Bogdan Logofatu

Michaela Logofatu

Dumitru Dan Burdescu

Doru Ursutiu

Cornel Samoila

RO-i2010, Promoting ICT in Education in Romania

Keywords:

This paper will present the RO-i2010 project based the partnership of three Romanian (RO) universities: University of Bucharest, University of Craiova and University Transilvania of Brasov. The project is aiming to increase the visibility of the RO scientific community and research programs in the field of ICT for education, in order to better correlate and integrate into European and international educational and research programs. The resulting activities of the RO-i2010 project are aiming at fighting the digital divide at the institutional level, together with promoting the digital inclusion at the individual level. These aims will contribute to a durable socioeconomic development of our country and will support the integration in the European and world value and skills circuits.

Presentation of national and international context within the mentioned thematic field

Recent statistical data, regarding years 2004 and 2005, shows that Romania is less prepared for digital technologies when compared to Bulgaria and other EU 15 countries. An even darker situation appears in contrast with other world regions: North America (8.38 index in 2005) and Western Europe (7.87 index in 2005)

In order to achieve a durable socio-economical development together with European integration, Romania needs training and education systems that are able to take full advantage of the digital technologies. The 'digital divide' concept comprises of two issues: (i) the poor quality of the infrastructure, or even lack thereof (computer access, internet access, etc), and

(ii) even where equipment exists, it remains unused or under-utilized due to socioeconomical or psycho-pedagogical reasons; human resources exist but do not possess skills and abilities required by a competitive and dynamical market economy.

The project aims to use the existent orientation, directions, priorities and objectives: (i) share partners' human and material resources at a national level; (ii) improve partners' research infrastructure, as well as the training and education ones; (iii) create modern training products and services that will contribute to the elimination of the digital divide at the institutional level and will promote digital inclusion at the individual level; (iv) contribute to the durable socio-economical development of our country and to help the inclusion in the world skills and values circuits.

Project's objectives

Promoting EU i2010 in Romania

This objective is focused on the recently launched EU initiative (June 2005) which aims to increase the degree of ICT use in socio-economic activities, in order to close the gap between the EU and US and Japan. In this respect, the project includes specific activities related to the establishment of the RO-i2010 consortium and to the definition of project management and implementation procedures: establishing the consortium, planning the project management, a website and forum, public presentations, seminars, conferences, printed materials (flyers, leaflets etc.).

Strengthening the long-term cooperation with EU partners in order to integrate in European and international research programs

The traditional partners are Carinthia Tech Institute, Villach, Austria and International Centre for Theoretical Physics in Trieste, Italy. We are happy to implement (2006)(2007)(2008)(2009) a SOCRATES ERASMUS CD project named BIT2010-Joint Bachelor Degree in Information Technology 2010. Also, we will contribute as co-organisers to the training seminar "Workshop on Rich-Media Webcasting Technologies for Science Dissemination" which will be organized by ICTP Science Dissemination Unit. We are open to new partnerships.

Increasing international visibility of the Romanian ICT community

The project will contribute to the fight against the digital divide at institutional level, and will promote the digital inclusion at individual level. Both these results will contribute to create better working and living conditions, learning opportunities, will contribute to the durable socio-economic development of our country and will sustain European integration. The project generates competitive capacity for future EU programs. The RO consortium will concentrate a critical mass of high quality ICT human and material resources, will allow national sharing of human and digital resources, and will encourage creativity and personal development. The implementation of the project will demonstrate the research-developmentinnovation abilities of the partner institutions and will help convince potential EU partners to accept us for future EU projects and programs. The project will generate sustainable operational results. The RO-i2010 consortium will allow the harmonization of partners on the subject of educational policies and ICT promotion. Digital training technologies will be implemented in the partner institutions ensuring the modernization of the operational systems. Each partner will try to bring other clients -private companies -from their respective areas, which will ensure the sustainability of the consortium beyond the timeframe of the financing.

Real results, UniBuc-VIR

The RO-i2010 Consortium took the opportunity to build a pilot project having as objective to set-up a virtual university platform: (i) to design, implement and develop a virtual university platform at the University of Bucharest, in pilot phase; (ii) to implement two Master programmes on this platform; (iii) to create a core for further development of a "virtual university"-like consortium with national coverage. The UniBuc-VIR platform will include: -the UniBuc Virtual Campus -hardware (computer networks, internet access points in faculties) and software (software applications) infrastructure which will provide the communications systems used by the "users" of this institution (students, teachers, researchers, administrative personnel); -computer based administration systems (information, planning, databases, student records, accounting and financial services, etc.) -communications services (email, discussion boards, chat, video-conferencing, wireless internet access); -educational "blended learning" services, flexibly combining face to face and virtual learning (e-Library, e-Books), eLearning resources, virtual and remote laboratories, subscriptions to electronic specialized newsletters, etc.

-research services in virtual (designed in this project) or actual (like BCUM -previously financed by the World Bank and the Romanian Government) laboratories for interdisciplinary and transdisciplinary study programs. This project is financed by the Ministry of Education in Romania with 1,000,000 EUR in 2007.

Author(s):


Hosam F El-Sofany

Fayed F M Ghaleb

Sameh S Daoud

Samir A El- Seoud

Ahmad M Hasna

Jihad M Jaam

XML and Databases for E-Learning Applications

Keywords: XML, XML Databases, Relational Databases, E-Learning

XML has become a standard format in information exchange and integration on the Web. Much research has been conducted in recent years on XML technology, which has led to new developments in this field. The effective combination of XML and relational databases for portable Internet data exchange and data management is the technology platform of choice for the Internet Applications. Our paper describes the relationship between XML and relational databases and its potential as an enabling technology to support e-learning. Also the paper discusses how relational databases and XML fit together and briefly illustrates their implication in the development of elearning systems. We introduce our e-learning model, as an example of this new developing methodology.

Introduction

An overall study of the XML enabled database systems, such as SQL Server, Oracle, and DB2 [START_REF]IBM. DB2 extender for XML[END_REF] leads to the fact that, they provide mechanisms to store and query XML data by extending the existing data model. This extension is created mainly by adding XML data type, so that a column of this data type can be defined and used to store XML data. In addition, a set of methods is associated with this new XML data type to process, manipulate and query stored XML data. This usually requires various mappings (e.g., schema mapping, data mapping and query mapping) to be performed between the two data models [START_REF] Fernandez | Silkroute: A framework for publishing relational data in XML[END_REF][START_REF] Lu | A new inlining algorithm for mapping XML DTDs to relational schemas[END_REF]. Therefore, the main issue is to develop efficient algorithms to perform these mappings.

One of the important features of XML documents is that we can perform operations based on their logical structures [START_REF] Tatarinov | Storing and querying ordered XML using a relational database system[END_REF]. Based on this feature, databases that manage XML documents have to support queries on their logical structures and on their contents. Considering access based on a logical unit and reusability, it is appropriate to decompose and store XML documents according to their tree structures. They are then stored in appropriate databases. In order to retrieve XML documents from such databases, we have used a dynamic application based on a W3C's Document Object Model (DOM), and database queries (typically in SQL) [START_REF] Sameh | A DOM-Based Approach of Storage and Retrieval of XML Documents Using Relational Databases[END_REF].

The potent combination of XML and databases for portable Internet data exchange and data management is the technology platform of choice for the Internet Applications. The main reason for this is that, XML provides a compelling environment for the integration of disparate forms of data in a platform independent manner. This paper discusses how relational databases and XML fit together and briefly illustrates their implication in the development of e-learning systems.

The paper is organized as follows: in section two we will give a brief overview about the XML database systems. In section three we introduce our E-learning model, that uses XML and relational databases for e-learning system development, while in section four, we will provide a comparison between four virtual learning environment systems. The paper is finally concluded in section five.

XML Database Systems

There are a number of options to manage XML data and documents. One way is to use traditional database systems such as relational databases or object-relational databases, the other way is to use a database system specially designed for handling XML documents. In this section, we introduce a brief overview for the XML databases types, and present some approaches used to store (and query) XML documents in relational databases.

Types of XML Databases

There are different views on what an XML database is. Some defines it as: "a database that stores XML in its native format". In general we have two types of XML databases:

1. If the XML document is not stored internally as XML data, then it is called an XMLenabled database. In such databases, XML data is stored as data records into relational tables. 2. If an XML document is stored as XML internally (i.e., by the same tree structure of XML document) then we call it a native XML database.

XML-enabled databases

We can use the existing database systems to manage XML. Relational databases were among the first that wanted to represent their data as XML. The storage of XML documents in relational databases means describing hierarchical, tree-type structures with relations. Therefore the first effort was directed towards enabling or extending the capabilities of these databases to support XML. This effort gave birth to the so-called XML-enabled database system. The idea behind such databases is to extend the existing databases in such a way that it would allow efficient storage, querying and publishing of XML in relational databases. These databases typically accept XML document, and insert it into database tables according to specified database schema. To retrieve the XML document, the data stored in XMLenabled database are collected back together again. In XML-enabled databases, the XML document schema (e.g., DTD or XML schema) is mapped to the database schema.

Storing XML Documents in a Relational Database

A database schema describes the structure of a database. It gives an abstract view over physical storage mechanisms used by a database. In relational databases, the data model organizes data in table structures. The XML data model, on the other hand, organizes data in highly nested, hierarchical structure. Due to the extreme differences between the two structures, different approaches for converting XML structure to relational structure have been developed. This section provides a brief overview of some approaches used to store XML documents in relational databases.

Conference ICL2007

September 26 -28, 2007 Villach, Austria 3(12)

The Edge Approach

Florescu and Kossmann in [START_REF] Florescu | A Performance Evaluation of Alternative Mapping Schemes for Storing XML Data in Relational Database[END_REF] proposed alternative ways to store XML data in a relational database, one of them is the called edge approach. This approach used a directed labeled graph data model.

Example 1: assume that, we want to export information (e.g. • The corresponding data graph of the above XML document is shown in Figure 1 [6]. • In mapping attributes: The entire attribute are stored in a single Edge table. The table has the following structure: Edge (source, ordinal, name, flag, target) source column is used to store the object identifiers (oids).

ordinal column is used to recover all attributes of an object in the right order and to carry out updates if objects have several attributes with the same name. name column is used to store the attribute name. • The following tables represent the Edge, and V type instance, corresponding to the XML document in example 1.

The Basic, Shared, and Hybrid inlining Approaches

Shanmugasundaram J. and et al. in [START_REF] Shanmugasundaram | Relational Databases for Querying XML documents: Limitations and Opportunities[END_REF] presented an approach to map XML to a relational database schema. This mapping scheme was developed to investigate the use of relational database systems to process queries on semi-structured documents. In this approach, the XML documents must have the associated DTDs to map them to a database schema. The database schema is created from the DTD, which is done by building a so-called DTD graph and constructing an element graph from it. • When the database schema is created from the XML DTD, several inlining techniques that destroy nested elements can be used to avoid the fragmentation of XML documents over several tables. The inlining technique keeps as many elements as possible into a single relation. • Shanmugasundaram J. [START_REF] Shanmugasundaram | Relational Databases for Querying XML documents: Limitations and Opportunities[END_REF] proposed three inlining techniques: [START_REF]IBM. DB2 extender for XML[END_REF] The basic inlining technique solve the fragmentation problem, by inlining as many descendants of an element as possible into a single relation. But this technique creates relations for every element because an XML document can be rooted at any element in a DTD.

Example: the author element in Figure 3 would be mapped to a relation with attributes firstname, lastname, and address. In addition, three relations would be created corresponding to these elements.

[2] The shared inlining technique is an improvement over basic inlining technique because it makes sure that an element node is represented exactly in one relation. It identifies the element nodes that are represented in multiple relations in basic inlining technique (e.g., firstname, lastname, and address), and creates a separate relations for them such that those elements can be shared.

[3] The hybrid inlining technique, is same as shared inlining except it inlines some elements that are not inlined in shared inlining technique.

2.2.1.3

The "New Inlining" Approach S. Lu., and et al. in [START_REF] Lu | A new inlining algorithm for mapping XML DTDs to relational schemas[END_REF] introduced an efficient algorithm which takes an XML DTD as input and produces a relational schema as output for storing and querying XML documents conforming to the input DTD. The algorithm features several significant improvements over the shared-inlining technique [START_REF] Shanmugasundaram | Relational Databases for Querying XML documents: Limitations and Opportunities[END_REF] explained in the previous section, including eliminating redundancies caused by shared elements, performing optimizations and enhancing efficiency.

• The new inlining algorithm contains the following three steps: 1. Simplifying DTDs: since a DTD expression might be very complex due to its hierarchical nesting capability, this step used to simplify the input DTD. 2. Creating and inlining DTD graphs: create the corresponding DTD graph based on the simplified DTD, and then inline as many descendant elements as possible to an XML element. In contrast to the shared-inlining technique, the new inlining rules eliminate the redundancy caused by shared elements in the generated relational schema and can deal with arbitrary input DTDs including those that contain arbitrary cycles.

DOM-Based Approach

In [START_REF] Sameh | A DOM-Based Approach of Storage and Retrieval of XML Documents Using Relational Databases[END_REF] We introduced a novel approach for storage and retrieval of XML documents using relational databases. In this approach, an XML document is decomposed into nodes based on its tree structure, and stored into relational tables according to the nodes types. Our approach enables us to store XML documents using a fixed relational schema without any information about XML schema, and DTD. For the processing of XML documents, we proposed two algorithms denoted by "XtoR" and "RtoX", where the first one is for converting XML data to relational data, and the second one for extracting data from a database and insert it into XML document. The application does not impose any extension of relational databases for storage and retrieval of XML documents. The data model we used for the data mapping algorithms is based on the W3C's DOM [START_REF]W3C site[END_REF].

• Proposed relational schema: Note that, it is needed to store XML document efficiently, to preserve the order of the document. Hence, we have mapped the XML DOM model onto the following relational database schema: -Figure 6 shows a database instance of the XtoR schema, that explains how the XML document given in Figure 5 is mapped into the relational database schema given in Figure 4, by using the XtoR algorithm introduced in [START_REF] Sameh | A DOM-Based Approach of Storage and Retrieval of XML Documents Using Relational Databases[END_REF].

Figure 6: A database instance of the XtoR schema storing the XML document in Figure 5 8(12)

Native XML Databases

The need to process and store XML data generates several new types of software tool, one of which is the "native XML database". This section explains the principles behind the native XML database.

What is a Native XML Database?

The term "Native XML Database" (NXD) understands in many ways. In fact many so-called NXDs aren't really standalone databases at all, and don't really store the XML in true native form (i.e. text). To get a better idea of what a NXD really is, let's take a look at the NXD definition offered by the XML: DB Initiative [START_REF]What is an XML database?[END_REF].

A native XML database: [START_REF]IBM. DB2 extender for XML[END_REF] Defines a logical model for an XML document. Stores and retrieves documents according to that model. At a minimum, the model must include elements, attributes, PCDATA, and document order.

Examples of such models are:

-The XPath data model, -The models implied by the DOM, that proposed by W3C, and -The SAX model (Simple API for XML) [START_REF] Fernandez | Silkroute: A framework for publishing relational data in XML[END_REF] Has an XML document as its fundamental unit of logical storage, just like a relational database has a row in a table as its fundamental unit of logical storage. [START_REF] Lu | A new inlining algorithm for mapping XML DTDs to relational schemas[END_REF] Is not required to have any particular underlying physical storage model. For example, it can be built on a relational, hierarchical, or object-oriented database; also it can use a proprietary storage format such as indexed, compressed files.

Native XML Databases Systems

A long list of native XML databases systems is available at [START_REF] Bourret | XML Databases Products: Native XML Databases[END_REF]. Some of these databases are open source or result of research projects, the rest are commercial native XML databases:

-Native XML databases resulting from research projects: such as, Lore from Stanford University and Timber from University of Michigan. -Open source native XML databases, such as: Oracle Berkeley DB XML, eXist XML database [START_REF]W3C site[END_REF], and ozone Database Project [12]. -Commercial native XML databases: such as Tamino XML Server (from Software AG).

The E-learning model

Our model Figure 7 provides the student with two kinds of contents, Learning content and Assessment content. Each content has different types of services such as:

Learning services: registration, online course, Interactive tutorial, course documents (is a repository for files that the instructor have made available to the student as a part of your course), announcements (displays information to the students that the instructors of the course want him to know), links (displays a list of useful URL links that have been identified by the course instructors), student papers (students can post/upload requests files to the instructor). Assessment services: provide exercises and quizzes for evaluation of the student knowledge.

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During the learning process, a dynamic selection presentation of both contents will be accomplished.

On the other hand, our model, allows teachers (professors, lecturers...) to create and admin course websites through a browser (Explorer, Netscape...). You can Publish documents in any format (Word, PDF, HTML, Video...) Admin public or private discussion forums Manage a list of links: displays a list of useful URL links that have been identified by the course instructors. Create student groups Compose exercises Structure an agenda with tasks and deadlines Make announcements: displays information to the students that the instructors of the course want him to know Have students submit papers: students can post/upload requests files to the instructor. The e-learning model uses XML, rather than the classical HTML language due to its flexibility and richness to deal dynamically with the questions. In fact, with HTML one must use the established tags, however, with XML we can create specific documents with the tags we need and the features we want. So, XML allows users to create their own specific language according to their needs. The first step is to create what is called the Document Type Definition (DTD) where the features are specified, and include the tags and attributes. Once the DTD is created, we can use the new tags for creating web pages. XML was chosen as a basis for system development because of the hierarchical data structure that auto-defines itself. The introduction of XML is performed by means of a DTD, which determines the question structure. The DTD defines the composition of each kind of question considering all the necessary fields [START_REF] Hosam | A Web-Based E-Learning System Experiment[END_REF][START_REF] Ghaleb | E-Learning Model Based On Semantic Web Technology[END_REF].

Common Features between Claroline and Moodle

5.1 Courses are broken down into component elements and then published to the site under separate areas (announcements, exercise, chat etc) 5.2 Courses can start with a limited number of resources, but grow in size and complexity. 5.3 Students have full flexibility in the order that they undertake the elements of the course. 5.4 Categorized links feature to manage relevant URLs. 5.5 Offers fully optional layout for course beneficiaries. 5.6 Ability of the course administrators to set exercise and assign completion deadlines. 5.7 Students can upload their own papers for peer review.

Common Features between Moodle and ATutor

6.1 Inbuilt glossary function 6.2 'Send course email' feature to allow all course students to be contacted simultaneously 6.3 Flexible assignment creator tool 6.4 Excellent documentation and help manuals 6.5 Dynamic site mapping feature that grows as more courses are authored on the system. • Integrates editor for creating content without the need for HTML knowledge.

• Print compiler tool-allows student to select pages of text and group them together on one page • Context sensitive help.

• Links to various other educational databases are included with the download package.

Blackboard:

• A Blackboard course consists of a navigation path, a button bar and content frames. • Faculty type or upload their course materials into Blackboard. Blackboard can accommodate text, graphics and audio mediums. • The course material does not have to be html documents. Faculty can load Word documents or even PowerPoint presentations. • The navigation path allows users to return to any page accessed between the main course page and the current pages. • The button bar links users to the available content areas and tools. The content frame displays web pages accessed through the button or navigation path. • Typical assessment methods used in determining learning outcomes include tests made up of short answer, multiple choice, True/False or combinations of all three.

Conclusion

Our paper described the relationship between XML and relational databases and its potential as an enabling technology to support e-learning. Also the paper discussed how relational databases and XML fit together and briefly illustrated their implication in the development of

Figure 1 :

 1 Figure 1: Data graph corresponding to the XML document in example 1.

Example 2 :

 2 The DTD graph corresponding to the DTD in Figure2is given in Figure3. Cycles in the DTD graph indicate the presence of recursion. <!ELEMENT book (booktitle, author)> <!ELEMENT article(title, author*, contactauthor)> <!ELEMENT contactauthor EMPTY> <!ATTLIST contactauthor authorID IDREF IMPLIED> <!ELEMENT monograph(title, author, editor)> <!ELEMENT editor(monograph*)> <!ATTLIST editor name CDATA#REQUIRED> <!ELEMENT author(name, address)> <!ATTLIST author id ID#REQUIRED> <!ELEMENT name(firstname?, lastname)> <!ELEMENT firstname(#PCDATA)> <!ELEMENT lastname(#PCDATA)> <!ELEMENT address ANY>

Figure 2 :

 2 Figure 2: A sample DTD

Figure 4 :

 4 Figure 4: The proposed schema for mapping the XML document to relational tables

Figure 5 :

 5 Figure 5: An XML instance of (books.xml) document

Figure7.

  Figure7. Framework for Our Web-Based e-learning system

  target column is used to store the oids of the target object of the attribute.-The key of the Edge table is {source, ordinal}

	Conference ICL2007	September 26 -28, 2007 Villach, Austria
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flag column is used to indicate in which Value table a value is stored, a flag can therefore takes values such as string, integer, data or ref . • In mapping values: two different schemes have been proposed: a. Storing values in a separate value table: V type (vid, value), and b. Storing values together with the attribute.

  [START_REF] Lu | A new inlining algorithm for mapping XML DTDs to relational schemas[END_REF]. Generating relational schemas: generate a relational schema based on the inlined DTD graph in step 2.Finally, the following eight relations will be generated after performing the four steps of generating relational schemas:

	publication(ID)
	conference(ID, name.ID)
	journal(ID,nodetype,name.ID
	name(ID, PCDATA)
	paper(ID, nodetype, ptitle, volume, number, year)
	person(ID, nodetype, pname, institute)
	techreport(ID, nodetype, title
	edge(parentID,childID, parentType, childType)
	Example 3: Consider the following input DTD for publications:
	<!DOCTYPE publication [
	<!ELEMENT publication (journal*, conference*)>
	<!ELEMENT journal (name, editors, paper+)>
	<!ELEMENT conference (name, paper+)>
	<!ELEMENT paper (ptitle, authors,(volume, number)?)>
	<!ATTLIST paper year CDATA>
	<!ELEMENT editors (person+)>
	<!ELEMENT authors (person+)>

<!ELEMENT person (pname, institute, techreport*)> <!ELEMENT techreport (title, references)> <!ELEMENT references (paper+)> <!ELEMENT institute (#PCDATA)> <!ELEMENT pname (#PCDATA)>

  -The core of the mapping is the Element entity, that contains the following attributes: o ElmId: A unique identifier for each element, o DocId: The unique key of the associated document, o ElmName: The name of this element, o EmlValue: The text or data of this element (NULL for none), o ParentID: The element which is parent to this element (NULL for the root element), o OrderId: Element id of the next element in scope order (NULL for last element), and o ElmDepth: The scope depth of this element. -The Element entity is supplemented with two other entities. The first one is the Document entity, that contains the following attributes:

	o	DocId: A unique identifier for each document, and
	o	DocName: The name of this document.
	-The second entity is the Attribute entity, that contains information about each attribute,
	such as:
		publisher>
		</book>
		<book>
		<author>Jack Herrington</author>
		<title>PHP Hacks</title>
		<publisher>O'Reilly</publisher>
		</book>
		</books>

7(12) o ElmId: The unique key of the associated element, o AttName: The name of this attribute, and o AttValue: The value of this attribute. Example 4: Consider the following XML document: <?xml version="1.0" encoding="ISO-8859-1" ?> <books> <book> <author> H.M. Deitel and P.J.Deitel </author> <title> C++ How To Program </title> <publisher> Prentice Hall Publishing Co. </

4 Virtual Learning Environment Comparison Virtual

  learning environments are software packages designed for helping educators to create and deliver online courses. Such e-learning systems are sometimes called a Learning Management System (LMS), Course Management System (CMS), or Virtual Learning Environments (VLE). The use e-learning in the majority of universities has begun with the introduction of a VLE and although this project is funded by the European Union.

	In this section we provide a comparison between four the VLE systems, Moodle [15],
	Claroline [16], ATutor [17], and Blackboard [18]. From the proposed comparison, one can
	decide easily, what is the suitable VLE he needs?
	1. Creation
	1.1 Claroline : Consortium of French University faculties
	1.2 Moodle	: PhD student called Martin Dougiamas.
	1.3 ATutor	: Toronto University in Canada
	1.4 Blackboard : Learning Management System, partially owned by Microsoft
	2. License Agreement
	2.1 Claroline, Moodle, and ATutor are Open source (GPL)
	2.2 Blackboard: Licensed annually
	3. Cost
	3.1 Claroline, Moodle, and ATutor are Freely downloaded, installed and distributed
	without charge
	3.2 Blackboard: approx. $8,600 each year
	4. Common Features
	4.1 Theme feature allows the change of look and feel of the VLE without a new style
	sheet.
	4.2 Agenda feature which allows authors to set weekly announcements in advance

  Chat facility for all users (text interface system) • Can upload video files for use as course recourses • Statistics function for course administrators to monitor number of courses, courses popularity, etc • Supports multi languages 8.2 Moodle : • Basic security features to limit customer access to particular courses • Journal feature to allow students to post questions, maintain a course diary, or aid revision. • Use XML metadata to describe e-learning content within the system. 8.3 ATutor : • Course construction broken down into a series of 'content pages' • Structure is built on a 'slide by basis' but would require HTML skills.

	7. Blackboard vs. Moodle
	7.1 Moodle advantages over Blackboard:
	• Providing individualized feedback easily to all assignments • Easier to track each student's activity in class
	7.2 Blackboard advantages over Moodle
	• More polished appearance • Better grade book • Threaded discussions easily differentiate between read and unread posts • Announcements are more prominently displayed upon entering the course
	8. Different Features between the four virtual learning environments
	8.1 Claroline:

•

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e-learning systems. We introduced our e-learning model, and the comparison between four VLE systems, as an example of this new developing methodology.


Nicola Festuccia

Agnès Dubois

Sandrine Vandormael-Pournin

Elena Gallego Tejeda

Adrien Mouren

Sylvain Bessonnard

Florian Mueller

Caroline Proux

Michel Cohen-Tannoudji

Pablo Navarro Gil

Pablo Navarro
email: pnavarro@pasteur.fr

Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network

Introduction

Embryonic Stem (ES) cell lines are derived from the pre-implantation blastocyst and can be maintained indefinitely in culture in an undifferentiated and pluripotent state [START_REF] Smith | Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides[END_REF] . Despite undergoing virtually infinite divisions, ES cells do not lose their biological identity nor do they compromise their developmental potential. This phenomenon, known as self-renewal, is strictly dependent upon a network of Transcription Factors (TFs) centred on Oct4, Sox2, Nanog and Esrrb [START_REF] Kim | An extended transcriptional network for pluripotency of embryonic stem cells[END_REF][START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF] . Although these factors act collaboratively [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF] , they also display unique functions. While Nanog -/-cells exhibit low selfrenewal efficiency [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF] , Oct4 -/-or Sox2 -/-undifferentiated cells cannot be maintained [START_REF] Nichols | Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[END_REF][START_REF] Niwa | Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells[END_REF][START_REF] Masui | Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells[END_REF] and pure undifferentiated Esrrb -/-cultures can be propagated only when differentiation is inhibited [START_REF] Festuccia | Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells[END_REF][START_REF] Martello | Esrrb is a pivotal target of the gsk3/tcf3 axis regulating embryonic stem cell self-renewal[END_REF] .

Moreover, whereas Oct4 and Sox2 up-regulation triggers the loss of pluripotency [START_REF] Niwa | Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells[END_REF][START_REF] Kopp | Small increases in the level of Sox2 trigger the differentiation of mouse embryonic stem cells[END_REF] , that of Nanog and Esrrb abolishes the ability of ES cells to differentiate [START_REF] Festuccia | Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells[END_REF][START_REF] Chambers | Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells[END_REF] . Therefore, even though Oct4 is perhaps the only obligatory and general regulator of pluripotency, the function of Esrrb and Nanog is more specifically dedicated to the control of self-renewal.

ES cells are highly proliferative and present an atypical cell cycle structure characterised by a short G1 phase lacking a G1/S checkpoint [START_REF] Stead | Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities[END_REF][START_REF] White | Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation[END_REF][START_REF] Faast | Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a)[END_REF][START_REF] Dalton | Linking the Cell Cycle to Cell Fate Decisions[END_REF] . Therefore, ES cells effectively exist only in two continuously alternating states of DNA replication and cell division. Generally, mitosis is accompanied by the eviction of TFs and global transcriptional silencing [START_REF] Martinez-Balbas | Displacement of sequence-specific transcription factors from mitotic chromatin[END_REF] . Given that pluripotency TFs are permanently required to promote self-renewal and prevent differentiation, their activity must be promptly reinstated after each division. Moreover, the inactivation of major chromatin regulators associated with the epigenetic inheritance of gene expression programmes does not have severe detrimental effects on the propagation of the undifferentiated state [START_REF] Tsumura | Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b[END_REF][START_REF] Leeb | Polycomb complexes act redundantly to repress genomic repeats and genes[END_REF][START_REF] Lehnertz | Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin[END_REF][START_REF] Tachibana | G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis[END_REF] . This large independence from classical epigenetic pathways has recently been recognised as one of the most intriguing hallmarks of ES cells [START_REF] De Los | Hallmarks of pluripotency[END_REF] , raising a major unsolved question: how do ES cells maintain the TF-based regulatory continuity from mother to daughter cells and preserve their transcriptional status over indefinite periods of frequent and rapid divisions?

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Increasing observations suggest that some transcriptional regulators can resist chromatin condensation and remain bound to their targets during mitosis. This process, known as mitotic bookmarking [START_REF] Michelotti | Marking of active genes on mitotic chromosomes[END_REF] , has been reported for general TFs [START_REF] Xing | The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action[END_REF] , coactivators [START_REF] Zaidi | Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells[END_REF] and chromatin modifiers [START_REF] Blobel | A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit[END_REF] , but also for sequence specific TFs such as Runx2 [START_REF] Young | Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2[END_REF] , Hnf1 27 , FoxA1 [START_REF] Caravaca | Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes[END_REF] and Gata1 [START_REF] Kadauke | Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1[END_REF] . Therefore, mitotic bookmarking by one or more pluripotency TFs could propagate efficiently the regulatory information required to maintain the transcriptional identity of ES cells.

Given the intimate connection existing between their expression and the efficiency of selfrenewal, we initially sought to determine whether Nanog or Esrrb could act as mitotic bookmarking factors. Using fluorescence microscopy we demonstrate that Esrrb, but not Nanog, is retained on mitotic chromosomes, both in ES cells and during early embryogenesis. Furthermore, we report the compendium of genomic locations bound by Esrrb during mitosis, and demonstrate that mitotic Esrrb retention relies on dynamic interactions with its cognate DNA binding motif. Finally, we identify the genes that are either rapidly activated or kept silent after mitosis in response to Esrrb activity.

Altogether, we propose that Esrrb may act as a mitotic bookmarking factor in ES cells.

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Results

Esrrb decorates the mitotic chromosomes in ES cells and early embryos.

To study whether Nanog and/or Esrrb are retained on mitotic chromosomes, we generated two cell lines expressing either Nanog-GFP or Esrrb-GFP fusion proteins. A Nanog-GFP expression vector was stably integrated into wild-type E14Tg2a ES cells; Esrrb-GFP was introduced in EKOiE ES cells, in which the two endogenous Esrrb alleles were previously deleted and Esrrb is expressed from a Dox-inducible transgene (Methods and Supplementary Fig. 1a online). After confirming that the transgenic GFP-fusions and Dox-inducible Esrrb proteins are functional (Supplementary Fig. 1b online), we monitored their mitotic behaviour. While Esrrb-GFP was systematically localised to mitotic chromosomes, Nanog-GFP was excluded (Fig. 1a, Supplementary Fig. 2a, b online). We then established an immunofluorescence protocol on metaphase spreads prepared from EKOiE cells cultured with Dox. We found that Esrrb was clearly associated to the metaphasic chromosomes (Fig. 1b). We did not observe any signal when Dox was withdrawn for 48h, demonstrating the specificity of this assay (Supplementary Fig. 2d online). Moreover, a similar coating of the chromosomes was observed in metaphase spreads prepared from EKOiE cells expressing an Esrrb Snap-tagged variant, further supporting our observations (Supplementary Fig. 2e online).

To exclude that the mitotic retention of Esrrb is due to the high levels of expression obtained either by Dox induction or by the CAG-driven Esrrb-GFP or Esrrb-Snap transgenes, we fused both endogenous Esrrb alleles to tdTomato (Methods online). We found that Esrrb-tdTomato decorates the chromosomes throughout all phases of mitosis (Fig. 1c, Supplementary Fig. 2c online), as also observed in time-lapse imaging of Esrrb-GFP (Supplementary Movie online). Moreover, when we performed immunofluorescence for Nanog and Esrrb using metaphase spreads prepared from E14Tg2a cells, we observed that Esrrb, but not Nanog, decorates the mitotic chromosomes in wild-type cells (Supplementary Fig. 2f online). Overall, we have documented the chromosomal retention of Esrrb in all mitotic ES cells observed so far using diverse microscopy tools.

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Next, since Esrrb mRNA is abundant in fertilised oocytes and is expressed during early embryogenesis 30 , we asked whether the mitotic retention of Esrrb also occurs in vivo when pluripotency is established. To do this, in vitro transcribed Esrrb-TdTomato and GFP mRNAs were coinjected in single blastomeres of 2 cell embryos. The embryos were let to develop in vitro for 48h, reaching the compacted morula stage. We observed that Esrrb-TdTomato coats the mitotic chromosomes of GFP positive cells (Fig. 1d). This indicates that the mitotic retention of Esrrb is operational during early embryogenesis.

Mitotic Esrrb is specifically enriched at individual genomic regions.

To determine whether Esrrb is enriched at specific genomic regions during mitosis, we established a ChIP-Seq assay in interphasic and mitotic ES populations obtained by nocodazole treatment and mitotic shake-off (Methods online). Binding profiles of Esrrb were produced in wildtype E14Tg2a and in transgenic EKOiE cells cultured with Dox. EKOiE cells depleted for Esrrb (-Dox for 48h) were used as negative controls. We found that Esrrb remains bound at a subset of its interphase targets during mitosis, suggesting that it acts as a bookmarking factor (Fig. 2a).

Using statistical and reproducibility parameters (Methods and Supplementary Fig. 3 online), we identified nearly 14.000 Esrrb binding regions in interphase and 1.980 in mitosis (Supplementary Table 1 online). Albeit at different levels (Supplementary Fig. 3 online), mitotic binding regions were also bound by Esrrb in interphase (thereafter, Bookmarked; Fig. 2b,c,d). Nine regions were used for validation by quantitative PCR and titration experiments, excluding that remnant interphase cells in our mitotic preparations (<5%; Methods online) may affect the observed Esrrb binding profiles (Supplementary Fig. 4

online).

We observed that Esrrb binding levels in mitosis followed a bi-modal distribution (Fig. 2b), with ~6.000 regions showing very low levels. At these regions Esrrb binding was never detected in mitosis (Supplementary Fig. 3 online), indicating that Esrrb is efficiently evicted (thereafter, Lost; Fig. 2e). The remaining ~8.000 regions include the 1.980 regions identified as Bookmarked. Excluding those, the 7/59 levels of mitotic Esrrb binding did not significantly differ from the negative controls and the detection of Esrrb failed in at least one of our four independent replicates (Supplementary Fig. 3 online). Even though a subset of these regions may be inefficiently bound in mitosis, we adopted a conservative approach and considered them as Lost for subsequent analyses.

We conclude that Esrrb remains robustly bound to 10-15% of its interphase targets (Bookmarked regions; Fig. 2c), and shows no or labile mitotic binding at other regions (Lost regions; Fig. 2c). Of note, ~55% of all ES-specific super-enhancers [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF] such as those of key pluripotency factors (Sox2, Tbx3 and Klf4), together with other regulatory regions of important genes are mitotically bound by Esrrb (Supplementary Fig. 5 online).

Relationships between mitotic Esrrb binding and chromatin status in interphase.

In mitosis, Esrrb binding is generally reduced at Bookmarked regions compared to interphase (Fig. 2b,c). Moreover, Bookmarked regions show higher Esrrb binding in interphase than Lost regions (Fig. 2c). Using published datasets [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF][START_REF] Brookes | Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs[END_REF] , both Bookmarked -B-and Lost -L-regions could be divided into two clusters (Fig. 3a) according to their chromatin status in interphase: either particularly enriched for binding of other TFs and active histone marks (B1 and L1) or not (B2 and L2; Supplementary Fig. 6a online). However, direct quantitative comparisons showed that, whilst B2/L2 display homogeneously low levels (Supplementary Fig. 6a online), B1 regions exhibit higher enrichment for TFs, the transcriptional machinery and euchromatin-associated histone marks than L1 regions (box plots in Fig. 3a). For instance, both B1 promoters and enhancers display higher levels of Nanog, RNAPII and H3K27ac than their L1 counterparts losing Esrrb in mitosis (Fig. 3b). Other marks of activity followed an identical trend (Supplementary Fig. 6b online). Conversely, although it is generally detected at very low levels, the repressive H3K27me3 mark is slightly more abundant in L1 than in B1 regions, particularly at promoters (Supplementary Fig. 6b online).

Gene ontology analysis (Fig. 4a) showed that genes associated with B1 regions tend to be expressed in undifferentiated cells of pre-implantation embryos. In contrast, genes associated with L1 8/59 regions are expressed in non-pluripotent extra-embryonic tissues. Genes associated to B2/L2 tend to be expressed at later stages of development. In accordance, analysis of hallmarks of transcriptional activity in published genome-wide datasets [START_REF] Brookes | Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs[END_REF][START_REF] Bulut-Karslioglu | Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells[END_REF] , indicates that genes located in the vicinity of Bookmarked regions are more actively transcribed than those located nearby Lost regions (B1 versus L1 and B2 versus L2; Fig. 4b).

Notwithstanding that Esrrb binds regions of different chromatin constitution (B1/2 an L1/2), mitotic Esrrb binding marks active enhancers and promoters of genes highly transcribed in ES cells and developmentally associated with pre-implantation pluripotency.

Mitotic Esrrb binding is driven by specific DNA binding to its motif.

Using de-novo motif discovery algorithms we found that the canonical extended estrogen receptor half-site characteristic of Esrrb was strongly enriched at Bookmarked regions, with nearly 90% of these regions containing at least one motif (Fig. 5). Conversely, only 32% of Lost regions had Esrrb motifs, suggesting that specific DNA-protein interactions are required for mitotic binding of Esrrb. To test this, we mutated 3 residues of Esrrb that mediate the principal base-specific interactions with DNA (Methods online). This binding-defective mutant (Supplementary Fig. 7b online) did not decorate the mitotic chromosomes to the extent of wild-type Esrrb-GFP (Fig. 5b and Supplementary Fig. 7c online).

An undescribed longer Esrrb motif extended by 3 bases (AGT) was prominent in Bookmarked regions (63%). In Lost regions, however, only 14% displayed a long motif and of lower quality (Fig. 5a and Supplementary Fig. 7a online). Hence, the long motif may play a specific role in Esrrb bookmarking.

To test this, we used CRISPR to turn the single long Esrrb binding motif present under the major Esrrb peak at the Esrrb enhancer into a short version (Supplementary Fig. 7d online), with no further modification of the locus. In this mutant cells, we could not measure any significant difference in levels of Esrrb binding in interphase and mitosis (Supplementary Fig. 7e online).

To directly test whether long and short motifs are sufficient to drive mitotic Esrrb binding, we used recombinase mediated cassette exchange (RMCE) to introduce a tandem array of 4 long or short 9/59 motifs in an identical genomic location (Methods online). We observed that Esrrb was efficiently recruited in interphase and its binding maintained during mitosis both for the long and short motifs (Fig. 5c,d) and irrespectively of the locus of integration (Clones #1 and #2). While the significance of the 3 extra-bases that we have identified remains unclear, these experiments demonstrate that the presence of Esrrb binding sites is sufficient to drive its mitotic retention. In agreement, the presence of any Esrrb motif in Bookmarked regions is associated with increased binding in mitosis and more efficient mitotic retention, as revealed in our ChIP-Seq results (Supplementary Fig. 7f, g online).

We conclude that the interaction of Esrrb with its cognate binding site correlates with efficient mitotic binding at endogenous loci and is sufficient to drive the mitotic retention at ectopic sites. For the 10% of Bookmarked regions lacking Esrrb binding sites, however, other mechanisms must be invoked; for example protein-protein interactions with other factors. In line with this, Bookmarked regions lacking a motif display unexpectedly high enrichment levels of other pluripotency TFs in interphase (Supplementary Fig. 7f, h online).

Mitotic Esrrb binding is dynamic.

The retention of Esrrb on mitotic chromatin could either result from the permanence of Esrrb protein already loaded on chromatin at the end of G2, or from de novo and dynamic binding of Esrrb in mitosis. To discriminate between these two possibilities, we performed FRAP experiments (Fig. 6a), a strategy that has been widely used to infer TF binding dynamics [START_REF] Farla | The androgen receptor ligand-binding domain stabilizes DNA binding in living cells[END_REF][START_REF] Sauer | C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity[END_REF][START_REF] Mueller | Quantifying transcription factor kinetics: at work or at play?[END_REF] : while a fast fluorescent recovery is associated with rapid ON/OFF binding transitions, a slow recovery results from a decreased turnover of photo-bleached and fluorescent molecules, indicating more stable interactions. In interphase, Esrrb-GFP signal promptly recovered after bleaching (80% recovery in 5s, Fig. 6a,b), indicating that Esrrb binding is dynamic. Strikingly, the specific spatial localisation of the GFP signal resulting from the decoration of mitotic chromosomes by Esrrb-GFP was also recovered after bleaching, and with faster dynamics than in interphase (80% in 2s, Fig. 6a,b). As a control, DNA binding Errb mutant showed even 10/59 faster GFP recovery rates (Fig. 6b), confirming that the delay in recovery observed for wild-type Esrrb results from its DNA binding ability both in interphase and mitosis.

These results indicate that Esrrb is able to bind de novo to mitotic chromatin and that the observed retention likely results from continuous cycles of transient and fast interactions of Esrrb with DNA. Together with the previous analysis of Esrrb binding sites, this suggests that Esrrb behaves as a canonical TF in mitosis in terms of its binding to DNA.

Mitotic Esrrb binding correlates with transcription activation in early-G1.

To explore the functional consequences of mitotic Esrrb binding on gene expression we introduced a new cell-cycle reporter into Dox-inducible EKOiE cells (Methods online). This reporter system is based on the fusion of residues 1-156 of murine Cyclin A2 (Ccna2) to GFP, driving its mitotic degradation starting from prometaphase [START_REF] Den Elzen | Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase[END_REF][START_REF] Geley | Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint[END_REF] . When used in conjunction with the live DNA stain Hoechst, this system allows sorting cells as they proceed through G1, S and G2, without compromising viability (Supplementary Fig. 8a online). Ccna2-GFP cells were grown in the presence or absence of Dox (24h withdrawal), isolated by FACS in early-G1, late-G1 and G2 fractions (Fig. 7a), and analysed by RNA-Seq using a pipeline enabling to quantify both mRNA and pre-mRNA levels (Methods and Supplementary Table 2

online).

Overall, around 500 genes were up/downregulated by Esrrb at both mRNA and pre-mRNA levels (Fig. 7b and Supplementary Table 3 online). 168 and 206 genes showed prompt positive or negative response already in early-G1 and were coherently affected by the loss of Esrrb during all cellcycle phases (Supplementary Fig. 8b online). The number of Esrrb-responsive genes increased during cell-cycle progression (Fig. 7b), with around half showing changes only in G2. Early-G1 responsive genes associated with a Bookmarked region include pluripotency TFs (Tfcp2l1, Tbx3 and Klf4) and regulators of differentiation (Id2-3), chromatin (Dnmt3L) and embryonic cell-cell interactions (Jam2; Fig. 6c).

Notably, Esrrb-up/downregulated genes in early-G1 are also up/downregulated in ground-state conditions of systematic self-renewal [START_REF] Marks | The transcriptional and epigenomic foundations of ground state pluripotency[END_REF] (Supplementary Fig. 8c online).

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Almost 75% of genes activated by Esrrb in early-G1 were located within topological associated domains [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF] (TADs) containing at least one Esrrb bookmarked region (Fig. 7d). In contrast, only 55% of the genes regulated by Esrrb only in G2 were located within bookmarked TADs, as observed for an equivalent set of random genes. Similarly, nearly 25% of the genes upregulated by Esrrb in early-G1 were within 25kb of a Bookmarked region. In contrast, genes downregulated by Esrrb tend to be further away from Bookmarked regions (Fig. 7d).

Overall, the integration of our ChIP-Seq and RNA-Seq results indicate that mitotic Esrrb binding transcriptionally activates a subset of key genes in early-G1, including pluripotency regulators that in turn may control additional genes during the cell-cycle. This strongly suggests that Esrrb behaves as a canonical bookmarking factor.

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Discussion

We report here that Esrrb, a major pluripotency regulator [START_REF] Festuccia | Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells[END_REF][START_REF] Martello | Esrrb is a pivotal target of the gsk3/tcf3 axis regulating embryonic stem cell self-renewal[END_REF][START_REF] Feng | Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb[END_REF] , decorates the mitotic chromosomes of ES cells, an ability not shared by Nanog. We further demonstrate that Esrrb is specifically enriched at a subset of its interphase targets. Recently, other sequence-specific TFs have been shown to bind mitotic chromatin, suggesting that the mitotic retention of TFs may have been generally overlooked. Several common observations can be identified among recent studies [START_REF] Verdeguer | A mitotic transcriptional switch in polycystic kidney disease[END_REF][START_REF] Caravaca | Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes[END_REF][START_REF] Kadauke | Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1[END_REF][START_REF] Lerner | Human mutations affect the epigenetic/bookmarking function of HNF1B[END_REF] .

First of all, only a subset of TF binding regions are effectively bound in mitosis, albeit at lower levels than in interphase. Whether this is due to technical reasons or reflects a particular behaviour of mitotic binding is unclear, but our FRAP analyses suggest that mitotic Esrrb is hyper-dynamic and likely less amenable to chemical cross-linking and robust detection by ChIP-Seq. Secondly, regions bound in mitosis tend to exhibit particularly robust binding in interphase and contain canonical DNA binding sites (Fig. 8). Similarly to Gata1, FoxA1 and Hnf1, Esrrb bookmarking requires its DNA-binding domain to interact with the mitotic chromatin. Moreover, we demonstrate that the experimental introduction of ectopic recognition motifs is sufficient to drive Esrrb binding in interphase and in mitosis. Additional mechanisms of mitotic retention may however exist, as we identified around 10% of mitotically bound regions lacking clear motifs. These regions exhibit high enrichment for other pluripotency TFs, suggesting that additional regulators may bind mitotic chromatin in ES cells, a possibility that needs to be comprehensively assessed (Fig. 8). Thirdly, the mitotic retention of TFs is associated with widespread coating of the chromosomes, which is hardly explained by binding at a subset of enhancers and promoters. The mutation of FoxA1 DNA binding domain leads to the loss of binding at regions containing binding sites, but not to the loss of the chromosome-wide coating [START_REF] Caravaca | Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes[END_REF] . Conversely, mutations in the Hnf1 DNA-binding domain abolish chromatid coating but not site specific DNA binding in interphase [START_REF] Lerner | Human mutations affect the epigenetic/bookmarking function of HNF1B[END_REF] . In the case of Esrrb, we show that the mutation of its DNA-binding domain abolishes both binding at specific loci in interphase and the general coating of mitotic chromosomes. Hence, the decoration of the mitotic chromosomes is mediated by different types of molecular interactions 13/59 depending on the TF under study. This differential behaviour of TFs needs to be directly investigated, as it calls into question the idea that mitotic chromosomes are intrinsically refractory to scanning by TFs. New understanding of the nature of mitotic chromatin needs to be attained should we want to uncover the exact nature and relevance of mitotic bookmarking.

We have observed that, in interphase, regions bound by Esrrb tend to be either highly enriched for signatures of active chromatin (B1/L1 regions) or not marked by any specific feature (B2/L2), a chromatin state reminiscent of naïve chromatin [START_REF] Soufi | Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome[END_REF][START_REF] Soufi | Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming[END_REF] . However, direct comparison of B1 and L1 regions clearly shows that mitotic Esrrb binding is associated with regions of particularly high activity in interphase. Most notably, H3K27ac and RNAPII enrichment show the highest fold difference between bookmarked and lost regions. That Esrrb interacts with several members of the basal transcription complex, coactivators and histone acetyl-transferases [START_REF] Van Den Berg | An Oct4-centered protein interaction network in embryonic stem cells[END_REF] may provide a molecular understanding of the consequences of mitotic binding. Together with previous reports [START_REF] Blobel | A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit[END_REF][START_REF] Verdeguer | A mitotic transcriptional switch in polycystic kidney disease[END_REF][START_REF] Caravaca | Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes[END_REF][START_REF] Kadauke | Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1[END_REF][START_REF] Lerner | Human mutations affect the epigenetic/bookmarking function of HNF1B[END_REF] , our findings indicate that mitotic binding of Esrrb and of other factors, enables the maintenance of active chromatin properties at key regulatory regions that, in turn, activate important genes during interphase. Esrrb binding in mitosis seems to result in the rapid upregulation of important pro-self-renewal genes (Klf4, Tfcp2l1 and Tbx3) as ES cells re-enter into interphase (Fig. 8). The fact that alterations in both transcriptional output and mature mRNA accumulation are evident already in early-G1 argues in favour of an almost immediate resumption of transcription. Therefore, Esrrb should be considered as a mitotic bookmarking factor [START_REF] Zaidi | Mitotic bookmarking of genes: a novel dimension to epigenetic control[END_REF][START_REF] Kadauke | Mitotic bookmarking by transcription factors[END_REF][START_REF] Zaret | Genome reactivation after the silence in mitosis: recapitulating mechanisms of development?[END_REF] contributing to the long term maintenance of pluripotency by jumpstarting the post-mitotic resuscitation of the pluripotency network and counterbalancing the inherent propensity of ES cells to undergo differentiation from G1 [START_REF] Dalton | Linking the Cell Cycle to Cell Fate Decisions[END_REF][START_REF] Sela | Human embryonic stem cells exhibit increased propensity to differentiate during the G1 phase prior to phosphorylation of retinoblastoma protein[END_REF][START_REF] Pauklin | The cell-cycle state of stem cells determines cell fate propensity[END_REF] .

As shown for Gata1 [START_REF] Kadauke | Tissue-specific mitotic bookmarking by hematopoietic transcription factor GATA1[END_REF] , taking into consideration only mitotic binding allows a contraction of the gene ontology associations of Esrrb binding regions to particularly relevant genes, those highly expressed during pre-implantation development when pluripotency is established. Hence, the observation that in early embryos mitotic Esrrb binding is operational opens the possibility of a direct involvement of this process when Esrrb expression becomes restricted to the pluripotent ICM 30,[START_REF] Boroviak | The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification[END_REF] .

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Similarly, the generation of induced pluripotent stem (iPS) cells has been shown to transit from a stochastic to a more deterministic process coinciding with the reactivation of endogenous Esrrb expression [START_REF] Buganim | Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase[END_REF] . Thus, naïve pluripotency may only be attained when the regulatory network becomes mitotically self-sustainable through Esrrb binding.

Whilst ES cells self-renew indefinitely, in the embryo pluripotency is a transient state that is rapidly extinguished upon differentiation. We speculate that Esrrb bookmarking might be a key phenomenon enabling the transient epigenetic maintenance of pluripotency in the early embryo without the need to establish mitotically heritable chromatin marks which, although reversible, impose further molecular constrains to the timely extinction of pluripotency. More generally, we believe that mitotic bookmarking by TF has enormous advantages over other systems of mitotic inheritance in transiently expanded cell types such as activated stem cells and progenitors. We excluded deletion of the genomic region extending beyond the PCR-screened region by qPCR. All lines carry homozygous mutations.

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End notes

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Regular culture conditions:

ES cells were cultured on 0.1% gelatine (SIGMA, G1890-100G) in DMEM+GlutaMax-I (Gibco, 31966-021), 10% FCS (Gibco 10270-098), 100µM 2-mercaptoethanol (Gibco, 31350-010), 1xMEM nonessential amino-acids (Gibco, 1140-035) and 10ng/ml recombinant LIF (MILTENYI BIOTEC, 130-099-895). Cells were passaged 1:10 every 2-3 days. Clonal assays were performed by plating 600 cells per well of a 6 wells plate and after six days an Alkaline Phosphatase kit (Sigma, 86R-1KT) was used to identify undifferentiated colonies using a stereomicroscope (NIKON-SMZ1500).

31/59 nocodazole (Sigma, M1404) for 4 hours, with addition of 100nM Hoechst-33342 for 15 min, and mitotic cells collected by shake-off and resuspended in a minimal volume of fresh medium. One drop was deposed on a glass slide and a glass coverslip gently deposed on top. Cells were imaged immediately (Supplementary Fig. 1). Image stacks (0.2µm gap) were acquired using a Nikon Eclipse X microscope equipped with: 63x oil immersion objective (N.A1.4); LUMENCOR excitation diodes; Hamamatsu ORCA- 10 frames were acquired before bleaching (20ms pulse using a 488nm laser) and then recovery was imaged for 2min (1 image each 110ms). Fluorescence recovery was analysed in Matlab as described [START_REF] Mueller | Monitoring dynamic binding of chromatin proteins in vivo by fluorescence recovery after photobleaching[END_REF] , with one modification: the bleached region was located automatically by subtracting the first prebleach image from the last post-bleach image and calculating the 2d convolution (conv2 function) of this image with a circle of the typical size of a bleach spot.

Time-lapse imaging: WT or Mutant Esrrb-GFP EKOiE ES were kept at 37°C in a humidified atmosphere containing 7% CO2 during the entire duration of the movie. Image stacks with a 0.3µm gap were acquired every 3min using a 63x oil immersion objective (N.A1.4) on a Zeiss Observer Z1 microscope 32/59 (CoolLED excitation system, Photometrics Evolve EMCCD camera, Metamorph software).

Deconvolution was performed as described above with 0.2539µm effective pixel size.

Karyotype staining: 5x10 6 cells were arrested in metaphase with Colcemid (4h; 100ng/ml; Gibco, 15212-012), trypsinised and resuspended in 10ml of hypotonic solution (NaCitrate 0.017M, KCl 0.03M) for 10mins at RT. The cells were resuspended in 2.5ml of ice-cold hypotonic solution. 5ml of cold fixative acetic acid-methanol 1:3 solution were added dropwise at 4°C. Cells were collected and resuspended in 2.5ml of the supernatant and the above procedure was repeated 3 times. Imaging of embryos: To prevent blastomere mitotic exit, microinjected embryos were incubated with 1µM MG132 6 hours prior to imaging. After 30min in 1µg/ml Hoechst-33342, live imaging was performed in microdrops of KSOM with 10mM HEPES and 1µM MG132 on glass-bottomed microwell dishes (MatTek) using a Leica TCS SP5 confocal microscope (700Hz, HC Plan Apochromat 20x/0.70NA immersion objective, 25% laser intensity, voxel size of 75nm, pinhole of 1 airy unit).

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Preparation of mitotic populations:

E14tg2a or EKOiE cells were grown to 70-80% confluency in 4 to 6 T150 flasks per preparation. Vigorous shake off was performed to detach debris, dying cells or poorly attached cells colonies, medium discarded and flasks washed twice in PBS. After 4 hours in medium containing 50ng/ml nocodazole (Sigma, M1404), flasks were gently washed with PBS. Gentle shake-off was performed in 10ml of medium, monitoring the process under a microscope to avoid detaching clumps of cells in interphase.

The cell suspension was filtered through a double layer of 20µm filters (Millipore, NY2004700) and spun down. Typically, 10 7 cells were obtained from each preparation. Purity of mitotic preparations were systematically checked under a fluorescence microscope after fixation, cytospinning and DAPI staining. Preparations with more than 5% contamination from interphase were discarded.

Chromatin preparation for Esrrb binding analysis:

In previous reports [START_REF] Van Den Berg | Estrogen-related receptor beta interacts with Oct4 to positively regulate Nanog gene expression[END_REF] describing Esrrb ChIP analysis a dual crosslinking strategy with DSG and FA was used. Therefore, we initially applied similar conditions. However, in order to prove that Esrrb binding is not necessarily indirect, as it may be the case when DSG is required, we established an alternative protocol using FA only and digested chromatin with MNase rather than by sonication. Similar ChIP-Seq profiles were observed (Bioinformatic analyses section).

Chromatin preparation (DSG-FA-Sonication): 10

RNA isolation and Reverse Transcription:

Total RNA was isolated in Trizol (Invitrogen, 15596026) and digested with DNase I (Qiagen, 79254).

Reverse transcription reactions were performed with random hexamers on 1µg of total RNA (First Strand cDNA kit, Roche, 04379012001).

Quantitative real-time PCR:

Real-time PCR reactions were performed in quadruplicate for pre-mRNA expression analysis and duplicate for ChIP analysis in 384-wells plates with a 480 LightCycler (Roche) using LightCycler 480 SYBR Green I Master (Roche, 04707516001). Standard and melting curves were generated to verify the amplification efficiency (>85%) and the production of single DNA species. PCR primer sequences are 36/59 listed in Supplementary Information Table 4. Values for gene expression were normalised to the levels of Tbp mRNA.

FACS:

Ccna-GFP EKOiE ES cells were incubated with 20µM Hoechst 33342 for 45min, filtered through a 40µm cell strainer and kept on ice. ES cells were sorted based on Hoechst and GFP levels in 3 populations (EarlyG1, LateG1 and G2) using a FacsARIA III (Becton-Dickinson), keeping samples and collection tubes at 4°C. Data was analysed using the FlowJo software suite (Tree Star). Around 10 6 cells were sorted per population before RNA extraction. For replating experiments, sorted cells were cultured for 7 hours, incubated 45 minutes with Hoechst, trypsinised and analysis with a LSR II Fortessa (Becton-Dickinson).

Bioinformatic analyses:

ChIP-Seq sequencing and alignment: ChIP-Seq libraries (Supplementary Information Table 5) were generated using a MicroPlex kit (Diagenode) and sequenced (50bp; HiSeq 2500 instrument, Illumina).

Reads were mapped to the mm9 genome assembly using Bowtie and allowing 3 mismatches using GeneProf Categorisation of DESEQ2 filtered peaks as Bookmarked or Lost regions: 14.559 regions passing DESEQ2 filter were divided in 5 classes (Fig. S3) according to statistical significance of their enrichment levels (versus -Dox) and the reproducibility of their identification in individual replicates:

Class_1 (450 peaks): Passed DESEQ only in Mitosis. However, most (437) were identified as peaks in at least two Interphase replicates and all but one were identified in at least one of our Interphase replicates or in an independent study 3 . Therefore, these peaks cannot be rigorously qualified as mitotic-specific. ChIP-qPCR on 3 Class_1 peaks, including the single peak that was not detected in any Interphase replicate (Fig. S4), confirmed this interpretation. S1.

ChIP-Seq binding profiles:

The ChIP-Seq profiles shown in Fig. 2 and Fig. S4 were obtained in SeqMonk designing 100bp windows every 10bp and quantitating the base coverage normalised to the total number of aligned reads of the pooled datasets. A smoothing window of 25 was then implemented.

The profiles shown in Supplementary Fig. 5 were obtained similarly but using 100bp windows sliding every 50bp.

Average profiles: All metaplots were calculated in SeqMonk using base coverage per million reads every 100bp. All Esrrb peaks overlapping with a +/-500bp region flanking mRNA 5'-ends were considered promoters, leaving the rest as enhancers.

Hierarchical clustering and heatmaps: The heatmaps shown in Fig. 3 and Fig. S3 were obtained in SeqMonk (RPKM quantification) using either 5kb-long regions centred on the middle of each peak or the exact peak coordinates, respectively. To generate B1, B2, L1 and L2 groups a euclidean clustering based on the smallest absolute difference between region quantification values was used.

40/59 (Supplementary Information Table 5). BAM/SAM files were generated for each aligned library and used as inputs for the iRNASeq pipeline [START_REF] Madsen | iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data[END_REF] . Raw read counts of exons (mRNA) and introns (pre-mRNA) were normalised and compared using edgeR as implemented in iRNASeq. Fold changes and associated FDR derived from multiple testing were generated for each annotated mRNA and pre-mRNA. We considered genes as upregulated in a given cell-cycle phase if displaying an exonic FDR < 0.1, an exonic mRNA FC > 0.5 and an intronic pre-mRNA FC > 0.3; for downregulated genes exonic FC < -0.5, intronic FC < -0.3 and exonic FDR < 0.1. From genes up/down-regulated in G2 we subselected those displaying a -0.6 < FC < 0.6 and FDR > 0.1 in both early and late G1 to identify genes responding to Esrrb levels in G2 only.

Correlation with Esrrb-binding regions: A TAD (http://chromosome.sdsc.edu/mouse/hic/download.html) was considered bookmarked when it included at least one Bookmarked peak. The fraction of Esrrb-responsive genes at each cell-cycle phase located in a bookmarked TADs or located within 25kb of a bookmarked peak was scored and compared to randomly selected genes (20 lists of 200 genes).

Statistics and Reproducibility:

The retention of Esrrb on mitotic chromosomes has been systematically observed by independent Contingency tables were analysed with a Fisher's exact test.

Figure 1 :

 1 Figure 1: Esrrb decorates the mitotic chromosomes. (a) Representative images of Esrrb-GFP (top) or

Figure 2 :

 2 Figure 2: Esrrb binds a subset of its interphase targets in mitosis. (a) Illustrative binding profile of

Figure 3 : 33 .

 333 Figure 3: Mitotic Esrrb binding at regions of high activity in interphase. (a) Hierarchical clustering ofEsrrb binding regions (numbers on the left) based on enrichment for the indicated marks or factors[START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF][START_REF] Brookes | Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs[END_REF] . The corresponding boxplots (median, 25 th /75 th percentiles) and respective whiskers (median

Figure 4 :

 4 Figure 4: Mitotic Esrrb binding occurs in the vicinity of key, highly transcribed genes, in vivo and in

Figure 5 :

 5 Figure 5: The Esrrb DNA binding motif drives mitotic binding. (a) The Esrrb consensus is shown

Figure 6 :

 6 Figure 6: Mitotic Esrrb binding is dynamic. (a) Esrrb-GFP signal before and after bleaching, at the

Figure 7 :

 7 Figure 7: Esrrb transcriptional control along the cell-cycle. (a) FACS profile of EKOiE cells expressing

Figure 8 :

 8 Figure 8: Potential Esrrb function as a mitotic bookmarking factor. This schematic diagram highlights

Flash 4 .

 4 0LT camera; NIS Elements 4.3 software. After photo-bleaching correction (EMBL Plugin CorrectBleach), deconvolution was performed with Volocity 5.4.0 (99% confidence and 23max iteration), using calculated PSFs and 0.11µm effective pixel size. Spinning Disk Fluorescence imaging for FRAP or acquisition of images showing exclusion of Nanog-GFP and Mutant Esrrb-GFP: WT or Mutant Esrrb-GFP EKOiE cells or Nanog-GFP E14Tg2a were kept at 37°C in a humidified atmosphere (7% CO2) during the entire duration of the experiments. Only for acquisition of images showing exclusion of Nanog-GFP and Mutant Esrrb-GFP, cells were incubated with 100nM Hoechst-33342 for 15 min before imaging. Images were acquired using a 63x oil immersion objective on a Zeiss AxioObserver Z1 microscope equipped with Yokagawa CSUX1 spinning-disk confocal setup, a Hamamatsu EMCCD ImageEM X2 camera and Volocity imaging software. For FRAP,

  Fixed cells were spread by dropping on pre-heated glass slides. Slides were incubated in PBST (PBS 0.1% Triton X-100; 10 mins), blocked in PBST/5% donkey serum (30 mins) and incubated (2h, RT) with primary antibodies (1:500 Esrrb from Perseus Proteomics H6-705-00 and/or 1:200 Nanog from Cosmobio RCAB0001P). After 3 washes with PBST, samples were incubated with goat anti-mouse or anti-rabbit secondary antibody (1:500; Alexa 488 or 594; Jackson ImmunoResearch, 715-545-151; 715-585-150; 711-545-152) over-night at 4ºC, washed, mounted (Vectashield; VectorLab, H1200) and imaged as for wide-field fluorescence. EKOiE cells ectopically expressing a SNAP-Esrrb fusion protein were cultured in the presence of 150nM SNAP-Cell TMR-Star (NEB, S9105S) over-night, washed and cultured for 2h in medium without TMR-Star before karyotype staining.

Class_2 ( 1216

 1216 ): Passed DESEQ filter both in Mitosis and in Interphase. They represent regions bound by Esrrb in Interphase and in Mitosis. Class_3 (314): Passed DESEQ only in Interphase but were identified as peaks in all 4 Mitosis replicates. These regions are characterised by robust Esrrb binding in Interphase and low but reproducible binding in Mitosis. Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network 38/59 Class_4 (6621): Passed DESEQ only in Interphase and were identified as peaks in the pooled Mitosis dataset but not in all Mitosis replicates. Class_5 (5958): Passed DESEQ only in Interphase and were never detected in any of the Mitosis replicates. They represent clear Interphase-specific Esrrb binding regions. Although Class_4 regions may represent weak binding in Mitosis, we followed a conservative approach and combined them with Class_5 regions into the list of Lost regions. In contrast, Classes_1,2,3 represent regions that bind Esrrb in Mitosis and Interphase (although at different degrees, see Figs. S3 and S4); they were combined into the list of Bookmarked regions. Coordinates and Class of each individual peak, together with the quantifications in all datasets are available in Table

  researchers (N.F, E.G.T, A.D and A.M). Statistical analyses of genome-wide studies were conducted with DESeq, EdgeR and Prism 5.00. Normality was assessed with D'agostino and Pearson test followed by one-way ANOVA using Kruskal-Wallis and Dunn's Multiple Comparison tests or Student t-tests.

Table 5 )

 5 [START_REF] Halbritter | analysis of high-throughput sequencing experiments[END_REF] . Aligned reads corresponding to Interphase, Mitosis and respective controls were .Identification of candidate peaks and statistical analysis: Peaks were called with MACS as implemented in SeqMonk using EKOiE -dox controls as background, a 10 -5 p-value threshold, and fragment sizes of

	separately pooled. Unless otherwise specified all analyses were performed with SeqMonk
	(http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). We generated 500bp-long
	windows sliding every 250bp and quantitated read counts per kb per million (RPKM) across the
	genome after excluding the Y chromosome (lost in EKOiE). Interphase_rep4 was a clear outlier in terms
	of fold-coverage and global correlation to other interphase replicates (Supplementary Information

Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network

Acknowledgements: We are grateful to Ian Chambers for allowing N.F and P.N to pursue observations made initially in his laboratory on the chromosomal retention of Esrrb during mitosis. We are grateful to A. Rada-Iglesias, G. Cecere, S. Tajbakhsh, M. Yaniv and members of our laboratory for critical reading of the manuscript. We acknowledge B. Jagla for running the iRNA-seq pipeline and P. Clerc for assistance with fluorescence microscopy. P.N acknowledges financial support to the Institut Pasteur, CNRS and the ANR Laboratoire d'Excellence Revive (Investissement d'Avenir; ANR-10-LABX-73). N.F was funded by an EMBO Long Term Fellowship (ALTF 876-2013) and a Marie-Curie Intra-European Fellowship (EFIMB -626705).

Data availability:

Primary NGS datasets have been deposited at Geneprof (gpXP_003043) and at the Gene Expression Omnibus (GEO) under accession number GSE75066. Previously published datasets are available under accession numbers GSE11431, GSE29218, GSE34518, GSE23943 and GSE57092. ChIP-Seq and RNA-Seq results as well as all primer sequences used in this study have been provided in Supplementary Tables 12345. All other data supporting our conclusions as well as the reagents that were generated are available upon reasonable request to P.N.

Author Contribution: This study was conceived by N.F and P.N. All experiments were carried out by N.F with the exception of embryo work performed by S.V.P, S.B and M.C.T. In addition, A.D, E.G.T and A.M provided technical help and F.M contributed to the analysis of FRAP assays. C.P performed next generation sequencing and P.N conducted the Bioinformatic analyses. All experiments were analysed and interpreted by N.F and P.N. The manuscript was written by P.N with inputs from N.F.

Competing Financial Interests:

The authors declare that they have no financial competing interests.

Supplementary Figures Legends

Supplementary Figure 1: Cell lines used in this study. (a) Esrrb immuno-fluorescence on EKOiE cells cultured in the presence or absence of Dox (24h withdrawal). Scale bars represent 30µm (b) The selfrenewal efficiency of wild-type E14Tg2a, TNG, EKOiE and the respective derivative cell lines was assessed by Alkaline Phosphatase staining of cell colonies obtained 6 days after clonal plating in the indicated conditions. The graph shows the average and s.d of two independent experiments, each performed in duplicate wells. E14Tg2a cells self-renew exclusively in the presence of LIF. Upon introduction of a Nanog-GFP transgene, self-renewal efficiency increases in the presence of LIF and is rescued in its absence. As expected, TNG cells (Nanog heterozygous) exhibit reduced self-renewal as compared to E14Tg2a [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF] . Upon knock-in of TdTomato at both Esrrb alleles, so that only Esrrb-TdT fusion protein is expressed in the cells, self-renewal efficiency is unaffected. EKOiE cells cultured without Dox show extremely severe defects in self-renewal efficiency as compared to Dox-treated cells, demonstrating the importance of Esrrb. In the presence of Dox, EKOiE cells also exhibit LIFindependent self-renewal. Upon the introduction of a constitutive Esrrb-GFP transgene, EKOiE cells are able to self-renew both in presence or absence of Dox; they also exhibit LIF-independent self-renewal.

We conclude from this analysis that all the GFP and TdT fusion proteins (Nanog and Esrrb) are functional. Note that across all regions the levels of Esrrb enrichment are significantly higher in interphase than in mitosis and all the controls. Also, note that in mitosis only Classes_1,2,3 show consistently high levels of enrichment. (e) Scatter plot representing enrichment levels in interphase (X-axis) versus mitosis (Yaxis) for the 5 classes of Esrrb binding regions. Note that Classes_1, 2 and 3 cluster together whilst Class_4 localises between Classes_1, 2 and 3 and Class_5. These analyses strongly support grouping 1: Esrrb enrichment at its binding regions. In this table the coordinates of all annotated peaks can be found along with the RPKM values calculated in each independent replicate.

Supplementary

Supplementary Information Table

Supplementary Information Table 2: Genome-wide expression analysis of EKOiE cells in early G1

, late G1 and G2. In this file the results of the RNA-Seq conducted for this study are presented in 3 tables (exonic counts, intronic counts, gene expression changes as established using edgeR within the iRNAseq pipeline). 3: Esrrb-dependent genes in early G1, late G1 and G2. In this file 6 tables describing the differentially expressed genes identified at each analysed cell-cycle phase are presented along with their associated FC and FDR. 4: Primer sequences. Sequences of all primer pairs used to validate the ChIP-Seq and RNA-Seq are provided.

Supplementary Information Table

Supplementary Information Table

Supplementary Information Table 5: Global statistics of NGS analyses.

In this file we provide global statistic parameters of our ChIP-Seq (2 tables) and RNA-Seq results (2 tables).

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Methods:

Cell lines:

No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. All the cell lines generated for this study were derived from E14Tg2a, tested for mycoplasma and their identity authenticated by several means including resistance to drug selection, PCR and sequencing of specific genomic regions.

EKOiE and EKOiE Mut:

The first exon of both Esrrb alleles of E14Tg2a cells was flanked by LoxP sites as described [START_REF] Festuccia | Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells[END_REF] and transfected with a CAG-rtTA-IRES-blasticidin R and a doxycycline inducible tdTomato transgenes flanked by heterotypic FRT sites. Flp recombinase mediated cassette exchange (RMCE) was then used to exchange tdTomato with an Esrrb-IRES-Puromycin R cassette (wildtype or mutant Esrrb, see below). Both Esrrb alleles were then deleted by Cre recombinase.

EKOiE derivatives: EKOiE cells were stably transfected with CAG promoter driven wild-type Esrrb-5xGly-GFP, mutant Esrrb-5xGly-GFP, SNAP-Esrrb or Ccna-5xGly-GFP-IRES-puromycin R transgenes and homogeneously expressing clones selected. In mutant Esrrb, Glu121, Lys124, and Lys128 are mutated to glycine [START_REF] Gearhart | Monomeric complex of human orphan estrogen related receptor-2 with DNA: a pseudo-dimer interface mediates extended half-site recognition[END_REF] . Ccna-GFP includes only the mitotic degron of murine Cyclin A2 (residues 1-156) [START_REF] Den Elzen | Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase[END_REF][START_REF] Geley | Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint[END_REF] .

Nanog-GFP: E14Tg2a cells were stably transfected with a CAG-Nanog-GFP-IRES-puromycin R transgene.

EtdT: tdTomato-IRES-Blasticidin R and tdTomato-IRES-Hygromycin R were knocked-in downstream of the Esrrb ORF at each allele of TNG cells [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF] .

ES cells carrying ectopic

Esrrb binding sites: Two E14Tg2a clones bearing an FRT-flanked doxycyclineinducible tdTomato transgene and showing high and homogeneous expression were selected.

Microinjection:

All experiments were conducted according to the French and European regulations (EC Directive 86/609, French Law 2001-486) and were approved by the Institut Pasteur ethics committee (n° 2012-0011). (C57BL/6xSJL/J) F1 female mice were purchased from Janvier and mated with CD1-IGS male mice purchased from Charles River Laboratories, France. Fertilized eggs were obtained from 6 superovulated 3 weeks old females submitted to intra-peritoneal injection of Chronogest PMSG (2.5units/mouse, Centravet) followed by Chorulon (5units/mouse, Intervet) 42-48h later and then mated overnight with 6 males. Single blastomere microinjection was performed in 2-cell stage embryos in M2 medium using an IMT-2 inverted microscope (Olympus), micromanipulators (Leica Microsystems) and a manual microinjection system. Embryos were co-injected with mRNAs for Esrrb-tdTomato (275 or 550ng/μl) and mGFP (350ng/μL) and cultured in KSOM+AA+ D-Glucose (EmbryoMax, Millipore) for 48h. No statistical method was used to predetermine sample size; the experiments were not randomized; the investigators were not blinded to allocation during experiments and outcome assessment.

Imaging:

Culture conditions: Unless otherwise specified, cells were plated on IBIDI hitreat plates coated overnight with Poly-L-ornithine 0.01% (Sigma, P4957) at 4°C, washed and coated 2h with laminin (Millipore, CC095) 10 µg/ml in PBS. Cells were transferred to red-phenol-free medium before imaging.

Wide-Field Fluorescence and deconvolution: EtdT and Esrrb-GFP cells were incubated with 100nM

Hoechst-33342 for 45min before imaging (Fig. 1). Alternatively, cells were cultured in 50ng/ml 39/59 Expression of genes associated to Esrrb binding sites: All 4 clusters of binding regions identified by hierarchical clustering (Fig. 3) were used as inputs for GREAT 58 using standard 'basal plus extension' parameters. The first 5 tissues showing expression of the selected genes according to the Mouse Gene Informatics database are shown in Fig. 3c. In Fig. 3d all genes located within 25kb of each Esrrb-binding region were selected. If a gene was near two or more peaks belonging to distinct clusters, it was associated to one only in a prioritized manner (B1>B2>L1>L2). Next, RPKM values were calculated in several datasets corresponding to nuclear RNA-Seq, Ser2-phosphorylated RNAPII and H3K36me3.

De-novo motif discovery:

The RSAT peak-motif tool [START_REF] Medina-Rivera | RSAT 2015: Regulatory Sequence Analysis Tools[END_REF] was used on a virtual machine (rsat-vm-2015-10) to identify motifs enriched at Bookmarked or Lost regions using 6, 7 or 8bp-long seeds on both strands.

The significance of the motifs was established using a background model of Markov order adapted to the length of the sequences. The corresponding TFs were identified on cisBP and JASPAR databases as implemented in RSAT. To search for putative Esrrb binding sites in all the regions a background model was used with a Markov order of 1.

RNA-seq sequencing and alignments: Libraries for RNA-Seq (Supplementary Information Table 5) were prepared using the TruSeq Stranded mRNA Sample preparation kit (Illumina) with rRNA-depleted samples (Ribo-Zero Gold kit -Illumina). Strand-specific sequencing (50bp) was performed on a HiSeq 2500 instrument (Illumina). Reads were aligned to the mm9 genome using Bowtie (allowing 50 hits per read; Supplementary Information Table 5).

RNA-Seq quantification and identification of differentially expressed genes: Read counts were quantified as RPKM in SeqMonk. High Pearson correlations were observed between all datasets