stem cells are intercalated between post-mitotic Paneth cells, which constitute the stem cell niche at the base of crypts [START_REF] Barker | Tissue-resident adult stem cell populations of rapidly selfrenewing organs[END_REF]. Apc loss-of-function in those cells causes the formation of hypertrofic/hyperproliferative intestinal crypts with impaired epithelial organization [START_REF] Sansom | Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration[END_REF][START_REF] Andreu | Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine[END_REF]. In both humans and mice, benign adenomas then progress toward malignant stages through the sequential accumulation of further genetic alterations [START_REF] Fearon | A genetic model for colorectal tumorigenesis[END_REF][START_REF] Drost | Sequential cancer mutations in cultured human intestinal stem cells[END_REF][START_REF] Matano | Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids[END_REF]. Beside those genetic alterations, the analysis of epigenetic profiles in CRC samples has initially revealed important remodelling of the DNA methylation profiles, distinguishing tumor tissues from their surrounding nontumoral mucosa [START_REF] Feinberg | Hypomethylation distinguishes genes of some human cancers from their normal counterparts[END_REF]. Indeed, cancer cells display a general genomic hypomethylation, although genomic regions associated with tumor suppressors are frequently hypermethylated, which leads to their long-term transcriptional silencing [START_REF] Kulis | DNA methylation and cancer[END_REF]. Recently, part of the alterations associated with CRC were shown to be already present in the intestinal adenomas found in Apc Min mice, as compared to the surrounding non-transformed tissue [START_REF] Grimm | DNA-methylome analysis of mouse intestinal adenoma identifies a tumour-specific signature that is partly conserved in human colon cancer[END_REF]. However, the extent and dynamics of DNA methylation changes occurring during early tumorigenesis, as well as whether they functionally contribute to tumor initiation, remain unclear. De novo patterns of methylation are established in unmethylated DNA regions by methyltransferases DNMT3A and DNMT3B, and then maintained through cell divisions by DNMT1 [START_REF] Bestor | The DNA methyltransferases of mammals[END_REF]. Importantly, de novo methyltransferases are preferentially expressed in the epithelial proliferative crypt compartment and overexpressed in tumor samples [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF], and their activity was reported to promote adenoma formation through the methylation of specific loci [START_REF] Lin | Suppression of intestinal neoplasia by deletion of Dnmt3b[END_REF][START_REF] Weis | Inhibition of intestinal tumor formation by deletion of the DNA methyltransferase 3a[END_REF][START_REF] Linhart | Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing[END_REF]. Importantly though, all these comparisons have been based on heterogenous biological samples (i.e. cancer samples at various stages vs. non-transformed mucosa) in which the proportions of the different cell types are significantly altered, whereas transcriptomic programs and DNA methylation profiles can vary extensively during cell differentiation, including in the intestinal epithelium [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Jackson | Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells[END_REF][START_REF] Smith | DNA methylation: roles in mammalian development[END_REF][START_REF] Meissner | Genome-scale DNA methylation maps of pluripotent and differentiated cells[END_REF][START_REF] Ji | Comprehensive methylome map of lineage commitment from haematopoietic progenitors[END_REF][START_REF] Bock | DNA methylation dynamics during in vivo differentiation of blood and skin stem cells[END_REF][START_REF] Cortese | Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse[END_REF][START_REF] Sheaffer | DNA methylation is required for the control of stem cell differentiation in the small intestine[END_REF].

Here, we investigated the earliest consequences of the loss of Apc function specifically in the predominant tumor cells-of-origin expressing the Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) CBC stem cell marker, early after the induction of Apc deletion. We then exploited the organoid culture system to dissect further the functional role of DNA methylation variations during the early tumorigenic process.

Materials and Methods

Extended experimental procedures are described in the Supplemental Information.

Animal strains and procedures. All animal experiments were approved by the French Agriculture and Forestry Ministry. All the mice were on a C57BL/6/J genetic background and maintained in an SPF animal facility.

Flow cytometry, fluorescence-associated cell sorting and cell cycle analyses. Single cell suspensions of the most proximal 1/3 of freshly isolated small intestine were used for the cell cycle analysis or FACS sorting of GFP + cells directly in RLT+ lysis buffer (Qiagen) for subsequent gDNA/RNA extraction, using a FACSAria (Becton Dickinson). Flow cytometry analyses were performed by using the FlowJo software (FlowJo LLC).

Organoid formation assay and organotypic culture methods. Culture of organotypic structures from Lgr5-GFP + cells or intestinal crypts was performed as previously described [START_REF] Sato | Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[END_REF].

To induce the Cre-mediated recombination of Apc in vitro, cells were cultured during 3 days in a medium supplemented with 4-OH-tamoxifen (200 nM) resuspended in ethanol. To inhibit the activity of DNA methyltransferases, medium was supplemented with Nanaomycin-A (5μM) reconstituted in DMSO or 5-Azacytidine (200 nM) (Sigma-Aldrich) administered at the same time than 4-OH-tamoxifen treatment and then maintained all along the culture. Lentiviralmediated transduction and antibiotic selection were performed as previously described [START_REF] Onuma | Genetic reconstitution of tumorigenesis in primary intestinal cells[END_REF], with minor modifications. Experimental procedures for the evaluation of growth kinetics, clonogenic potential and responsiveness to R-spondin depletion or BMP stimulation are detailed in the supplemental information. gDNA and RNA methods. DNA and RNA of FACS-sorted eGFP + cell and intestinal organoids were isolated using the Allprep DNA/RNA Micro (GFP + cells) or Mini (organoids) kit (Qiagen) according to the manufacturer's instructions and used for next generation sequencing, RT-PCR, RT-qPCR and McrBC-qPCR methylation assay.

Data Access. The accession number for the sequencing data reported in this work is GenBank:

GSE123006

Fluorescent immunohistochemistry on paraffin-embedded tissue and organotypic cultures. Tissue dissection and immunohistochemistry on 5-μm-thick sections of paraffinembedded tissue and organotypic cultures were performed essentially as described previously [START_REF] Gerbe | Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium[END_REF] . All experiments were performed on formalin-fixed tissues with epitope retrieval in boiling 10 mM sodium citrate (pH 6.4).

Results

Apc loss-of-function leads to the expansion of the Lgr5 + stem cell compartment without increasing the rate of stem cell division.

To investigate the cellular and molecular dynamics of the ISC compartment during early tumorigenesis, we used the Lgr5-Cre ERT2 -ires-eGFP mouse line that allows specific identification and genetic manipulation of the intestinal CBC stem cell population [START_REF] Barker | Identification of stem cells in small intestine and colon by marker gene Lgr5[END_REF]. In this model, the variegated expression of the eGFP and the Cre ERT2 recombinase leads to two distinct populations of intestinal crypts, expressing or not the knocked-in allele under the control of the Lgr5 promoter. When in combination with Apc LoxP/+ or Apc LoxP/LoxP alleles, the administration of tamoxifen results in deletion of one or both Apc alleles in Lgr5-expressing ISCs. Such recombined ISCs will be called hereafter Apc HET and Apc KO , respectively, whereas nonrecombined ICSs will be named Apc WT . As early as fifteen days after tamoxifen administration, the loss of a single Apc allele did not produce significant changes in the size of the eGFP + stem/progenitor cell compartment, whereas the biallelic loss, resulting in Wnt activation in most of the GFP-expressing intestinal crypts, produced a mean 9-fold expansion of eGFP + cells as compared to Apc WT controls (Fig. 1A,B). Consistently with this observation, a clonogenic assay performed by seeding FACS-sorted eGFP + cells led to the development of significantly more multicellular organotypic structures from Apc KO eGFP + cells as compared to Apc WT eGFP + cells, thus confirming the increased proportion of eGFP + cells endowed with stem ability in Apc KO crypts. (Fig. 1C). Indeed, we observed an increase in the number of cells expressing the bona fide ISC marker Olfm4 in Apc KO crypts compared to their Apc WT counterparts (Fig. 1D), as well as cells expressing Sox9 (Fig. 1E), a marker shared by ISCs and Paneth cells in the intestinal epithelium. Moreover, quantification of eGFP + ISCs on intestinal sections using the very restrictive anatomic criterion of a direct contact with the Lyzozyme-expressing Paneth cell niche denoted an accumulation of ISCs following Apc deletion, associated with the concomitant increase of Paneth cell numbers in Apc KO crypts (Supplementary Fig. S1 A,B). Together, these results indicate that Apc inactivation induces a rapid expansion of the ISC compartment.

The Wnt signaling pathway is considered as a major driving force of intestinal epithelial proliferation. Accordingly, deletion of its transcriptional effector Tcf7l2/Tcf4 causes proliferation arrest whereas aberrant constitutive activation of the Wnt pathway following Apc loss-of-function results in the accumulation of proliferating cells and formation of hypertrophic crypts [START_REF] Korinek | Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4[END_REF][START_REF] Van Es | A Critical Role for the Wnt Effector Tcf4 in Adult Intestinal Homeostatic Self-Renewal[END_REF]. As expected, Apc-deficient ISCs gave rise to crypts with increased numbers of actively proliferative BrdU + cells (Fig. 1F-G). To evaluate the direct contribution of ISCs proliferation in the expansion of Apc-deficient crypts, we assessed the cell cycle status of ISCs following Apc deletion. Surprisingly, the incorporation of BrdU after a pulse of 2 hours (Fig. 1F) indicated a slight but significant reduction of BrdU + eGFP + cells upon deletion of Apc (Fig. 1H), instead of an increased proliferation of Apc KO ISCs. We confirmed that Apc deletion is not directly linked to increased proliferation rates in ICSs by flow cytometry quantification of the proportion of eGFP + cells found in the S phase using propidium iodide staining of cellular DNA (Fig. 1I). In addition, no significant difference was found between numbers of Apc KO and Apc WT BrdU + cells when we only considered ISCs, defined as eGFP + cells in direct contact with Lyz + Paneth cells (Fig. 1J). Of note, the proportion of eGFP + cells expressing markers of terminal differentiation into Paneth (Lyz + ), enteroendocrine (Chga + ) or tuft (Dclk1 + ) postmitotic cell lineages was slight (<3%) for both genotypes (Supplementary Fig. S1C-F).

Moreover, analysis of the post-mitotic tuft cell surface marker Siglec-F (Supplementary Fig. S1G), revealed a physiological representation of tuft cells (∼0.4%, Supplementary Fig. S1H) within the epithelial samples, with minimal and comparable co-localization of this marker with eGFP in Apc WT and Apc KO sorted cells (Supplementary Fig. S1I). Together, these data rule out the possibility that the increased proportion of non-dividing cells in the total Lgr5-GFP + Apc KO compartment may reflect a larger proportion of terminally differentiated cells with residual eGFP protein with our cell-sorting strategy. We thus concluded that Apc deletion causes the accumulation of eGFP + cells with ISC features in Apc KO intestinal crypts, which cannot be ascribed to an augmented rate of cell division in ISCs.

ISC accumulation following Apc loss-of-function relies on an impaired molecular commitment towards differentiation.

To identify the mechanisms underlying the rapid ISC accumulation following Apc loss-offunction, we next investigated the molecular consequences associated with Apc disruption, specifically in eGFP + cells comprising ISCs and their most direct progeny in the transitamplifying (TA) crypt compartment. The comparison of gene expression profiles of Apc WT and Apc HET FACS-sorted eGFP + cells by RNA-seq showed that the first genetic hit in the Apc locus, i.e. the loss of a single Apc allele (Apc HET ), significantly altered the expression of more than 400 transcripts (FDR-adjusted p<0.01) as compared to Apc WT eGFP + cells (Fig. 2A, results listed in table S1).

Quantification of the residual loxP-flanked exon-15 showed an average 83% reduction of this exon in cells from Apc LoxP/LoxP ;Lgr5-Cre ERT2 (Apc KO ) mice (Supplementary Fig. S2A), therefore confirming the efficient loss of the full-lenght Apc transcript in the majority of sorted cells in our experimental setting. Remarkably, such a biallelic disruption of Apc, resulting in constitutive Wnt activation, caused dysregulation of approximately 5000 transcripts (Fig. 2A) in Apc KO eGFP + cells, highlighting a dramatic rewiring of gene expression in ISCs and their immediate progeny associated with the constitutive activation of Wnt signaling. Remarkably, a subset of genes was exclusively expressed in either one or the other genetic condition. Among these markers, the endoderm-specific SRY-box 17 (Sox17) transcription factor (Supplementary Fig. S2B) was undetectable in both Apc WT and Apc HET eGFP + cells and expressed in Apc KO eGFP + cells. Immunohistochemical detection confirmed that this gene is ectopically expressed by scattered Apc KO eGFP + cells within the ISC zone as early as day 6 post-recombination, and then intensely expressed in the transit amplification compartment of Apc KO crypts (Supplementary Fig. S2C).

To gain further insight into the changes occurring upon the disruption of Apc in ISCs we performed gene set enrichment analyses (GSEA). The analyses on the representation of wide collections of gene sets related to chemical and genetic perturbations (c2.cgp.v6.1) and KEGG pathways (c2.cp.kegg.v6.1.symbols) in Apc WT and Apc KO gene expression profiles revealed an overlap with the results from other studies on the outcome of Apc inactivation in mouse models of early tumorigenesis and human adenomas (reported in Tables S2,S3; examples of sets related to early tumorigenesis are shown in Supplementary Fig. S2G). Consistently with Apc abrogation, GSEA interrogation of our data confirmed an exacerbated Wnt signaling in Apc KO eGFP + cells, with an increased expression (negative correlation with Apc WT gene expression, NES WT vs KO=-1.57) of the set of genes representing β-catenin transcriptional targets (Fig. 2B). In line with the cell cycle analyses of ISCs from Apc WT or Apc KO mice, indicating absence of increased cell proliferation (Fig. 1D-F), no preferential enrichment was found in Apc KO or Apc WT eGFP + cell profiles for the set of genes associated with progression through the cell cycle (Fig. 2B). Concomitantly, the gene signature associated with ISC identity (32) (Table S4) showed a significant enrichment (NES WT vs KO=-1.6) in the transcriptomic profiles of Apc KO eGFP + cells (Fig 2B). Conversely, the signature associated with intestinal epithelial differentiation [START_REF] Merlos-Suárez | The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse[END_REF] was depleted in the gene expression profiles associated with Apc KO cells (Fig 2B, NES WT vs KO =2.95), whereas no significant enrichment was detected for the set of genes associated with progenitor cells from the transit-amplification compartment in one or the other conditions (Fig. 2B). Indeed, a subset of bona fide ISC markers, including Lgr5, Axin2, Musashi RNA Binding Protein 1 (Msi1), and others, were up-regulated in Apc KO eGFP + cells (Fig. 2C), whereas early markers of intestinal epithelial commitment (Atoh1, Spdef) and differentiation into enterocytes (Elf3, Krt20), goblet (Cdx2, Muc2), enteroendocrine (Insm1, Chga), tuft (Pou2f3, Dclk1) and M (Spi-B) epithelial cell lineages were down-regulated (Fig. 2D). The most important exception was represented by markers associated with Paneth cell maturation, which is known to rely on Wnt signaling [START_REF] Andreu | Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine[END_REF][START_REF] Van Es | Wnt signalling induces maturation of Paneth cells in intestinal crypts[END_REF]. Indeed, we could observe the expression of transcription factors involved in lineage commitment in eGFP + CBC cells and, more frequently, in eGFP + cells immediately above the CBC cell zone in control mice (Fig. 2E-H). We found Apc WT -eGFP + cells clearly expressing markers involved in lineage commitment such as Elf3 (an enterocyte specification marker [START_REF] Ng | Inactivation of the transcription factor Elf3 in mice results in dysmorphogenesis and altered differentiation of intestinal epithelium[END_REF]) and Cdx2 (a pan-epithelial marker involved in enteroendocrine cell commitment [START_REF] Roman | Distinct Processes and Transcriptional Targets Underlie CDX2 Requirements in Intestinal Stem Cells and Differentiated Villus Cells[END_REF]) or, less frequently, Insm1 (enteroendocrine cells (37)) or Pou2f3 (tuft cells specification [START_REF] Gerbe | Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites[END_REF]). Overall, the expression of these markers in the self-renewal compartment supports the early lineage specification occurring in ISCs and their daughter TA cells, which is impaired by the inactivation of the gate-keeper Apc gene.

We then decided to examine the possible involvement in these findings of the transcriptional regulator Myc, an important Wnt target gene previously described as a key mediator of the epithelial phenotype consequent to Apc loss [START_REF] Bettess | c-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium[END_REF][START_REF] Sansom | Myc deletion rescues Apc deficiency in the small intestine[END_REF]. As expected, the size of the Myc + compartment expanded during time following Apc inactivation, as Myc was expressed by the increasing number of cells showing β-catenin translocation in Apc KO crypts compared to Apc WT control crypts (Supplementary Fig. S2D). However, constitutive activation of Wnt signaling in Apc KO Lgr5-GFP cells did not further increase the expression of Myc in individual FACS-sorted stem/progenitor cells (Supplementary Fig. S2E). Together, these observations suggest that the specific effects of Apc loss and exacerbated Wnt signaling found in ISCs do not rely on an altered level of Myc expression. Interestingly, the expression of the Myc paralogue Mycn, which is known to be preferentially expressed by differentiated cells in the intestinal epithelium [START_REF] Bettess | c-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium[END_REF], was significantly reduced in Apc KO cells (Supplementary Fig. S2F). Overall, these results revealed that the primary outcome of the oncogenic Wnt pathway hyper-activation in Apc KO ISCs consists in their impaired commitment toward differentiation, which explains their rapid accumulation in spite of the absence of increased ISC division rate.

Exacerbated Wnt activation is accompanied by rapid and defined alterations in the DNA methylation of the self-renewal compartment.

We then investigated if the impairment in the ability of Apc-deficient ISCs to commit toward differentiation is accompanied by rapid alterations of DNA methylation profiles. Some instructive changes in the DNA methylation patterns have been shown to occur during homeostatic intestinal differentiation [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Sheaffer | DNA methylation is required for the control of stem cell differentiation in the small intestine[END_REF]. Moreover, alterations in the DNA methylation patterns have been reported in advanced stages of CRC and, more recently, in early adenomas [START_REF] Feinberg | Hypomethylation distinguishes genes of some human cancers from their normal counterparts[END_REF][START_REF] Kulis | DNA methylation and cancer[END_REF][START_REF] Grimm | DNA-methylome analysis of mouse intestinal adenoma identifies a tumour-specific signature that is partly conserved in human colon cancer[END_REF][START_REF] Berman | Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains[END_REF][START_REF] Forn | Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression[END_REF]. However, whether these alterations are immediate consequences of the oncogenic loss of Apc function remains unclear. We therefore performed reducedrepresentation bisulfite sequencing (RRBS) on FACS-sorted Apc WT , Apc HET and Apc KO eGFP + cells. Hierarchical clustering of the methylation scores provided fair discrimination of the Apc KO from the Apc WT and Apc HET samples, indicating a specific impact of Apc inactivation on the methylome (Fig. 3A). Overall, RRBS analyses revealed significant discrete rearrangements of the DNA methylation profiles in Apc HET and Apc KO eGFP + cells as compared to Apc WT controls (Fig. 3B). We identified 58 differentially methylated regions (DMRs) in Apc HET eGFP + cells compared to eGFP + cells from control mice, and 790 DMRs in Apc KO eGFP + cells (detailed in Table S5). In line with the notion of a general DNA hypomethylation in CRC samples [START_REF] Feinberg | Hypomethylation distinguishes genes of some human cancers from their normal counterparts[END_REF][START_REF] Kulis | DNA methylation and cancer[END_REF], 75% of the DMRs were hypomethylated in Apc KO cells (Fig. 3C). These DMRs were reproducible between replicates, highlighting the consistent effect exerted by Apc loss on the DNA methylation profiles (Fig. 3D). Both hypo-and hypermethylated DMRs identified by RRBS were generally distant from gene transcription start sites (TSSs), with only 57 out of 790 DMRs located within 2000 bps from the most proximal TSSs (Fig. 3E), and are preferentially located within intronic and inter-genic regions (Supplementary Fig. S3A). This is in accordance with previous findings obtained by comparing the DNA methylation in adenoma samples and in ISCs [START_REF] Forn | Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression[END_REF]. Importantly, the DNA methylation changes occurring 15 days after Apc deletion remained focal and less extensive than the general DNA remodeling described in colon cancer [START_REF] Feinberg | Hypomethylation distinguishes genes of some human cancers from their normal counterparts[END_REF][START_REF] Berman | Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains[END_REF]. For instance, repeated elements belonging to endogenous retrotransposon families, such as intrascisternal-A-particles (IAPs), are severely methylated in normal cells and frequently hypomethylated in cancer cells [START_REF] Howard | Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice[END_REF][START_REF] Bakshi | Retrotransposon-based profiling of mammalian epigenomes: DNA methylation of IAP LTRs in embryonic stem, somatic and cancer cells[END_REF]. However, the methylation of these regions was not affected 15 days following Wnt constitutive activation (Fig. 3F). This result was confirmed experimentally for IAPs by qPCR on genomic DNA fragments obtained after digestion with the McrBC methylation-sensitive restriction enzyme (Supplementary Fig. S3B).

Taken together, these observations indicate that the DNA methylation status of a defined subset of genetic loci changes very rapidly at tumor initiation following loss of Apc function.

To investigate the direct involvement of altered DNA methylation in the impaired ability of ISCs to commit towards differentiation we first interrogated the RRBS profiles for the enrichment of epithelial stem and post-mitotic signatures (Tables S4), by analyzing the methylation of promoter regions (encompassing -1000 bps to +500 bps from the TSS). GSEA results did not show global tendencies in the methylation of the TSSs associated to genes belonging to those sets (Fig. 3G). Even restricting the analysis to the list of TSSs associated with the identified DMRs (Tables S5) did not show preferential hypermethylation of genes associated with post-mitotic commitment (Fig. 3G), therefore indicating that altered DNA methylation of loci directly associated with stemness/maturation does not represent an instructive mechanism for the imbalanced fate program dictated by Apc loss. As ISC identity and commitment are regulated by opposite gradients of Wnt and BMP/TGF-β signaling along the crypts-villus axis (a schematic representation is provided in Supplementary Fig. S3C), we focused our attention on these two signaling pathways. Indeed, secretion of Wnt ligands and BMP/TGF-β inhibitors co-operate to the formation of a stem-permissive niche in both homeostatic crypts and adenomas [START_REF] Vermeulen | Wnt activity defines colon cancer stem cells and is regulated by the microenvironment[END_REF][START_REF] Whissell | The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression[END_REF][START_REF] Qi | BMP restricts stemness of intestinal Lgr5 + stem cells by directly suppressing their signature genes[END_REF]. Thus, forcing BMP inhibition concomitantly to Wnt constitutive activation leads to the expansion, or even to the ectopic formation of an adenoma stem cell compartment [START_REF] Haramis | De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine[END_REF][START_REF] Davis | Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche[END_REF], whereas increasing secretion of BMP ligands along the villus axis was shown to promote epithelial differentiation [START_REF] Whissell | The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression[END_REF]. Combining the data from RRBS and RNA-seq analyses revealed an altered extent of methylation and expression of four loci associated to components of the Wnt pathway (Axin2, Nfatc2, Prkca, Vangl1) and three loci associated with the TGF-β/BMP signaling pathway (Inhbb, Bmp7, Smad6) in Apc KO eGFP + cells (Supplementary Fig. S3D,E). The Smad6 locus, coding for an intracellular inhibitor of the BMP signaling pathway, was used for experimental validation using gDNA and RNA samples from an independent cohort of Apc WT and Apc KO eGFP + cell samples from intestinal epithelia. McrBC-qPCR and RT-PCR analyses confirmed the hypomethylation (Supplementary Fig. S3F) and increased expression (Supplementary Fig. S3G) of Smad6 in Apc KO ISCs.

Overall, these observations suggested that rapid, defined, changes in the methylation patterns of specific loci may be implicated in impaired cell fate decisions at tumor initiation.

Inhibition of de novo DNA methyltransferases partially compensates the consequences of constitutive Wnt signaling after Apc inactivation in intestinal organoids.

We next evaluated the contribution of DNA methylation changes in the altered behaviour of Apc KO ISCs. We first re-assessed the expression patterns of de novo methyltransferases Dnmt3a and Dnmt3b in the normal and Apc KO epithelium. RNAseq data showed no significant differences in the expression of Dnmt3a between Apc WT and Apc KO Lgr5-GFP cells (4A), whereas the expression of Dnmt3b was slightly but significantly increased upon Apc-loss in those cells (4C), probably reflecting ISC accumulation within the compartment. Specific immunostainings confirmed that these enzymes are preferentially expressed in the in normal intestinal crypts, with Dnmt3a signal spanning from ISCs to TA cells at crypt/villus border, whereas Dnmt3b staining was accentuated in ISCs and immediate daughter cells in the normal crypt (Fig 4B and4D, upper panels). Those patterns were clearly expanded upon Apc deletion in transgenic crypts, in accordance with the outgrowth of cells displaying β-catenin translocation (4B, 4D central panels). Furthermore, these enzymes were expressed in virtually all Apc KO epithelial cells in well-developed adenomas from Apc ∆14/+ mice harbouring a germline heterozygous mutation on Apc, and spontaneously developing clonal adenomas from stem cells in which the remaining wild type Apc allele has been mutated or silenced (bottom panels).

Having shown that the dynamics of de novo Dnmt patterns coincide with the expansion of the intestinal self-renewal compartment, we then investigated whether the precocious changes in DNA methylation that we observed in ICSs following constitutive Wnt activation are functionally involved in such an expansion at the expense of homeostatic commitment to differentiation. To this end, we assessed the outcome of de novo methyltransferases inhibition in intestinal organotypic cultures (organoids) derived from the intestinal epithelium of Apc LoxP/LoxP ;VillinCre ERT2 mice. Such organoids are normal (Apc WT ) until they are treated with tamoxifen, which results in the deletion of both Apc alleles (Apc KO ) in all epithelial cells. RT-qPCR quantification confirmed a substantial reduction (>400-fold change) in the LoxP-flanked exon-15 upon treatment with 4-OH-tamoxifen, therefore validating the effective abrogation of the functional full-length Apc transcript (Supplementary Fig. S4A). Indeed, we constantly observed a switch toward a spheroid morphology (Supplementary Fig. S4B), an increased proportion of actively proliferating (Ki67 + ) cells (Supplementary Fig. S4C) consistent with increased global crypt proliferation in mice (see Fig. 1G), overall confirming the Wnt oncogenic activation in Apc KO intestinal organoids. All those phenotypic changes were associated to the accumulation of cells expressing the ISC maker Sox9 (Fig. 4E) in Apc KO organoids, which is consistent with the increased stemness described in the figure 1.

Apc LoxP/LoxP ;VillinCre ERT2 organoids were lentivirally transduced with shRNAs directed against Dnmt3a and Dnmt3b transcripts or, as a control, with lentiviral vectors containing length-paired non-target sequences. Immunohistochemical detection confirmed the reduced amount of nuclear Dnmt3a and Dnmt3b enzymes (Supplementary Fig. S4D-E). The knock-down of the two enzymes did not affect the general rate of proliferation of Apc WT models (total average of 59.9% ± 7.1 vs. 51.2% ± 5.70 of Ki67 + cells in replicates from two NT-control and two doublesh clones respectively, Fig. 4F). As expected, after Apc deletion the general extent of cell proliferation was increased in control organoids expressing non-target shRNAs (total average of 77.1% ± 2.5 in two clones). In sharp contrast, the combined knock-down of Dnmt3a/Dnmt3b reduced the general proliferation of Apc KO organoids to an extent comparable to Apc WT organoids (total average of 53.9% ± 3.4 in two clones, Fig. 4F), supporting the notion of a requirement of de novo methyltransferase function in the phenotype resulting from Apc lossof-function. Compared to Apc KO organoids transduced with non-target shRNA, Apc KO organoids with Dnmt3a/Dnmt3b knock-down also displayed a reduced sphere formation ability in 3/3 replicates when replating was performed by dissociating a number of organoids equivalent to an identical number of cells (Fig. 4G), therefore suggesting a decreased stemness, as defined by the ability of a single cell to proliferate and generate a multicellular organoid, of Apc KO organoid cells with reduced Dnmt3a/b expression. To confirm these findings with an independent approach, we tested the effect of de novo methyltransferases inhibition by treating organoids with the specific Dnmt3b inhibitor Nanaomycin-A, or with the demethylating agent 5-Azacytidine [START_REF] Kuck | Nanaomycin A selectively inhibits DNMT3B and reactivates silenced tumor suppressor genes in human cancer cells[END_REF]. Remarkably, the administration of either inhibitor concomitantly with the deletion of Apc resulted in reduced proliferation rates in three independent organotypic models (Fig. 4H), at extents comparable to those of isogenic Apc WT organoids, in which cell division rate was not affected by the presence of inhibitors, although increased cell-death was observed early after administration of 5-Azacytidine. Moreover, when we examined the expression of Wnt-related genes (Axin2, Nfatc2, Prkca and Vangl1) and BMP-related genes (Smad6, Inhbb and Bmp7) that were differentially methylated and expressed in Apc KO CBC (see figure S3D-E), we found that those genes were also differentially expressed in Apc KO organoids compared to Apc WT controls (figure 4I), thus confirming the relevance of organotypic models in the study of homeostatic disruption upon Apc inactivation. Taken together, these results indicate that the activity of de novo methyltransferases is required for the increase in the proportion of actively proliferating cells able to form growing organoids following Apc loss-of-function.

De novo DNA methyltransferase activity contributes in altering Wnt and BMP signaling driving ISC accumulation upon Apc inactivation.

Next, we sought to test whether de novo methyltransferase activity is implicated in the responsiveness of Apc KO epithelial cells to environmental Wnt and BMP stimuli, regulating the homeostatic choice between self-renewal and differentiation. Intestinal organoids are routinely maintained in presence of R-spondin 1 (Rspo), which cooperates with Wnt signals to define ISC properties [START_REF] Yan | Non-equivalence of Wnt and Rspondin ligands during Lgr5(+) intestinal stem-cell self-renewal[END_REF], and becomes dispensable following constitutive Wnt activation [START_REF] Sato | Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[END_REF][START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF]. Indeed, we observed a loss of spheroid formation ability and reduction of spheroid size in Apc KO organoids with combined Dnmt3a and Dnmt3b knock-down. Strikingly, these effects were further accentuated upon withdrawal of Rspo from the culture medium (Fig. 5A,B). This confirmed the reduction in the sphere-formation ability and proved the recovered dependence to Rspo upon de novo Dnmts knock-down in Apc KO organoids. We then assessed the responsiveness of Apc KO organoids to the stimuli that modulate the BMP signaling. Stimulation of organoid cultures with recombinant BMP2 after withdrawal of the BMP inhibitor Noggin from the medium dramatically increased the formation of crypt-like structures in Dnmt3a/Dnmt3b shRNA-expressing Apc KO organoids as compared to the non-target shRNA control Apc KO organoids (Fig. 5C). This indicated a restauration of the ability of epithelial cells to morphologically self-organize the formation of distinct crypt and differentiated compartments. Indeed, control individually seeded Apc KO cells were largely more prone to form spheroids than Nanaomycin-A-treated cells in the presence of rBMP2 stimulation (Fig. 5D), supporting the recovered responsiveness to differentiation stimuli orchestrated by BMP signaling upon de novo Dnmt inhibition. As in the case of FACS-sorted eGFP + cells from Apc WT and Apc KO crypts (Supplementary Fig. S3E), Apc KO organoid cultures displayed an upregulation of the BMP inhibitor Smad6 mRNA as compared to Apc WT organoids. Moreover, treatment with 5-azacytidine reduced the expression level of this gene in 2/2 independent Apc KO organoid cultures (Supplementary Fig. S5A) and Nanaomycin-A exerted comparable effect in 2/3 independent cultures (Supplementary Fig. S5B). To further elucidate the responsiveness to BMP signaling as involved in the Apc phenotype, we therefore decided to test the implication of the Alk-pSmad axis physiologically regulated by Smad6 during homeostasis. Strikingly, BMP inhibition resulting from the addition of the Alk-pSmad inhibitor LDN193189 abolished the rescue exerted by Dnmt3b inhibition mediated by Nanaomycin-A treatment, as demonstrated by the size (Fig. 5E-F, quantification from two independent models) and proliferative rate (Supplementary Fig S5C ) of spheroids compared to controls and Nanaomycin-A treated organoids. Together, these results confirm that de novo DNA methylation occurring upon Apc inactivation is critically involved in the increase in the proportion of actively proliferating cells within the intestinal epithelium by dictating a reduced responsiveness of ISCs to the different stimuli regulating the proliferation-to-differentiation balance (summarized in Fig. 5G).

Discussion

We investigated the early consequences of oncogenic Apc loss-of-function, the most common genetic initiating event in CRC, on the behavior of ISCs. Importantly, we show that this tumor suppressor does not operate by negatively controlling the rate of cell division of intestinal stem cells but rather represents a critical regulator of the ability of ISCs to engage towards differentiation. In line with the rapid accumulation of ISCs observed early upon Apc deletion, the analysis of ISC gene expression profiles clearly confirmed the impaired ability of stem cells and their progeny to commit toward homeostatic differentiation, with a reduced expression of several markers for the different post-mitotic cell lineages. Recently, single cell RNA-seq analyses from purified Lgr5 + cells revealed a relatively homogeneous stem cell population mixed with rare secretory cells, likely reflecting early fate commitment decision in some progenitor cells [START_REF] Grün | Single-cell messenger RNA sequencing reveals rare intestinal cell types[END_REF]. Indeed, we show that some markers of lineage commitment are expressed in the self-renewal compartment, supporting a role of Apc in controlling early homeostatic fate decision in ISCs. Moreover, transcriptomic profiles provided us with a list of early markers of Wnt oncogenic activation that are exclusively expressed in Apc KO cells, as we show in the case of Sox17, which is undetectable in the Apc WT epithelium, including in ISCs, and could be used to trace aberrant Wnt activation.

Of note, the over-activation of Wnt signaling in ISCs led to an increased number of Mycexpressing cells without increasing the cellular expression of Myc, a key mediator of the phenotype associated with Apc loss-of-function in intestinal epithelium [START_REF] Sansom | Myc deletion rescues Apc deficiency in the small intestine[END_REF], whose expression coincides with β-catenin signaling in both normal and pre-tumoral crypts. This finding allowed us to conclude that Myc is expressed in cells with active Wnt signaling and, reminiscently to its role during development [START_REF] Bettess | c-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium[END_REF], co-operates to the establishment of a crypt-like phenotype in the Apc KO epithelium, but its role in ISCs is not exacerbated upon oncogenic Wnt overactivation.

Alterations in DNA methylation have extensively been proven in advanced tumor stages, and were suspected to play a pivotal role during intestinal tumorigenesis [START_REF] Feinberg | Alterations in DNA methylation in human colon neoplasia[END_REF][START_REF] Timp | Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host[END_REF]. However, the confounding cellular heterogeneity of the tissues used for comparison between tumor and healthy profiles has hampered precise conclusions about its actual contribution to cancer initiation. The RRBS profiling of ISCs and early progenitors provided evidences that the oncogenic Wnt activation does not trigger an immediate extensive remodeling of the DNA methylation landscape in the ISC compartment. Instead, Apc disruption rapidly produces discrete DNA methylation changes, indicating that focal remodeling of DNA methylation profiles initiates as early as the first oncogenic event, i.e. loss of Apc function, before becoming more generalized in later stages of the disease. The characterization of CpG-rich genomic regions methylation showed that these changes are rarely found in the close proximity to gene TSSs. Of note, methylation of non CpG-rich regions, as well as other epigenetic marks, may also contribute to the transcriptional and functional dysregulation observed in Apc KO ISCs. However, we show that genes implicated in the signaling pathways governing intestinal cell fate decisions represent preferential targets for altered DNA methylation during early tumorigenesis. The short timing in our experimental design rules out the possibility that these alterations represent the result of a clonal selection, and rather suggests the existence of a specific program associated with the loss of Apc function and Wnt constitutive activation.

Several studies have reported the impact of DNA methylation in controlling cell differentiation [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Jackson | Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells[END_REF][START_REF] Smith | DNA methylation: roles in mammalian development[END_REF][START_REF] Meissner | Genome-scale DNA methylation maps of pluripotent and differentiated cells[END_REF][START_REF] Ji | Comprehensive methylome map of lineage commitment from haematopoietic progenitors[END_REF][START_REF] Bock | DNA methylation dynamics during in vivo differentiation of blood and skin stem cells[END_REF][START_REF] Cortese | Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse[END_REF][START_REF] Sheaffer | DNA methylation is required for the control of stem cell differentiation in the small intestine[END_REF], and the activity of de novo DNMTs was shown to regulate hematopoietic multipotency and stemness both in homeostasis and cancer [START_REF] Challen | Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells[END_REF]. The specific role of Dnmt3a and Dnmt3b in intestinal homeostasis awaits further elucidation. However, the fact that these factors are expressed in the ISC compartment, together with the expansion of their pattern of expression in early lesions prompted us to investigate their functional implication in ISCs during intestinal tumorigenesis. Importantly, we show that Dnmt3a/Dnmt3b knockdown/inhibition in Apc KO intestinal organotypic cultures reduces the proportion of actively proliferating cells to a homeostatic level comparable with Apc WT organoids, therefore restraining the uncontrolled expansion of Apc KO organoids. Moreover, functional organotypic assays suggest that the activity of de novo methyltransferases may contribute to ISC accumulation by impairing their responsiveness to exogenous stimuli controlling the homeostatic balance between self-renewal and differentiation, such as Wnt and BMP signaling upon Apc inactivation. BMP signaling has been recently shown to play a crucial role in the commitment of Lgr5 + ISCs by repressing the signature associated with stemness without affecting Wnt signaling, therefore preventing ISC outgrowth during homeostasis and regeneration [START_REF] Qi | BMP restricts stemness of intestinal Lgr5 + stem cells by directly suppressing their signature genes[END_REF]. However, we show that Wnt constitutive activation rapidly impairs the responsiveness of ISCs to BMP signals, hence demonstrating an interaction between these pathways in early oncogenesis. More investigation is needed to identify specific and overlapping genomic targets of the two de novo DNMTs in the Lgr5 + compartment both during homeostasis and tumorigenesis, as recently accomplished in hematopoietic and epidermal stem cells [START_REF] Challen | Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells[END_REF][START_REF] Rinaldi | Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis[END_REF]. This question might be addressed by combining conditional Dnmt3(a/b) Flox

and FIGURE 1 .

 1 FIGURE 1. Expansion of the self-renewal compartment consequent toApc lossof-function in Lgr5 + ISCs. A, Representative flow cytometry analyses of GFP content in dissociated intestinal epithelium from Apc +/+ : , Apc LoxP/+ : or Apc LoxP/LoxP : Lgr5-Cre ERT2 -ires-eGFP or control mice 15 days after the initial administration of tamoxifen and B, flow cytometry quantification of the % of eGFP + cells; bars represent mean ± SEM of n= 6 Apc +/+ , 5 Apc LoxP/+ and 7 Apc LoxP/LoxP mice; *p<0.05. C, clone formation ability of GFP + isolated cells quantified as the number of resulting multicellular organoids; bars represent mean ± SEM of n=16 wells seeded with equal number of Apc WT or Apc KO FACS-sorted GFP + cells from an Apc +/+ or Apc LoxP/LoxP mouse; **p<0.01. D, representative immunofluorescence fields of proximal intestines showing the increased proportion of Olfm4 + cells and E, Sox9 + cells in Apc LoxP/LoxP -crypts as compared to control crypts from Apc +/+ ; Lgr5-Cre ERT2 -ires-eGFP mice (scale bar = 10µm) . F, representative immunofluorescence fields of proximal intestines from Apc +/+ or Apc LoxP/LoxP Lgr5-Cre ERT2 -ires-eGFP mice (scale bar = 10µm) used to quantify the %

FIGURE 4 .

 4 FIGURE 4. De novo DNA methylation can rescue the impaired ISC commitment and proliferation in Apc KO mini-guts. A and C, expression of Dnmt3a and Dnmt3b as by RNAseq data and B and D, representative immunofluorescence fields of Apc WT -(top panels) or Apc KO-Lgr5-Cre ERT2 crypts and adenomatous (bottom panels) Apc ∆14/+ intestinal epithelium, showing the pattern of expression of Dnmt3a and Dnmt3b during homeostatic disruption and adenoma formation (scale bar = 10µm). E, representative immunofluorescences showing the expansion of the Sox9 + ISC compartment in Apc KO organoids (scale bar = 10µm). F, proliferation of Apc WT and Apc KO organoids expressing control-or double-ShRNA as the % of Ki67 + cells; bars represent mean ± SEM of integration of multiple organoids from 2 independently-derived clones per condition; n=19 (9 and 10 organoids) control-Apc WT , 27 (12 and 15) double Sh-Apc WT , 21 (6 and 15) control-Apc KO , 16 (6 and 10) double Sh-Apc KO oganoids; ***p<0.001. G, Representative clonogenic assay performed by seeding an equivalent number of organoids corresponding to 40 000 Apc KO cells transduced with NT/NT-or Dnmt3a/b-shRNA. H, proliferation of Apc WT and Apc KO organoids treated with Nanaomycin-A or 5-Azacytidine, expressed as the % of KI67 + cells; bars represent mean ± SEM of integration of multiple organoids from independent organoid models per condition; n= 8 (5 and 3 organoids) untreated control, 7 (4 and 3) 5-Azacytidine-, 7 (4 and 3) Nanaomycin-A-treated Apc WT and 27 (6, 6, 15) untreated, 18 (5, 4, 9) 5-Azacytidine-treated, 15 (4, 5, 6) Nanaomycin-A-treated Apc KO organoids; p-value as determined by t-tests (with Welch's correction when needed), **p<0.01, ***p<0.001. I, RT-PCR products in representative organotypic models per genotype confirming the dynamics of the expression of genes belonging to Wnt (Axin2, Nfatc2, Prkca and Vangl1) and BMP signaling pathways (Smad6, Inhbb and Smad6) in Villin-Cre ERT2 miniguts in response to Apc deletion. Actb expression is presented as an endogenous loading control.

Acknowledgments

This work was supported by ITMO Cancer (Plan Cancer 2009-2013 EPIG201311 to P.J. and M.W.), SIRIC Montpellier Cancer (grant INCa_Inserm_DGOS_12553 to P.J), ARC (SL220110603456 to P.J.), ANR (ANR-14-CE14-0025-01 and ANR-17-CE15-0016-01 to P.J.), INCa (2014-174 and INCA_2018-158 to P.J.), the Labex EpiGenMed (an "Investissements d'avenir" program ANR-10-LABX-12-01 to P.J.), the PJ team is "Equipe Labellisée Ligue contre le Cancer", and the European Research Council (Consolidator grant n°615371 to M.W.). M.B. was supported by Ligue Nationale contre le Cancer. We acknowledge M. Zenati, S. Barahoui and E. Sidot for contribution to the collection of data, J. Pannequin and T. Bouschet for reagents, protocols and advices, C. Perret (Cochin Institute, France) for sharing the Apc-mutated mice, the team of S. Fre (Curie Institute, France) for protocols of organotypic culture, F. Gallardo and D. Greuet in the iExplore facility for maintenance of mouse colonies, and C. Duperray in the Montpellier Ressources Imagerie (MRI) facility.

Apc Flox alleles with the Lgr5-Cre ERT2 -Ires-EGFP model. Overall, our findings establish a critical effector role for DNA methylation in ISCs, at the onset of the tumor phenotype resulting from Apc disruption, and paves the way towards the design of novel strategies suitable to target the tumor stem cell compartment. of G, total crypt BrdU + cells or H, % of BrdU + /GFP + . I, quantification of the flow cytometry analyses on the distribution of GFP + cells in the cell cycle according to the amount of DNA; bars represent mean ± SEM of integration of multiple technical replicates from 3 (7, 7, 5 replicates) Apc +/+ -, 4 (4, 5, 5, 2 replicates) Apc LoxP/+ -or 4 (5 replicates each) Apc LoxP/LoxP -mice respectively; ***p<0.001 J) BrdU + /GFP + cells in physical contact with at least 1 Lyz + Paneth cell per intestinal crypt; nuclei are counterstained with Hoechst; bars for G, H and J represent integration mean ± SEM of n=43 [START_REF] Bestor | The DNA methyltransferases of mammals[END_REF][START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Bestor | The DNA methyltransferases of mammals[END_REF], 45 [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF] and 41 [START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF][START_REF] Feinberg | Hypomethylation distinguishes genes of some human cancers from their normal counterparts[END_REF][START_REF] Kaaij | DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus[END_REF]